Immunogenic proteins and nucleic acids encoding the same

Abstract

The invention relates to proteins and nucleic acids for immunization regimens, in particular, immunogens that can prime rare bnAb precursor B cells and guide their maturation towards bnAbs capable of neutralizing diverse HIV strains, modifications thereof, and / or development of nanoparticles, and / or development of membrane-anchored immunogens, and methods of making and using the same. The invention also encompasses cell surface trimers that bind to the broadly neutralizing antibodies and / or nucleic acids encoding the same.

Description

IMMUNOGENIC PROTEINS AND NUCLEIC ACIDS ENCODING THE SAMEINCORPORATION BY REFERENCE

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 642,550, filed May 3, 2024, and U.S. Provisional Patent Application No. 63 / 642,203, filed May 3, 2024, the entire contents of which are incorporated herein by reference in their entirety. All documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.FEDERAL FUNDING LEGEND

[0002] This invention was made with government support under grants number UM1 AI144462, UM1 A1100663, and R01 AH47826 awarded by the National Institute of Allergy and Infectious Diseases (NIAID). The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The invention relates to proteins and nucleic acids, such as, for example, mRNAs. for immunization regimens, modifications thereof, and / or development of nanoparticles, and / or development of membrane-anchored immunogens, and methods of making and using the same.BACKGROUND OF THE INVENTION

[0004] A vaccine for HIV-1 is urgently needed, as there are approximately 1.5 million new infections each year as of 2020 (www.unaids.org / en / resources / fact-sheet). The target of HIV neutralizing antibodies, the trimeric envelope (Env) spike, varies substantially in sequence across different HIV-1 isolates, indicating that a vaccine should induce ‘broadly neutralizing antibodies’ (bnAbs), antibodies capable of neutralizing diverse isolates (Burton and Hangartner, 2016, Annu Rev Immunol 34, 635-659). Potent HIV bnAbs develop in a small percentage of infected individuals, typically over an extended course of infection (Burton and Mascola, 2015, Nat Immunol 16, 571-576; Kwong and Mascola, 2018, Immunity 48, 855-871). Passive immunizationwith HIV-1 bnAbs has been shown to protect against simian / human immunodeficiency virus challenge in non-human primates (Pegu et al., 2019, Immunity 48, 855-871 ; Pegu et al., 2017, Immunol Rev 275, 296-312) and to be capable of protecting humans against HIV-1 infection by neutralization-sensitive isolates (Corey et al., 2021, N Engl J Med 384, 1003-1014). Vaccine induction of bnAbs is regarded as having potential to protect against HIV, but bnAb elicitation in humans has not yet been achieved.

[0005] HIV bnAbs target at least five major epitopic regions on the Env trimer: V2-apex, V3- glycan, CD4 binding site, gp!20 / gp41 interface, and membrane proximal external region (MPER). Here, Applicants focus on germline-targeting immunogens for initiating the induction of bnAbs, in particular, designing immunogens that can prime rare bnAb precursor B cells and guide their maturation towards bnAbs capable of neutralizing diverse HIV strains such as targeting the CD4 binding site or MPER. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3 -dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. Germline-targeting priming in particular can aid in the development of vaccines to induce several different classes of bnAbs targeting different epitopes in order to achieve optimal neutralization coverage coverage, increasing the need for effective HCDR3- dominant germline-targeting.

[0006] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.SUMMARY OF THE INVENTION

[0007] The present invention relates to non-naturally occurring proteins, which may be involved in forming immunogenic proteins of the present invention.

[0008] The invention relates to a non-naturally occurring protein, which may comprise any one of the sequences in Tables 1-4. In some embodiments, the non-naturally occurring protein can comprise any one of the sequences in Table 2. In some embodiments, the non-naturally occurring protein can comprise any one of the sequences in Table 3. In some embodiments, the non-naturally occurring protein can comprise any one of the sequences in Table 4.

[0009] The protein may have at least 90% or 95% homology or identity with the sequence of the non-naturally occurring protein(s) of the invention.

[0010] The invention also encompasses trimers, which may comprise any one of the non- naturally occurring protein(s) of the invention.

[0011] The invention also encompasses nucleic acids encoding the non-naturally occurring protein(s) of the present invention, including nucleic acids that encode protein(s) that may have at least 90% or 95% homology or identity with a nucleotide encoding the sequence of the non- naturally occurring protein(s) of the invention.

[0012] The invention also encompasses eliciting an immune response which may comprise systemically administering to an animal in need thereof an effective amount of any one of the non- naturally occurring protein(s) or any one of the nucleic acids encoding the non-naturally occurring protein(s) of the present invention, including nucleic acids that may have at least 90% or 95% homology or identity with a nucleotide encoding the sequence of the non-naturally occurring protein(s) of the invention. Advantageously, the nucleic acid is formulated in lipid nanoparticles (LNPs). The animal may be a mammal, advantageously a human.

[0013] The invention also relates to a non-naturally occurring mRNA, which may encode a protein having the sequence of any one of the sequences in Tables 1-4. In some embodiments, the non-naturally occurring mRNA can encode a protein having any one of the sequences of Table 2. In some embodiments, the non-naturally occurring mRNA can encode a protein having any one of the sequences of Table 3. In some embodiments, the non-naturally occurring mRNA can encode a protein having any one of the sequences of Table 4.

[0014] The mRNA may encode a protein that has at least 90% or 95% homology or identity with the sequence of any one of the sequences in Tables 1-4. In some embodiments, the mRNA may encode a protein that has at least 90% or 95% homology or identity with the sequence of any one of the sequences in Table 2. In some embodiments, the mRNA may encode a protein that has at least 90% or 95% homology or identity with the sequence of any one of the sequences in Table 3. In some embodiments, the mRNA may encode a protein that has at least 90% or 95% homology or identity with the sequence of any one of the sequences in Table 4.

[0015] The invention also encompasses eliciting an immune response which may comprise systemically administering to an animal in need thereof an effective amount of any one of the non- naturally occurring mRNAs of the present invention, including mRNAs that may have at least 90% or 95% homology or identity with the non-naturally occurring mRNA(s) of the invention.Advantageously, the mRNA is formulated in lipid nanoparticles (LNPs). The animal may be a mammal, advantageously a human.

[0016] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112(a)) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant s) in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

[0017] It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of’ and “consists essentially of’ have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0018] These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0020] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0021] Figure 1. Design and characterization of the core-g28v2 60mer boost immunogen. (A) Shown is the iterative immunogen design workflow diagram to improve upon the starting HxB2 core-e-2cc N276D immunogen. (B) Shown is a surface representation of a computational model of core-g28v2 monomer colored with CD4bs (yellow), N276D and T278M mutations (orange), resurfaced residues (pink), natural glycosylation sites (green), engineered glycosylation sites (blue), and mutations to add potential CD4+T helper cell epitopes conserved with the HIV env trimer (TH6 residues, purple). (C) KD values were measured by SPR for mAbs elicited by eOD-GT8 60mer protein in humans and SE09 mice for first-boost immunogen candidates eOD- GT6v2-cRSF, core-g28v2, and 191084-N276D. Thick lines indicate median values, boxes show 25 and 75% percentile values. *The low-capture IgG SPR method may include some avidity for trimeric analytes.

[0022] Figure 2. Comparison of protein boost immunogens. (A) Shown is the immunization scheme for evaluating boost immunogen candidates delivered as proteins plus adjuvant in SE09 mice. (B) The frequency of antigen++MBCs among total MBCs was analyzed by flow cytometry. Each group was sorted with matched antigens, except for the PBS placebo group which was sorted with the core-g28v2 probe. (C to E) Shown is the frequency of VRCOl-class MBCs among antigen++MBCs (C), the frequency of VRCOl-class MBCs among total MBCs (D), and the frequency of VRCOl-class MBCs with human VK1-33 light chains among total MBCs (E). The blue dashed bar in (E) indicates the overall frequency of VRCOl-class MBCs with human VK1-33 light chains among total MBCs within each group. Red bars indicate medians and each point represents an individual mouse for (B to E). (F and G) The median percent aa SHM in the VH gene (F) and in the VK / VL genes (G) is shown for all VRCOl-class MBCs. (H and I) The median percent aa SHM in the VH gene (H) and in the VK / VL genes (I) is shown for non-VRCOl -class MBCs. Each point represents the median per mouse and the red bars indicate the median of medians for panels (F to I). Statistical comparisons were made by Kruskal -Wallis test followed by Dunn’s test for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001; ns, not significant.

[0023] Figure 3. mAb SPR and serum antibody binding responses after eOD-GT8 60mer priming and core-g28v2 boosting. (A) Monovalent KD values were measured by SPR of VRCOl- class mAbs from naive SE09 mice, eOD-GT8 60mer primed sE09 mice, and eOD-GT8 60mer primed, core-g28v260mer boosted SE09 mice for eOD-GT8, core-g28v2, and core-g28v2-N276+. Each point represents the KD of a single antibody. Red bars indicate median affinities and includenon-binders. Geomean affinities were calculated among binders only. (B) Shown is serum IgG ELISA binding to eOD-GT8, eOD-GT8-KO, core-g28v2, and core-g28v2-KO for SE09 mice primed with eOD-GT8 60mer protein (purple) alone or SE09 mice primed with eOD-GT8 60mer protein and boosted with core-g28v260mer protein (pink). Each point represents the half-maximal effective dilution (ED50) of serum per mouse. Red bars indicate median ED50 values. Statistical comparisons were made by Kruskal -Wallis test followed by Dunn’s test for multiple comparisons. **p<0.01, ***p<0.001; ns, not significant.

[0024] Figure 4. Key VRCOl-class heavy chain residues after protein immunization. (A) Shown is a ribbon diagram of the VRC01 variable fragment (Fv) with key VRCOl-class heavy chain residues colored green for non-paratope residues and pink for paratope residues. (B) A molecular surface representation of HIV Env shows contact residues of key VRCOl-class heavy chain paratope residues (red). (C) Shown is the 90thpercentile number of key VRCOl-class heavy chain residues elicited during immunization experiment described in Fig. 2A. Each point is the 90thpercentile number of key VRCOl-class heavy chain residues for each mouse with the red bars indicating the median of the 90thpercentile values for each group. Statistical comparisons were made by Kruskal -Wallis test followed by Dunn’s test for multiple comparisons. **p<0.01; ns, not significant. (D) Shown are key VRCOl-class heavy chain residues for all VRCOl-class sequences recovered for placebo and core-g28v2 boost groups. Numbers at top indicate positions within the antibody, colored as in (A); column at left indicates data from each mouse in a different color; each row indicates a single VRCOl-class sequence, with red boxes indicating the presence of nongermline key VRCOl-class heavy chain residues. Residue numbers in panels (A), (B), and (D) use the Kabat antibody numbering scheme (T.T. Wu et al., 1970). (E to G) KD values between core- g28v2 and mAbs isolated after core-g28v2 60mer boosting were correlated with number of key VRCOl-class heavy chain residues (E), percent Vn SHM (F), and percent VK SHM (G). In (E to G), solid lines show correlations, dashed lines show 95% confidence internal, S represents slope, and p represent the P-value from a simple linear regression test, ns, not significant.

[0025] Figure 5. Immune response elicited using mRNA / LNP immunization. (A) Shown is the immunization scheme for evaluating immunogens delivered using mRNA / LNPs in SE09 mice. (B) The frequency of antigen++MBCs among total MBCs was analyzed by flow cytometry. (C to E) Shown is the frequency of VRCOl-class MBCs among antigen++MBCs (C), the frequency of VRCOl-class MBCs among total MBCs (D), and the frequency of VRCOl-class MBCs withhuman VK1-33 light chains among total MBCs (E). Red bars indicate medians and each point represents an individual mouse for panels (B to E). Statistical comparisons were made by Kruskal- Wallis test followed by Dunn’s test for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant.

[0026] Figure 6. Key VRCOl-class heavy chain residues, mAh SPR, and serum antibody binding after mRNA / LNP immunization. (A) Shown are monovalent KD values measured by SPR of VRCOl-class mAbs from naive SE09 mice, eOD-GT8 60mer mRNA / LNP primed SE09 mice, and eOD-GT8 60mer mRNA / LNP primed, core-g28v2 60mer mRNA / LNP boosted SE09 mice for eOD-GT8, core-g28v2, and core-g28v2-N276+. Red bars indicate median affinities and include non-binders. Geomean affinities were calculated among binders only. (B) Shown is serum IgG ELISA binding to eOD-GT8, eOD-GT8-KO, core-g28v2, and core-g28v2-KO for SE09 mice primed with eOD-GT8 60mer mRNA / LNPs (magenta) only or primed and boosted with core- g28v2 60mer mRNA / LNPs (green). Each point represents the ED50 value of serum per mouse. Red bars indicate median EDso values. (C) Shown are the 90thpercentile values for key VRCOl- class heavy chain residues elicited during mRNA / LNP immunization experiment described in Fig. 5 A. Each point is the 90thpercentile key VRCOl-class heavy chain residues for each mouse with the red bars indicating the median of the 90thpercentile values for each group. Statistical comparisons in (B and C) were made by Kruskal -Wallis test followed by Dunn’s test for multiple comparisons. *p<0.05, **p<0.01, ****p<0.0001; ns, not significant. (D) Key VRCOl-class heavy chain residues for all VRCOl-class sequences recovered for placebo and core-g28v2 boost groups. Numbers at top indicate positions within the antibody, colored as in (Fig. 4A); column at left indicates data from each mouse in a different color; each row indicates a single VRCOl-class sequence, with red boxes indicating the presence of non-germline key VRCOl-class heavy chain residues. Residue numbers use the Kabat antibody numbering scheme (T.T. Wu et al., 1970). (E to G) KD values between core-g28v2 and mAbs isolated after core-g28v2 60mer mRNA / LNP boosting were correlated with number of key VRCOl-class heavy chain residues (E), percent VH SHM (F), and percent VK SHM (G). In (E to G), solid lines show correlations, dashed lines show 95% confidence internal, S represents slope, and p represent the P-value from a simple linear regression test.

[0027] Figure 7. Post core-g28v2 mAbs bind N276(-) native-like HIV Env trimers and neutralize corresponding pseudoviruses. (A) Shown are apparent KD values measured by SPRof VRCOl-class mAbs elicited in SE09 mice after priming with eOD-GT8 60mer mRNA / LNPs and boosting with core-g28v2 mRNA / LNPs, for wildtype and N276(-) native-like HIV Env trimers. (B) Shown are apparent KD values measured by SPR of VRCOl-class mAbs elicited in SE09 mice after priming with eOD-GT8 60mer protein + adjuvant and boosting with core-g28v2 60mer protein + adjuvant, for wildtype and N276(-) native-like HIV Env trimers. Thick lines indicate median values, boxes show 25 and 75% percentile values (A and B). (C) Pseudoviruses neutralization was tested using a panel of 15 VRCOl-class mAbs elicited in SE09 mice after eOD- GT8 60mer mRNA / LNP priming and core-g28v2 60mer mRNA / LNP boosting. Murine leukemia virus (MLV) was used as a negative control. No neutralization: NN; ICso > 50 pg / mL.

[0028] Figure 8. Characterization of the Vnl-2JH2 / VKl-33hTdT(SE09) mouse model. (A) Illustration of genetic modifications in the Igh and Igk locus of the Vnl-2JH2 / VKl-33hTdT(SE09) rearranging mouse model. The mouse VH81X was replaced with the human VH1-2 and the mouse JHS were replaced with the human JH2. The intergenic control region 1 (IGCR1) regulatory element in the Vn-to-D intervening region was deleted. The mouse VK3-2 was replaced with human VKI- 33 plus a CTCF-binding element (CBE) 50 bp downstream of its recombination signal sequence. The human TdT gene was knocked into mouse Rosa locus. (B) Frequency of VRCOl-class naive B cells in humans (Rantalainen et al., 2020; Ofek et al., 2010; Correia et al., 2014) and in SE09 mice after sorting with eOD-GT8. Solid red lines indicate overall frequency (total VRCOl-class naive B cells among all samples divided by total number of naive B cells sorted among all samples). (C) Frequency of VRCOl-class naive B cells with human VK1-33 LCS in humans (Rantalainen et al., 2020; Ofek et al., 2010; Correia et al., 2014) and in SE09 mice after sorting with eOD-GT8. Solid red lines in (B and C) indicate overall frequency. Dashed blue lines in (B and C) indicated median frequencies. (D) LCDR3 amino acid logo plots for VRCOl-class BCRs. (E) Monovalent KD values measured by SPR of VRCOl-class naive precursors isolated from humans and SE09 mice for binding to eOD-GT8. (F) Monovalent KD values measured by SPR of VRCOl-class naive precursors with human VK1-33 light chains isolated from humans and SE09 mice for binding to eOD-GT8. Red lines in (E and F) denotes the median.

[0029] Figure 9. Sequence alignment of immunogens. (A) Immunogens aligned to HxB2 reference sequence with CD4bs (green), stabilization (red), glycan masking (blue), and TH6 (purple) mutations highlighted. (B) Table showing the number of mutations within the CD4bs epitope and the corresponding percent native.

[0030] Figure 10. Biophysical characterization of nanoparticle immunogens. (A) Normalized SEC profiles for core-g560mer, core-g2860mer, and core-g28v260mer plotting normalized milli-absorbance unit (mAU) versus elution volume. (B) Computational model of core-g28v2 60mer showing lumazine synthase (pink), N-linked glycans (blue), CD4bs (yellow), and gpl20 peptide (green). (C) Negative stain electron micrograph of purified core-g28v2 60mer. (D) Differential scanning calorimetry for assessing stability of core-g5 60mer, core-g28 60mer, and core-g28v2 60mer (red: raw data; blue: 2-state model fit). (E) Nanoparticle yield data following Galanthus nivalis lectin (GNL) affinity chromatography and SEC. (F) SPR data for non- VRC01 -class mAbs from IAVI G001 samples. (G) SPR affinity data for core-g28v2, showing affinity gradient of human VRC01 -class naive precursors, eOD-GT8 induced VRC01 -class mAbs (IAVI G001), and VRCOl-class bnAbs. (H) SPR affinity data for VRCOl-class naive precursors with human VK1-33 light chains isolated from SE09 mice binding to eOD-GT8 and core-g28v2. (I) Glycan occupancy data for purified core-g28v2 60mer as determined by mass spectrometry. Red lines indicate medians in (F to H).

[0031] Figure 11. SPR affinity measurements for VRCOl-class mAbs elicited after priming with eOD-GT8 60mer protein in SE09 mice and humans. (A) KD values were measured by SPR for mAbs elicited by the indicated first boost candidates. Data from Fig. 1C are included here to facilitate easy comparison among all booster immunogen candidates. Thick lines indicate median values, boxes show 25 and 75% quantiles. *Low-capture IgG SPR method many include some avidity for trimeric analytes. (B) Table listing immunogens with valency and CD4bs epitope mutations.

[0032] Figure 12. Representative FACS gating scheme for the isolation of antigen specific MBCs and BCR sequencing workflow.

[0033] Figure 13. Comparison of additional protein booster immunogens. (A) Shown is the immunization scheme for evaluating additional boost immunogen candidates delivered as adjuvanted proteins in SE09 mice. (B) The frequency of antigen++MBCs among total MBCs was analyzed by flow cytometry. Each group was sorted with matched antigens. (C to E) Shown is the frequency of VRCOl-class MBCs among antigen++MBCs (C), the frequency of VRCOl-class MBCs among total MBCs (D), and the frequency of VRCOl-class MBCs with human VK1-33 light chains among total MBCs (E). (F and G) The median percent amino acid SHM in the VH gene (F) and in the VK / VL genes (G) is shown for all VRCOl-class MBCs. (H and I) The median percentamino acid SHM in the VH gene (H) and in the VK / VL genes (I) for all non-VRCOl -class MBCs. Each point represents the median per mouse and the red bars indicate the median of medians for panels (B to I). The core-g28v2 60mer protein data from Fig. 2 is included in panels B through I to facilitate comparison. Statistical comparisons were made by Kruskal-Wallis test followed by Dunn’s test for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001,

[0034] ****p<0.0001; ns, not significant.

[0035] Figure 14. Key VRCOl-class heavy chain residues in VRCOl-class bnAbs and elicited after eOD-GT8 60mer protein immunization. (A) Key VRCOl-class heavy chain residues among VRCOl-class bnAbs. (B) Key VRCOl-class heavy chain residues among GT8 protein primed.

[0036] Figure 15. SHM analysis of VRCOl-class BCRs induced by mRNA / LNP immunization. (A) Median percent amino acid SHM in the VH gene among VRCOl-class MBCs induced after mRNA / LNP immunization. (B) Median percent amino acid SHM in the VK / VL genes among VRCOl-class MBCs induced after mRNA / LNP immunization.

[0037] Figure 16. Comparison of protein plus adjuvant immunization versus immunization with mRNA / LNPs. Select data repeated from Fig. 2D, 2E, and 2F; Fig. 4C; Fig. 5C and 5D; Fig. 6A; and Fig. 15 A. (A) The frequency of VRCOl-class MBCs among total MBCs after priming with eOD-GT8 60mer in a protein (pt) or mRNA / LNP format. (B) The frequency of VRCOl-class MBCs with human VK1-33 light chains among total MBCs after priming with eOD- GT8 60mer. (C) Median percent amino acid SHM in the VH gene among all VRCOl-class MBCs after priming with eOD-GT8 60mer. (D) 90th percentile key VRCOl-class heavy chain residues elicited after priming with eOD-GT8 60mer. Statistical comparisons were made by Mann- Whitney. *p<0.05, **p<0.01; ns, not significant.

[0038] Figure 17. LCDR3 logo plots for VRCOl-class VK1-33 BCRS. (A and B) Logo plots are shown for BCRs analyzed after eOD-GT8 mRNA priming (A) and after core-g28v2 mRNA boosting (B).

[0039] Figure 18. Comparison of protein plus adjuvant immunization versus immunization with mRNA / LNPs. Select data repeated from Fig. 2D, 2E, and 2F; Fig. 4C; Fig. 5C and 5D; Fig. 6A; and Fig. S8A. (A) The frequency of VRCOl-class MBCs among total MBCs after priming with eOD-GT8 60mer and boosting with core-g28v2 60mer. (B) The frequency of VRCOl-class MBCs with human VK1-33 light chains among total MBCs afterpriming with eOD-GT8 60mer and boosting with core-g28v2 60mer. (C) Median percent amino acid SHM in the VH gene among all VRC01 -class MBCs after priming with eOD-GT8 60mer and boosting with core-g28v2 60mer. (D) 90th percentile key VRCOl-class heavy chain residues elicited after priming with eOD-GT8 60mer and boosting with core-g28v2 60mer. Statistical comparisons were made by Mann-Whitney. *p<0.05, **p<0.01; ns, not significant.

[0040] Figure 19. (A) Key VRCOl-class heavy chain residues among GT8 mRNA primed. (B) GT8 mRNA primed followed by placebo boost (sorted with GT8). (C) Key VRCOl-class heavy chain residues among GT8 mRNA primed followed by GT8 mRNA boost (sorted with GT8). (D) Key VRCOl-class heavy chain residues among GT8 mRNA primed followed by GT8 mRNA boost (sorted with core).

[0041] Figure 20. SPR / BLI analyses of VRCOl-class mAh specificity. (A) Monovalent KD SPR / BLI analyses of VRCOl-class mAb specificity. (A) Monovalent KD isolated from SE09 mice after boosting with core-g28v2 60mer mRNA / LNPs. (B) VRC01 Fab competition BLI for core- g28v2 versus VRCOl-class (VRCOlc) and non- VRCOl-class (nonVRCOlc) mAbs elicited after boosting with core-g28v2 60mer mRNA / LNPs. Data shown across four plots for ease of viewing.

[0042] Figure 21. SPR analysis of non-VRCOl-class mAb affinity and specificity. (A) Monovalent KD values measured by SPR for eOD-GT8, eOD-GT8-KO, and core-g28v2 to non- VRCOl-class mAbs isolated from SE09 mice after priming with eOD-GT8 60mer mRNA / LNPs. (B) Monovalent KD values measured by SPR for core-g28v2 and core-g28v2-KO to non-VRCOl- class mAbs isolated from SE09 mice after priming with eOD-GT8 60mer mRNA / LNPs and boosting with core-g28v2 60mer mRNA / LNPs. Black lines indicate medians.

[0043] Figure 22. SHM analysis of non-VRCOl-class BCRs induced by mRNA / LNP immunization. (A) Median percent amino acid SHM in the VH gene among non-VRCOl-class MBCs induced after mRNA / LNP immunization. (B) Median percent amino acid SHM in the VK / VL genes among non-VRCOl-class MBCs induced after mRNA / LNP immunization. Each point represents the median per mouse and the red bars indicate the median of medians.

[0044] Figure 23. Pseudovirus neutralization activity of polyclonal IgGs isolated from serum after eOD-GT8 60mer mRNA / LNP priming or boosting with core-g28v2 mRNA / LNPs. No neutralization: NN; IC50 > 50 pg / mL.

[0045] Figure 24. SPR studies of boost candidate interactions with VRCOl-class mAbs. (A) SPR affinity data for VRCOl-class mAbs elicited by eOD-GT8 60mer protein immunizationin the Vnl-2 mouse model (Verkoczy et al., 2011). (B) SPR affinity data for VRCOl-class mAbs elicited by eOD-GT8 60mer protein immunization in humans binding to gp!20 core variants with or without the T278M mutation. Red lines indicate medians.

[0046] Figure 25. Detection of BG18-like responses in rhesus macaques. (A) The definition of a BG18 type I HC precursor that was used for searching naive B cells plotted in (B) for humans (HuPrel) and macaques (RmPrel and RmPre2), where prel and pre2 refer to different alleles. The position of the FG(V / L) was allowed to start at position 7, 8, or 9 of the HCDR3. The alignment shows the FG(V / L) starting at position 8. The BG18-inferred germline (BGI8 GL0) HCDR3 sequence is shown for comparison. Black indicates templated regions of the HCDR3; blue and red indicate junction regions. (B) Frequency of BG18 type I HC sequences in 14 human donors and 60 rhesus macaques. (C) Immunization schedule of NHPs (n=8) with N332-GT5 + SMNP and sampling time points for which sorting and BCR sequencing were carried out. ED, escalating dose; LN FNA, lymph node fine needle aspirate; PBMC, peripheral blood mononuclear cells. (D) Frequency of Env+GC B cells as a percentage of total CD20 B cells. The gray area is set from 0.001% to the median frequency of Env+GC B cells observed in the pre-immunization samples. Lines indicate the median + / - interquartile range. (E and H) Number of BG18 type I sequences identified in (E) GC or (H) memory B cells at different time points, with each circle representing a different animal. (F and I) Frequency of BG18-like sequences among Env+B cells from (F) GC or (I) memory B cells. (G and J) Frequency of BG18-like sequences among (G) GC or (J) memory B cells. (E - J) Bars indicate the median + / - interquartile range for the responders. For (E and F) “All” indicates the result of combining all sequences from weeks 3 to 13. In (G) “All” indicates the median frequency among the responders from weeks 3 to 13 at the time points for which responses were detected. In E-G, MD39 group (n=4) indicates Env+sequences isolated from GC B cells at weeks 3, 4, 7 and 10 after priming with BG505 SOSIP MD39 trimer.

[0047] Figure 26. BG18 type I lineages and structural analysis. (A) Gene segment assignment and representative HCDR3 sequence of 38 BG18 type I lineages. The D3-41 gene is colored blue and two critical contact residues are colored red. D3-41 in alternate reading frame is colored green. (B)-(E) upper panels show cryo-EM models of (B) BG18 GL0 Fab in complex with N332-GT2 (PDB ID: 6DFH), (C) canonical BG18 type I macaque Fab RM N332 03 in complex with N332-GT5 (KD, 16 pM), (D)BG18 type IFab RM_N332_36 with D3-41 in alternate reading frame in complex with N332-GT5 (KD, 38 pM), (E) BG18 type I Fab RM_N332_32 withkappa light chain in complex with N332-GT5 (KD, 81 pM). (B - E) lower panels show magnified view of HCDR3 interacting with gpl20 for each structure; gp!20 is colored gray and the N332 epitope (N332-GT2 residues Gly324, Asp325, Val326, Arg327, Met328, Ala329, His330, Ile415, Leu416 and Pro417 or the equivalent position in N332-GT5) colored red; HCDR3s are colored blue, orange, purple, green for BG18_GLo, RM_N332_03, RM_N332_36, and RM_N332-32, respectively.

[0048] Figure 27. Affinity maturation of BG18 type I and other N332-GT5-elicited antibodies. (A) SHM of Vn genes in BG18 type I and other Env+sequences from GC B cells. (B) SHM of VH genes in BG18 type I and other Env+sequences from memory B cells. (C) SHM of Vi. genes in BG18 type I and other Env+sequences from GC B cells. (D) SHM of Vi. genes in BG18 type I and other Env+sequences from memory B cells. For (A-D), each dot represents the median SHM for one animal, and the median of the medians + / - interquartile range for all animals is plotted. (E) SPR KDS for BG18 type I Fabs isolated at weeks 3, 4, 7 and 10 post prime from GC B cells binding to N332-GT5 or N332-GT5-KO. The dotted line at 2 xl O'5M indicates no binding at the highest concentration tested. The dotted line at 1.6x10-11M represents the approximate upper affinity limit of the instrument. Lines indicate the median with interquartile range. Each data point is representative of 1 to 4 technical replicates. (F) SPR KDS for BG18 type I Fabs compared to other epitope-specific Fabs binding to a panel of boost candidates that are more similar to a native trimer than N332-GT5. Data points represent 1 technical replicate. The dotted line at 2 xlO'5M indicates no binding at the highest concentration tested. The dotted line at 1.6xl0’nM represents the approximate upper affinity limit of the instrument.

[0049] Figure 28. Identification of a broader class of BG18-like antibodies. (A) HCDR3 length distribution of Env+GC B cells and epitope-specific memory B cells isolated at wk 12 after filtering out the BG18 type I sequences. (B) SPR KDS of Fabs derived from activated B cell supernatants that were positive for binding to BG505 B23 by ELISA. Each data point represents 1 technical replicate. The dotted line at 2 xl0‘5M indicates no binding at the highest concentration tested. The dotted line at 1.6xl0'nM represents the approximate upper affinity limit of the instrument. (C) HCDR3 sequence and gene segment assignment of two Fabs that bind with high affinity to the BG505_B23 trimer for which cryo-EM structures were determined. (D) Cryo-EM model of N332-GT2 in complex with BGI8 GL0 (PDB ID: 6DFH). (E) Cryo-EM model of Fab RM_N332_07 in complex with N332-GT5. (F) Cryo-EM model of Fab RM_N332_08 in complexwith N332-GT5. (G) Diagram showing definition of latitudinal, longitudinal, and HC-LC twist angles. (H) Latitudinal, longitudinal, and HC-LC twist angles are shown for BG18 (PDB id: 6DFG), BG18_iGLo (6DFH), HMP1 (6NF5), HMP42 (6NFC), RM N332 03, RM N332 32, RM N332 36, RM N332 07, RM N332 08 in yellow circles and six other N332-dependent bnAbs (PGT122 [4TVP], DH270.6 [6UM6], BF520.1 [6MN7], PGT128 [5ACO], QA013.2 [7N65], PGT135 [4JM2]) in cyan circles.

[0050] Figure 29. Comparison of BG18 precursor frequency in macaques and humans.(A) Comparison of HCDR3 length distributions from human BCRs (J.M. Steichen et al., 2019; B. Briney et al. 2019) and macaques IgMs shows that humans have a higher frequency of longer HCDR3s. (B) HCDR3 length distribution separated by sequencing method. Human, all human BCR HC sequences from (J.M. Steichen et al., 2019; B. Briney et al. 2019); RM all, 98 datasets of rhesus macaque BCR HC sequences from multiple studies (see methods); RM all IgM (not this study), IgM sequences from “RM all“ with the 4 data sets from this study removed; RM 5’ RACE IgMs, RM IgM sequences using 5 ’RACE and constant region primers; RM 5’UTR IgMs, RM IgM sequences using 5’UTR primers and an IgM constant region primer; RM SP IgMs, RM IgM sequences using signal peptide region primers and constant region primers; RM (this study), the 4 animals that were sequenced in this study. (C) Difference in D gene usage frequency between humans and macaques shows a slightly higher frequency of D3-3 usage in the human repertoire compared to D3-41 usage in macaques. (D) Comparison of frequencies between humans and macaque naive sequences across multiple sequence definitions show that NHPs have lower frequencies of BG18-like precursors at all levels. (E) Criteria used to search for BG18 precursors in humans and RMs.

[0051] Figure 30. ELISA response. (A) Schematic of vaccination and time points of plasma collection. ELISA area under the curve showing plasma response at the indicated time points to(B) N332-GT5 trimer or (C) N332-GT5-KO captured by C-terminal His-tag. (D) The difference between the N332-GT5 and N332-GT5-KO responses. (E) Area under the curve ELISA response to the MD39-V3 peptide. Area under the curve ELISA response to (F) BG5O5 SOSIP MD39 or (G) BG505 SOSIP MD39 BB with mutations that block binding to the bottom of the trimer captured by PGT128 Fab. (H) The difference in area under the curves between MD39 and MD39 BB showing plasma response to the bottom of the trimer. For B-H, lines indicate the median with interquartile range (n=8).

[0052] Figure 31. Analysis of GC B cells. (A) Gating strategy for longitudinal analysis of GC B cells. N332-GT5-binding B cells are gated as N332-GT5-BV650+ / N332-GT5-BV42P dual binders, and termed “N332-GT5++” or “Env++” hereafter. (B) Quantification of GC B cell kinetics. The dotted line separates post-prime and post-boost timepoints. (C) Frequency of N332 epitopespecific (Env++K0‘) GC B cells among Env++GC B cells. (D) Frequency of Env GC B cells as a percentage of total CD20+B cells. The gray area is set from 0.001% to the median frequency of Env++GC B cells observed in the pre-immunization samples. (E) Frequency of N332 epitopespecific GC B cells as a percentage of total CD20+B cells. The gray area is set from 0.0001% to calculated limit of detection. (F) Quantification of Env++GC B cells per million lymphocytes recorded. They gray area is set from 1 to the median number of epitope-specific GC B cells observed in the pre-immunization samples. (G) Number of N332 epitope-specific GC B cells per million lymphocytes recorded. The gray area is set from 1 to the median number of epitope-specific GC B cells observed in the pre-immunization samples. Each circle represents a 1 mb aliquot of FNA cells. Data from FNAs from both the left and right side are graphed individually for each animal. Lines indicate the median with interquartile range.

[0053] Figure 32. Memory B cells sorting. (A) Gating strategy for analysis of week 10 IgD' memory B cells. (B) Antigen-specific (N332-GT5-AF647+N332-GT5-BV421+) memory B cells (CD20+IgD ) from Fig. 32A were backgated to assess surface immunoglobulin (Ig) expression by IgD versus IgG MFI signals. The gate revealed that most of the antigen-specific memory B cells expressed IgG, indicating successful identification of antigen-specific memory B cells. (C) Representative flow plot of antigen-specific memory B cells (gated as in Figure 32A) from a preimmunization PBMC sample compared to the post-immunization timepoint (week 12) from the same animal, confirming specificity of N332-GT5 and N332-GT5-KO probe staining. (D) Gating strategy for analysis of week 12 IgG+memory B cells. (E) Frequencies of N332-GT5++B cells among IgD' (week 10) or IgG+(week 12) memory B cells. (F) Frequency of N332 epitope-specific B cells among IgD' (week 10) or IgG+(week 12) memory B cells. (G) Frequency of N332 epitopespecific B cells among N332-GT5++B cells in IgD' (week 10) or IgG+(week 12) memory B cells. For E-G, lines indicate the median with interquartile range (n = 8).

[0054] Figure 33. Schematic for sorting, B cell activation, functional screening and sequencing memory B cells. (A) Overall strategy for sorting memory B cells, functional characterization and sequencing. (B) ELISA OD405 for screening supernatants of activatedmemory B cells for binding to N332-GT5. BG18 is a positive control mAb and Den3 is a negative control mAb.

[0055] Figure 34. Clonal lineage trees of BG18-like B cells. Each tree denotes a clone, with clone ID, animal origin, and V, D, and J gene calls. Branch lengths are scaled by estimated heavy chain nucleotide mutations (1 scale unit = 5 mutations). Tree tips are colored by week of sampling. Representative heavy chain and light chain CDR3 sequences are provided for each clone.

[0056] Figure 35. Frequency of Glu at the (D3-41)+2 position in macaque antibodies. (A) Frequency of glutamate positioned 2 aa past the end of the D3-41 gene when D3-41 is present in the BG18 reading frame and position (+ / - 1 aa) in antibodies with HCDR3s >22 aa compared to antibodies with HCDR3s <22 aa and D3-41 positioned anywhere in sequences isolated from GC B cells. (B) as in (A) but sequences were derived from memory B cells.

[0057] Figure 36. Angles of approach for N332 / V3 glycan antibodies. (A) Latitudinal, (B) longitudinal, and (C) HC-LC rotation angle and (D) 3D scatter plot for BG18 (6DFG), BG18_iGLo (6DFH), HMP1 (6NF5), HMP42 (6NFC), RM N332 03, RM N332 32, RM N332 36, RM_N332_07, RM_N332_08 in yellow circles and six other N332-dependent bnAbs: PGT122 (4TVP), DH270.6 (6UM6), BF520.1 (6MN7), PGT128 (5ACO), QA013.2 (7N65), PGT135 (4JM2) in cyan circles.

[0058] Figure 37. Epitope footprints of BG18-like antibodies. The epitope footprint for nine BG18-like antibodies (defined as atoms within 5 A of the Fab). The yellow oval encompasses the footprint of BGI8 GL0 and is mapped onto the other eight structures for comparison. gp!20 is colored gray. The five structures from this study are RM_N332_36, RM_N332_03, RM_N332_32, RM N332 07 and RM N332 08. The four structures from are BG18 GL0 (PDB ID: 6DFH), BG18 Mat (PDB ID: 6DFG), HMP1 (PDB ID: 6NF5) and HMP42 (PDB ID: 6NFC).

[0059] Figure 38. Other BG18-like antibodies. (A) Cryo-EM structure of N332-GT2 in complex with BG18 GL0 (PDB ID: 6DFH). Inset shows the interactions of the HCDR3 to the conserved residues in gp!20. (B) Cryo-EM structure of Fab RM_N332_07 in complex with N332- GT5. Inset shows HCDR3 interactions to gpl20. (C) Cryo-EM structure of Fab RM N332 08 in complex with N332-GT5. Inset shows HCDR3 interactions to gpl20.

[0060] Figure 39. Glycan analysis of VI modified trimers. (A) VI loop sequences of VI loop modified trimers. All five trimers have identical sequences outside of the region shown. Differences are highlighted green. (B) Glycan analysis of VI modified trimers. Green indicateshigh mannose glycans, Pink indicates complex type glycans and Gray indicates unoccupied glycosylation sites.

[0061] Figure 40. Site-specific glycan compositions of BG505 MD65 B23. LC-MS analysis of the glycosylation of BG505 MD65 B23. Oligomannose-type glycans are colored green, hybridtype in hashed pink, complex-type in pink, and the proportion of unoccupied PNGS in grey. The table represents the grouping of the bar graphs, with any glycan composition containing at least one fucose or sialic acid (NeuAc) shown.

[0062] Figure 41. Neutralization assay of VI modified pseudoviruses against three BG18 type I antibodies. Three N332-GT5 elicited Rhesus macaque antibodies were tested for neutralization activity against BG505 pseudovirus and four variants of BG505 containing the N332-GT5 VI loop with or without adding the N133, N137, or both N133 and N137 glycosylation sequons. BG18 and PGT121 are positive controls and Den3 is a negative control.

[0063] Figure 42. Genetic features of non-BG18 epitope-specific BCRs isolated from weeks 7 and 10 post prime. (A) HCDR3 lengths for 45 non-BG18-like competitor antibodies isolated from GC B cells at weeks 7 and 10. (B) VH gene usage for the non-BG18-like competitors. (C) VL gene usage for the non-BG18-like competitors.

[0064] Figure 43. Structure of immunogenic residues in unliganded N332-GT5. The BG18 epitope on unliganded N332-GT5 is shown with solvent exposed VI loop aa side chains (K137 and R139) that are critical for binding to non-BG18-like competitor antibodies indicated. VI loop, green; V3 loop, yellow. The three mutations in the B23 trimer relative to N332-GT5 are A135T, K137N and R139T.

[0065] Figure 44. No indication of a lambda3-based BG18 type II response in rhesus macaques. HCDR3 length distribution of antibodies that use VL3 light chains from all epitopespecific GC B cells combined with and without BG18 type I antibodies removed.

[0066] Figure 45. ELISA binding to activated B cell supernatants from N332-GT5+ B cells isolated 2 weeks post boost. Each symbol represents the ELISA signal from one well and they are ordered based on the HCDR3 length of the BCR sequence identified in each well. BG18 type II antibodies would be found in wells that contained BCRs with HCDR3s > 20 aa (shaded gray). The plot on top shows ELISA reactivity to N332-GT5 and N332-GT5-KO. The bottom plot shows ELISA reactivity to the B23 trimer. The BG18 type I antibodies are indicated on the plots

[0067] Figure 46. Amino acid sequence alignment of trimers used in this study. Green highlight indicates amino acid changes relative to BG505 MD39 The immunogen used in this study, referred to as N332-GT5, is labelled BG505_MD65_congly_N332-GT5 in the sequence alignment. Congly indicates glycosylation sites at positions 241 and 289 were introduced.

[0068] Figure 47. Affinity of boostl candidates for NHP-elicited BG18-class antibodies isolated post-N332-GT5 prime measured by SPR.

[0069] Figure 48. A) NHPs (groups of 6) were primed with N332-GT5+SMNP escalating dose and boosted at week 10 with 1 of 4 heterologous trimers. At week 44 animals were boosted with either N332-GT5 (control group 1) or SF162P3_MD64_B20.1 (groups 2-4). PBMC blood draws were taken at the indicated time points (red circles). B) Memory B cells were sorted and BCR sequenced and BG18 type I sequences were quantified. C) Flow cytometry data indicates whether each BG18 sequence came from a B cell that was stained positive for the boost 1 immunogen or the boost 2 immunogen as indicated on the graph.

[0070] Figure 49. Affinity of NHP-elicited BG18-class antibodies isolated 2 weeks post boost 1 (week 12) for the boost 1 and boost 2 candidates measured by SPR. The DU156 MD39 is a stabilized “wild type“ trimer.

[0071] Figure 50. A) 6 NHPs were immunized with N332-GT6 mRNA-LNPs, boosted at week 8 with Bl l mRNA-LNPs and boosted again at week 16 with SF162P3-B20 mRNA-LNPs. B) Affinity of NHP elicited BG18-class Abs isolated 2 weeks post boost 1 (week 10) for prime and boost immunogens, and other HIV trimers measured by SPR.

[0072] Figure 51. 10E8-class broadly neutralizing antibodies (bnAb) precursors are present in most humans. (A) Schematic of epitope scaffold design showing antibody 10E8, the Envelope protein, including the membrane-proximal region (MPER) that was grafted onto an unrelated epitope-scaffold, (B) Frequency of 10E8-class IgH precursors in 14 NGS datasets of heavy chains from HIV- seronegative humans defined as sequences with VH genes closely related to 10E8 and HCDR3-lengths of 21-24 aa with YxFW motif at the correct position. Lines indicate median and 25 and 75% quantiles

[0073] Figure 52. 10E8-GT immunogens bind diverse 10E8-class precursors. (A) SPR- measured monovalent KD values for the scaffold without germline-targeting mutations (MPER) and various 10E8-GT scaffolds (10E8-GT9.2 to 10E8-GT12) binding to mature 10E8, GL-reverted 10E8 (10E8-iGL3), the proposed 10E8 UCA and multiple NGS-derived 10E8-class humanprecursor heavy chains paired with the GL-reverted 10E8 light chain (NGS). Each symbol represents a different antibody. LOD: limit of detection. NB: no binding. (B) SPR-measured monovalent KD values for 10E8-GT10.2, 10E8-GT11 and 10E8-GT12 binding to different antibodies containing the indicated number of 10E8-class mutations, including fully GL-reverted 10E8 (10E8-iGL3), partially mature 10E8-class antibodies (intermediates) and mature 10E8.

[0074] Figure 53. 10E8-GT immunogens mimic the interaction of 10E8 with the MPER. Structures, from left to right, of 10E8 bnAb bound to MPER peptide (Huang et al, 2012), 10E8 bnAb bound to T117v2 scaffold (Irimia et al., 2017), 10E8-iGLl bound to 10E8-GT4 scaffold, 10E8 bnAb bound to 10E8-GT10.2 scaffold, NGS precursor 10E8-NGS-03 bound to 10E8- GT10.2 scaffold, and 10E8-iGLl bound tolOE8-GTl 1 scaffold, in which the previously published MPER peptide and T117v2 complexes with 10E8 are shown for comparison. Top, structures shown as cartoon diagrams, aligned on the MPER, with antibody heavy chain in white or yellow, light chain in grey and the scaffold in blue. Antibody constant regions are omitted for clarity. Bottom, interaction between the HCDR3 YxFW motif (sticks) and the engineered Dn-gene binding pocket on the scaffold. All structures were determined by crystallography, except for the complex of 10E8 bnAb with 10E8-GT10.2, which was determined by cryo-EM and included a scaffoldspecific off-target Fab (not shown) to facilitate image reconstruction.

[0075] Figure 54. 10E8-GT scaffolds engage 10E8-class HCDR3s in human blood. (A) Representative flow cytometry staining of 10E8-GT12 double-positive (10E8-GT12++, signifying binding to two probes with different fluorochromes, left) and epitope-specific 10E8-GT12++10E8- GT12'KO'(right) naive B cells (CD20+CD27 gD+IgG‘) from HIV-seronegative donors. (B) Frequency of 10E8-GT9++(n=3 donors), 10E8-GT10.1++(n=3), and 10E8-GT12++(n=6) cells among CD20+IgG’ naive B cells for 10E8-GT9 and 10E8-GT10, or CD20+CD27TgD+IgG’ naive B cells for 10E8-GT12 that were sorted from HIV-seronegative donors and their B cell receptor (BCR) sequenced using either a Sanger sequencing method (squares) or a lOx Genomics sequencing method (cross). (C) Percentage of 10E8-GT9++, 10E8-GT10.1++and 10E8-GT12++naive B cells (CD20+IgG- B cells or CD20+CD27 gDHgG' B cells, as in b) that are epitopespecific (10E8-GT9++10E8-GT9-K0‘, 10E8-GT10.1++10E8-GT10.1-K0’, or 10E8-GT12++10E8- GT12-KO ). (D) HCDR3 length distribution for human naive BCRs sorted by 10E8-GT9, 10E8- GT10.1 and 10E8-GT12 (average of all donors in each case). NGS datasets (n=14) of heavy chains from HIV-seronegative humans served as unsorted controls where indicated (Briney et al., 2019;Steichen et al., 2019). The targeted HCDR3 length range (21-24 aa) is highlighted in grey. Exact HCDR3 length for the 10E8 bnAb is indicated by a tick mark at 22 aa. (E) Percentage of 10E8- class HCDR3s (with length 21-24 aa and YxFW at correct position within HCDR3) among epitope-specific (10E8-GT9++10E8-GT9-K0’, n=3 donors; 10E8-GT10.1++10E8-GT10.1-K0-, n=4 donors; 10E8-GT12++10E8-GT12-K0 n=6 donors) IgM+BCRs compared to unsorted controls defined in d. *p=0.03, ***p=0.0004, Kruskal-Wallis test with Dunn’s multiple comparison correction. (F) Percentage of 10E8-class HCDR3s among all naive IgM+B cells compared to unsorted controls as in e. (G) Percentage of 10E8-class and LN01 -class IgH precursors among naive IgM+B cells sorted with 10E8-GT12 or unsorted controls defined in (D). Lines indicate median.

[0076] Figure 55. 10E8-class B cells function in vivo. (A) Flow cytometry analysis and quantification of B220+TCRb‘ B cells, B220 TCRb+total T cells, CD4+CD8‘ T cells, CD4 CD8 T cells, CD2 CD24hiT0 / T1 B cells, CD2110CD2410follicular B cells, CD21hiCD24hiCD23- T2 B cells, and CD21hlCD24hlCD23+ marginal zone B (MZB) cells in the spleen of MPER-HuGL 18Hmice (n=7) compared to wild-type (WT) C57BL / 6 mice (n=4). Symbols represent individual animals, error bars indicate SD. (B) Frequency of CD38loCD95+GC B cells among total B220+B cells (left) and CD45.2+B cells among CD38loCD95+GC B cells (right) at day 21 after immunization with 10E8-GT10.2 12mer(n= 14) or control 10E8-GT9-KO 12mer (n=8) in CD4WT recipient mice adoptively transferred with 200,000 CD45.2 MPER-HUGL18HB cells. Symbols represent individual animals; bars indicate mean ± SD. ****p<0.0001, two-sided Mann-Whitney test. (C) Frequency of 10E8-GT10++cells among CD381OCD95+CD45.1 CD45.2+MPER- HUGL18HGC cells as in (B).

[0077] Figure 56. inRNA-LNP delivery of 10E8-GT12 nanoparticles primes diverse 10E8-class B cells. (A) Percentage of 10E8-GT10.1++10E8-GT10.1-K0‘ (epitope specific) CD19+IgD+naive B cells with 10E8-class HCDR3s for humans and IID3-3 / JH6 mice. (B) Percentage of 10E8-GT9-K0++10E8-GT9; 10E8-GT10.1++10E8-GT10.1-K0’ or 10E8- GT12++10E8-GT12-KO epitope specific IgG+BCRs with 10E8-class HCDR3s from day 42 postimmunization of hD3-3 / JH6 mice with 10E8-GT9-KO 12mer (n=3), 10E8-GT12 12mer (n=12), 10E8-GT12 12mer (n=5) or 10E8-GT1224mer (n=12) delivered as protein in SMNP, respectively, or 10E8-GT12 24mer delivered by mRNA (n=l l). Symbols represent individual animals; bars indicate median values. (C) Percentage of epitope-specific IgG+BCRs as in b with 10E8-classHCDR3s and at least one proline in position +7 or +8 relative to the YxFW motif from 1LD3-3 / JH6 mice with >100 sequences. Sequences of Du genes of mature 10E8 and iGL are shown with the YxFW motif in green, and the targeted prolines are colored red. **p=0.006, two-sided Mann- Whitney test.

[0078] Figure 57. 10E8-GT immunogens induce 10E8-class responses in non-human primates. (A) Alignment of known rhesus macaque homologues of the human DH3-3 gene. Differences within the critical YxFW binding motif (orange) are highlighted in red. (B) HCDR3 length distribution for 10E8-GT10.2++10E8-GT10.2-K0- epitope-specific CD20+IgG" naive B cells sorted from unimmunized macaques (n=9) compared to 10E8-GT10.2- non-binding BCRs from the same macaques and the human naive BCRs. (C) Percentage of 10E8-class HCDR3s among CD20+IgG' naive B cells from unimmunized macaques (n=9), compared to human naive BCRs (n=3) and to rhesus macaque sequences from the Observed Antibody Space repository (OAS, n=8) 49. (D) Macaque immunization schedule for an escalating dose of 10E8- GT 10.2 12mer (n=8) or 10E8-GT12 12mer (n=indicating analysis of lymph node (LN) fine needle aspirates (FNA) at week -2, 3 and 10 and analysis of blood at week 6 and 11 or week 10 for macaques immunized with 10E8-GT10.2 12mer and 10E8-GT12 12mer, respectively. (E) Percentage of 10E8-class HCDR3s among 10E8-GT10.2++10E8-GT10.2-K0' or 10E8- GT12 10E8-GT12-KO’ epitope-specific IgG+BCRs from macaques after immunization as in (D) in the GCs and PBMCs at all timepoints indicated in d combined for each, compared to macaques immunized with stabilized soluble HIV envelope protein50 (Control) at week 3, 4, 7 and 10 post-immunization. Open symbols, macaques lacking a permissive DH3-41 allele. **p=0.004, two-sided Mann- Whitney test. (F) Percentage of 10E8-class VH among IgG+CD20+IgD' memory BCRs with 10E8- class HCDR3s or among IgG+CD20+IgD" memory BCRs lacking the YxFW motif (Non-10E8), from macaques post-immunization as in d. *p<0.05, Kruskal -Wallis test with Dunn’s multiple comparison correction. P value (control vs. 10E8-class induced by 10E8-GT10 12mer): 0.03, p value (control vs. 10E8-class induced by 10E8-GT1212mer): 0.02. (G) Percentage of LNOl-like HCDR3s among 10E8-GT10.2++10E8-GT10.2-K0- or 10E8-GT12++10E8-GT12-K0- epitopespecific IgG+BCRs from macaques after immunization as in (D) **p=0.04, two-sided Mann- Whitney test. (H) Crystal structure of a 10E8-GT10.2-induced macaque antibody with YxIW motif from week 3 in complex with 10E8-GT10.2 (right), with structure of 10E8 bound to peptide (Huang et al., 2012) (left) shown for reference. Top, structures shown as cartoon diagrams, alignedon the MPER, with antibody heavy chain in white or yellow, light chain in grey and the scaffold in blue. Antibody constant regions are omitted for clarity. Bottom, interaction between the HCDR3 YxFW motif (sticks) and the engineered Dn-gene binding pocket on the scaffold.

[0079] Figure 58. 10E8-class BCRs induced by 10E8-GT nanoparticles bind epitopescaffold 10E8-B1 containing a near-native 10E8 peptide epitope. (A) SPR-measured monovalent KD values for 10E8-B1 binding to different antibodies containing the indicated number of 10E8-class mutations, including the 10E8 UCA, 10E8-class human naive precursors isolated by human B cell sorting (human naive), artificial partially mature 10E8-class antibodies (intermediates), and mature 10E8. Each symbol represents an antibody; overlapping data are staggered along the x axis. (B) Crystal structure of 10E8-B1 in complex with 10E8 bnAb (right), with structure of 10E8 bound to peptide (Huang et al., 2012) (left) shown for reference. Top, structures shown as cartoon diagrams, aligned on the MPER, with antibody heavy chain in white or yellow, light chain in grey and the scaffold in blue. Antibody constant regions are omitted for clarity. Bottom, interaction between the HCDR3 YxFW motif (sticks) and the engineered Dn-gene binding pocket on the scaffold. (C) SPR-measured monovalent KD values for 10E8-B 1 binding to inferred-germline antibodies (iGL) or antibodies recovered after immunization of hD3-3 / Ju6 mice or macaques with 10E8-GT nanoparticles (post-prime). Each symbol represents a different antibody. LOD: Limit of detection. **p=0.004, Kruskal-Wallis test with Dunn’s multiple comparison correction.

[0080] Figure 59. Design and properties of immunogens. (A) Overview of 10E8-class and LN01 -class antibody categories. HCDR3 motifs are shown as regular expressions that were used to query the database. If multiple amino acids were allowed at the same position, they are shown in square brackets; positions in which all amino acids were allowed are indicated as (B) Schematic of the development of MPER-GT scaffolds. (C) Schematic overview of nanoparticle formation by genetic fusion of the immunogen (T2983-GT) to each terminus of the 3- dehydroquinase nanoparticle from Thermus thermophilus (NP) via flexible linkers containing exogeneous T-help peptides derived from Aquifex aeolicus lumazine synthase. The epitope scaffold is shown in light blue, the MPER graft in purple, the linker in green, the nanoparticle in red and glycans in dark blue. (D) SEC-MALS traces of 10E8-GT NPs. Normalized UV280 absorptions are shown as dotted lines and protein molecular weights of main peaks are shown as solid lines. (E) DSC measurements of the indicated monomers and nanoparticles with results froma fit indicated in light grey. (F) Amino acid sequences of 10E8-GT epitope scaffolds through generation 7, none of which had the circular permutation present in later generations. All sequences are succeeded by a 6x His-tag, unless the protein ends in stop codons (denoted by symbol *). 10E8- GT8.2 through 10E8-GT12 are preceded by a mammalian secretion signal. (G) Resurfaced T298v2 sequences compared to previously published T29831. Colors as in (F). (H) Amino acid sequences of monomeric immunogens based on resurfaced circularly permutated T298v2, with colors as in f. (I) Amino acid sequences of multivalent nanoparticles. Sequences are wrapped over multiple lines. The 3-dehydroquinase is shown in purple; additional T help epitopes are brown; epitope KO mutations are cyan; and all other colors are shown as in (F).

[0081] Figure 60. Glycosylation sites on 10E8-GT nanoparticles vary in occupancy. Sitespecific glycan analysis was measured using the single site glycan profiling (SSGP) and DeGlyPHER methods. Positions of N-linked glycosylation sites are indicated as relative positions within the epitope-scaffold (ES) or the nanoparticle (NP, indicated with a box). The 24mer contains two independent copies of the scaffold, which cannot be distinguished by either method and therefore averaged values are shown.

[0082] Figure 61. Data collection, refinement and validation. (A) Data collection and refinement statistics of x-ray crystallography. Numbers in parentheses refer to the highest resolution shell; CCI / 2 = Pearson correlation coefficient between two random half datasets; From MolProbity64. (B) Summary statistics of data collection, refinement and validation of the cryo- EM reconstruction of 10E8-GT10.2 in complex with 10E8 and W6-10 Fabs. (C) Fourier Shell Correlation, (D) angular sampling and (E) map colored according to local resolution (units Angstrom) of the cryo-EM reconstruction of 10E8-GT10.2 in complex with 10E8 and W6-10 Fabs.

[0083] Figure 62. Ex vivo evaluation of 10E8-GT scaffolds of Fig. 53 (A) Representative gating scheme. (B) Enrichment of HCDR3 lengths among epitope-specific B cells over unsorted controls as in Fig. 53d. (C) Frequency of long (>=20aa) HCDR3s among epitope-specific B cells (sorted) as in (B), or among total IgM+naive B cells (unsorted). Symbols represent n=3 (GT9), n=4 (GT 10.1), n=6 (GT 12) or n=14 (unsorted controls) independent donors. * p=0.046, **** p<0.0001, Kruskal-Wallis test with Dunn’s multiple comparison correction. (D) Percentage of HCDR3s containing the YxFW motif among epitope-specific B cells as in c. * p=0.03, *** p=0.0001, Kruskal-Wallis test with Dunn’s multiple comparison correction. (E) Percentage of 10E8-class VH among epitope-specific BCRs with 10E8-like HCDR3s (with length 21-24 aa andYxFW at correct position within HCDR3) within datasets obtained using the lOx Genomics sequencing method as in c. **p=0.002, two-sided Mann-Whitney test. (F) Percentage of TGVL3 family light chains among 10E8-GT12-sorted BCRs that are either 10E8-class (10E8-class H3) or lack the YxFW motif (non-YxFW). n=6 independent donors, ns not significant, two-sided Wilcoxon test. (G) Percentage of 10E8-GT12-specific naive IgM+BCRs with 10E8-class or LNOl-class HCDR3s. Symbols represent n=6 (GT12) or n=14 (unsorted controls) independent donors. (H) Frequency of 10E8-class or LNOl-class B cells among IgM+naive B cells, detected through 10E8-GT12 sorting as in (G). (I) SPR-measured monovalent KD values for 10E8-GT9, 10E8-GT10.2, and 10E8-GT12 monomer binding to 10E8-class and non-10E8-class (competitor) antibodies isolated by the respective scaffolds. Symbols represent different antibodies; lines represent median values.

[0084] Figure 63. Poly- and Auto-reactivity of 10E8-class precursors. (A) Polyspecificity reagent binding as measured by ELISA. PGT121, VRC01 and PGT128 served as negative controls, MPER bnAb 4E10 as a positive control. NGS-1 through -22 correspond to human NGS precursors described in the main text. These are 10E8-class heavy chain (HC) precursors identified from searching next-generation sequencing (NGS) datasets of primarily naive IgM HCs from 14 HIV-seronegative human donors (Steichen et al., 2016; Irimia et al., 2017) paired with the inferred- germline 10E8 light chain (LC); I0E8-HuGL: bona fide HC / LC pairs isolated by epitope-specific sorting of naive human B cells (n=25). (B) Mean fluorescence intensity (MFI) of antibodies as in (A) in a HEp-2 cell autoreactivity assay. (C) raw images of data shown in (B).

[0085] Figure 64. Immunization of MPER-HuGL18HB cell adoptive transfer recipient mice with 10E8-GT10.2 timers. (A) Flow cytometry analysis of bone marrow cells from WT (n=4) and MPER-HUGL18H(n=7) mice; gating strategy shown on the left. B-cell progenitors (B220+) were divided into immature (CD43+) and mature (CD43‘) cells. Early (CD43+) B-cell progenitors were subdivided into Hardy populations A (CD24 BP-1 ), B (CD24+BP-L), and C (CD24+BP1+). Late (CD43') B-cell progenitors were subdivided into Hardy populations D (IgM' IgD'), E (IgM+IgDint), and F (IgM+IgD+). Right bars represent quantifications of these populations, error bars indicate SD. (B) Frequency of CD45.2+B cells among splenic B cells, one day after adoptive transfer of 200,000 CD45.2+MPER-HUGL18HB cells into CD45.H WT mice. Symbols represent individual animals, error bars indicate SD. (C) Germinal center (GC) response to immunization in CD45.1+WT mice adoptively transferred with 2 x 105 CD45.2+MPER-HUGL18HB cells on Day 21 after immunization with 10E8-GT10.2 12mer or negative control 1 OE8-GT9-KO 12mer (KO). Left column shows the frequency of total GC (CD38loCD95+) among B cells gated from SSL; right column shows the frequency of CD45.2 B cells among total GC.

[0086] Figure 65. Generation and characterization of 1ID3-3 / JH6 mice. (A) Illustration of genetic modifications in hD3-3 / Jn6 mice (not drawn to scale) with hD3-3 and hJn6 segments replacing mouse DQ52 and JH1-4 segments. Sequences of hD3-3, JH6, and flanking regions are shown below the diagram. (B) Characterization of B220+B cell and CD3+T cell populations among lymphocyte / live cell / single cells from homozygous D3-3 / J6 mouse spleens by flow cytometry compared to a wild-type 129SVE mouse (WT). (C) Characterization of IgM+IgDhlnaive B cells among B cells as in (B). (D) Characterization of CD23hlCD21lowfollicular (FO) B cells and CD23lowCD21hlmarginal zone (MZ) B cells among lymphocyte / live cell / single cell / B220+CD93lowmature B cells, as in b. (E) Characterization of Igk+and Igl+B cells among B cells as in (B). (F) Fraction of productive IgH rearrangements that contain hD3-3 or mouse D (mD), based on repertoire analysis of three homozygous IID3-3 / JH6 mice. The hD3-3 usage value represents average ± standard deviation. Since 1IJH6 is the sole JH segment in homozygous hD3- 3 / JH6 mice, all V(D)J recombination events contain hJn6. Therefore, 16.3% productive rearrangements in homozygous 1ID3-3 / JH6 mice contain both hD3-3 and hJn6. (G) HCDR3 length distribution of productive IgH rearrangements that contain hD3-3 and JH6 as in (F). Error bars indicate SD of three independent mice.

[0087] Figure 66. Immunogenicity of 10E8-GT nanoparticles in hD3-3 / Ju6 mice. (A) Representative gating strategy of splenic B cells sorted for sequencing of BCRs from hD3-3 / Jn6 mice six weeks after immunization with 10E8-GT12 24mer. (B) Percentage of 10E8-GT -binding cells (10E8-GT9-KO++, 10E8-GT10.1++or 10E8-GT12++) among IgM IgD B cells, 42 days after immunization of hD3-3 / Jn6 mice with 10E8-GT9-KO 12mer (control, n=3), 10E8-GT12 12mer (n=12), 10E8-GT12 12mer (n=5) or 10E8-GT12 24mer (n=12) delivered as protein in SMNP, respectively, or 10E8-GT1224mer delivered by mRNA (n=l 1). Each symbol indicates an animal, lines indicate median values. (C) Percentage of 10E8-class HCDR3s among all IgM IgD’ B cells after immunization as in (B). (D) Enrichment ratio for HCDR3 amino acid (aa) length distribution for epitope-specifi c ( 10E8-GT 10.1++10E8-GT 10.1 -KO’ or 10E8-GT 12++10E8-GT 12-KO ) IgG+B cells from animals immunized with the indicated 10E8-GT immunogens, relative to HCDR3 amino acid length distribution for epitope-specific (10E8-GT9-KO++10E8-GT9-) IgG+B cellsfrom animals immunized with 10E8-GT9-KO 12mer as in b. HCDR3 lengths longer than 22 were only found in the 10E8-GT-immunized groups, precluding calculation of enrichment scores for longer HCDR3s. (E) Frequency of long (>=20 aa) HCDR3s among epitope-specific IgG+B cells as in d. (F) Percentage of HCDR3s containing the YxFW motif among epitope-specific IgG+B cells as in (C). *p<0.05, Kruskal-Wallis test with Dunn’s multiple comparison correction. P value (control vs. GT10 12mer): 0.02, p value (control vs. GT12 24mer): 0.01. (G) Fold-change in SPR- measured KD for 10E8-GT immunogens binding to 10E8-iGL3 upon addition of the indicated HCDR3 mutations. (H) Percentage of epitope-specific (10E8-GT9-K0++10E8-GT9-,10E8- GT10.1++10E8-GT10.1-K0; or 10E8-GT12++10E8-GT12-K0’) with 10E8-class or LNOl-class HCDR3s among IgG+BCRs from day 42 after immunization of hD3-3 / Jn6 as in (B). Symbols represent individual animals; bars indicate median values.

[0088] Figure 67. Gating strategy used to assess immune responses to 10E8-GT immunogens in NHPs. (A) Gating scheme for 10E8-GT10.2-specific (10E8-GT10.2++) and 10E8- GT10.2 epitope-specific ( 10E8-GT 10.2 I 0E8-GT 10.2-KO-) naive B cells. (B) Gating scheme for 10E8-GT10.2-specific and 10E8-GT10.2 epitope-specific GC B cells. (C) Gating scheme for 10E8-GT10.2-specific and 10E8-GT10.2 epitope-specific PBMC-memory B cells.

[0089] Figure 68. Immunogenicity of 10E8-GT NPs in rhesus macaques. (A) Percentage of GC B cells (CD38’CD71+) among all B cells (CD3 CD20+) in fine needle aspirate samples of rhesus macaques 3 or 10 weeks post immunization with 10E8-GT10.2 12mer, as described in Fig. 55 Each symbol represents an independent lymph node (n=16 from n=8 independent animals). (B) Frequency of epitope-specific (10E8-GT10.2++10E8-GT10.2-K0’) GC B cells (CD38-CD71+) observed as a percentage of total B cells (CD3 CD20+) 2 weeks prior to or 3 or 10 weeks post immunization with 10E8-GT10.2 12mer, as described in Fig. 55. Each symbol represents an independent lymph node (n=8 for week -2, n=16 for week 3 and 10). (C) Median percent amino acid (aa) mutations in the VH of epitope-specific (10E8-GT10.2++10E8-GT10.2-K0- or 10E8- GT12++10E8-GT12-KO ) BCRs sorted from macaques immunized with 10E8-GT10.2 12mer or 10E8-GT12 12mer at the indicated time-points from GC B cells (CD38'CD7 I ) or PBMC-memory B cells (CD20+IgD ) among BCRs with 10E8-class HCDR3s (10E8-class) compared to BCRs lacking the YxFW motif (Competitor). Symbols represent individual animals, bars indicate median values. (D) SPR-measured monovalent KD values for 10E8-GT10.2 binding to selected antibodies with 10E8-class HCDR3s (10E8-class) or lacking the YxFW motif (Competitor) induced by 10E8-GT10.2 12mer isolated from the GCs at week 3 (W3) or 10 (W10) post immunization, compared to available matching inferred-germline (iGL) antibodies. Each symbol represents one antibody. (E) SPR-measured monovalent KD values for 10E8-GT10.2 binding to selected mutated or unmutated antibodies with 10E8-class HCDR3s (10E8-class) or lacking the YxFW motif (Competitor) induced by 10E8-GT10.2 12mer isolated from PBMC-memory (CD20+IgD‘). (F) Frequency of 10E8-class IgH among PBMC-memory B cells (CD20+IgD ) at week 11 (GT10.2, n=6) or week 10 (GT12, n=5) post immunization compared to macaques immunized with stabilized soluble HIV envelope protein (control, week 10, n=4). For comparison, dotted lines indicate median frequencies of VRC01 -class precursors 8 weeks after high dose (HD) or low dose (LD) immunization with eOD-GT8 60mer in the IAVI G001 human phase 1 clinical trial (Jardine et al. 2013), with the caveat that frequencies in G001 were measured among IgG+B cells rather than IgD’ B cells. Open symbols indicate macaques lacking a permissive DH3-41 allele. Lines indicate median values.

[0090] Figure 69. Antigenicity and immunogenicity of epitope-scaffold 10E8-B1 containing a near-native 10E8 peptide epitope. (A) SPR-measured monovalent KD values for 10E8-B1 binding to different antibodies containing the indicated number of 10E8-class mutations, including the 10E8 UCA, 10E8-class human naive precursors isolated by human B cell sorting (human naive), partially mature 10E8-class antibodies (intermediates), and mature 10E8. Overlapping data are staggered along the x-axis. (B) Crystal structure of a 10E8-B1 in complex with 10E8 (right), with structure of 10E8 bound to peptide (left) shown for reference. Colors are as in Fig. 52 (C) SPR-measured monovalent KD values for 10E8-B1 binding to antibodies recovered after immunization of IID3-3 / JH6 mice or NHPs with 10E8-GT NPs (post-prime) or inferred germline antibodies (iGL). Each symbol represents a different antibody. LOD: Limit of detection. **p<0.01, ns: not significant, Kruskal-Wallis test with Dunn’s multiple comparison correction. (D) IID3-3 / JH6 mice were immunized with PBS or lOpg of 10E8-GT12 24mer in 5 pg SMNP at day 0 and boosted on day 42 with PBS or 10E8-B1 24mer. Epitope-scaffold-specific IgMVIgD' B cells were sorted and their BCRs were sequenced by lOx Genomics at day 84. Symbols represent percentage of epitope-specific IgG+BCRs with 10E8-class HCDR3s among IgM IgD’ B cells for individual animals, bars indicate median values. *p<0.05, Kruskal-Wallis test with Dunn’s multiple comparison correction.

[0091] Figure 70. Immunogenicity of 10E8-B1 24mer in NHPs. (A) NHP immunization schedule. Group 1 (n=6 animals) received a dose-escalation regimen of 10E8-GT10.2 24mer totaling 100 pg 10E8-GT12 24mer and 750 pg SMNP distributed over 7 immunizations over 14 days, followed by boosting with a bolus immunization of 100 pg 10E8-B1 24mer in 750 pg SMNP at week 8. Group 2 received two bolus immunizations with 100 pg 10E8-GT12 24mer mRNA LNP at weeks 0 and 8, respectively, followed by 100 pg 10E8-B1 24mer mRNA LNP at week 16. All animals were analyzed two weeks post 10E8-B 1 24mer boosting. (B) Frequency of antigenspecific (1OE8-B1+) cells as a percentage of total memory B cells. (C) Frequency of epitopespecific (10E8-B1-KO ) cells among antigen-specific (10E8-Bl+) memory B cells. (D) Frequency of epitope-specific (10E8-B1+10E8-B1-KO‘) cells among memory B cells. (E-F) Cells binding epitope-specifically to priming or boosting immunogen (10E8-GT12+10E8-GT12-K0‘ and / or 10E8-B1+10E8-B1-KO’) were sorted and their BCRs were sequenced. Symbols indicate different animals. (E) Frequency of epitope-specific BCRs with 10E8-class HCDR3s among memory B cells. (F) Frequency of cross-reactive cells that bind epitope-specifically to both 10E8-GT12 and 10E8-B1 (10E8-GT12+10E8-GT12-KCT or 10E8-B1+10E8-B1-KO’) among memory B cells with 10E8-class HCDR3s.

[0092] Figure 71. Timeline for an eOD-GT8 60mer priming, core-g28v2 boosting (Boost#l) at week 5, N276(-) membrane-bound trimers (Boost#2) at week 12 and WT boost (Boost#3) at week 18. Humanized Vnl-2 / VKl-33hTdTmice were initially primed with eOD-GT8 60mer delivered by mRNA / LNPs and boosted with core-g28v2 60mer mRNA / LNP.

[0093] Figure 72. Frequencies of antigen specific MBCs after boosting with N276(-) membrane-bound trimers. The frequencies of VRCOl-class memory B cells were determined by antigen-specific cell sorting and single cell lOx Genomics VDJ sequencing conducted six weeks after boosting. A subset of mice was boosted again at week 18 with corresponding wildtype membrane-bound trimers delivered by mRNA / LNPs matched to those received at week 12.

[0094] Figure 73. Frequencies of VRCOl-class MBCs after boosting with N276(-) membrane-bound trimers. Boosting with stabilized membrane-bound HIV trimers lacking the N276 glycan at week 12 elicited VRCOl-class memory B cells.

[0095] Figure 74. Frequencies of antigen specific MBCs after boosting with WT membrane-bound trimers. Subsequent boosting with wildtype membrane-bound HIV Env trimers elicited VRCOl-class memory B cells that bind wildtype trimers.

[0096] Figure 75. Sequential vaccination induces VRCOl-class B cells that bind WT trimers. Subsequent boosting with wildtype membrane-bound HIV Env trimers elicited VRCOl - class memory B cells that bind wildtype trimers.

[0097] Figure 76. Post 001428-WT Boost3 VRCOl-class mAbs neutralize autologous WT virus. VRCOl-class memory B cells that bind wildtype trimers elicited via boosting with wildtype membrane-bound HIV Env trimers have BCRs capable of neutralizing the autologous wildtype HIV pseudovirus and heterologous pseudoviruses lacking the N276 glycan when expressed as recombinant IgGs.

[0098] Figure 77. Post 001428-WT Boost3 VRCOl-class mAbs bind to heterologous WT trimers (Boost#4 candidates). The recombinant IgGs bind to wildtype heterologous trimers with moderate affinity.DETAILED DESCRIPTION OF THE INVENTION

[0099] The invention relates to improved HIV antigens, including germline-targeting designs, trimer stabilization designs, combinations of those two, trimers designed with modified surfaces helpful for immunization regimens, other types of trimer modifications (see, for example, examples of trimers with combined germline-targeting mutations and stabilization mutations and additional trimer modifications that add functionality and that can be combined with other types of modifications as described herein) and on development of trimer nanoparticles and membranebound trimers. The invention also encompasses combinations of any of the herein described modifications, such as but not limited to, combinations of stabilization and modified surfaces with nanoparticles or membrane-bound trimers.

[0100] The invention further relates to mRNAs encoding improved HIV antigens, including germline-targeting designs, trimer stabilization designs, combinations of those two, trimers designed with modified surfaces helpful for immunization regimens, other types of trimer modifications (see, for example, examples of trimers with combined germline-targeting mutations and stabilization mutations and additional trimer modifications that add functionality and that can be combined with other types of modifications as described herein) and on development of trimer nanoparticles and membrane-bound trimers.

[0101] The HIV envelope protein trimer is the target of broadly neutralizing antibodies (bNAbs). The high mannose patch, including the N332-linked glycan at the base of the V3 loopof gpl20, is frequently targeted by bnAbs during natural infection and hence is an appealing vaccine epitope. Germline targeting has potential to initiate the elicitation of N332-dependent bnAbs by vaccination, but no immunogen has been reported to bind germline-reverted precursors of N332-dependent bnAbs.

[0102] VRCOl-class antibodies are defined as those with a Vnl-2 gene in the heavy chain and a five amino acid CDR3 in the light chain. The VH1-2 mouse employed here was originally developed by Ming Tian in the Fred Alt lab at Harvard and was first reported in Tian et al. Cell 2016. It is a stringent model system for inducing VRCOl-class responses, in which Applicants have measured a VRCOl-class precursor frequency of approximately 1 in 1 million naive B cells, which is similar to the frequency measured in humans as reported in Jardine et al. Science 2016 and Havenar-Daughton et al Science Translational Medicine 2018. eOD-GT8 60mer and derivatives are the only immunogens reported to be capable of priming VRCOl-class responses in this model (Tian et al. Cell 2016; Duan et al Immunity 2018).

[0103] Some sequences contain a leader sequence (MGILPSPGMPALLSLVSLLSVLLMGCVAETG) (SEQ ID NO: 1) that is cleaved during expression / secretion and is not present in the final expressed protein product. The embodiments contained herein are not limited to this particular leader sequence as different leader sequences could be used to serve the same purpose.

[0104] The invention also encompasses a protein having at least 90% homology or identity with the sequence of the protein of any one of the trimers disclosed herein. The invention also encompasses a protein having at least 95% homology or identity with the sequence of the protein of any one of trimers disclosed herein.

[0105] The invention also encompasses any nucleic acid encoding the protein of any one of the immunogens disclosed herein. The invention also encompasses a nucleic acid having at least 90% or 95% homology or identity with the sequence of said nucleic acid.

[0106] The invention also encompasses a mRNA encoding an immunogen. The invention also encompasses an mRNA that encodes a protein having at least 90% or 95% homology or identity with the sequence of said protein.

[0107] The invention also encompasses eliciting an immune response which may comprise systemically administering to an animal in need thereof an effective amount of any one of the non- naturally occurring protein(s) or any one of the nucleic acids encoding the non-naturally occurringprotein(s) of the present invention, including nucleic acids that may have at least 90% or 95% homology or identity with a nucleotide encoding the sequence of the non-naturally occurring protein(s) of the invention. In one embodiment, the nucleic acid may be a RNA, advantageously a mRNA. Advantageously, the nucleic acid is formulated in lipid nanoparticles (LNPs). The animal may be a mammal, advantageously a human.

[0108] The invention also encompasses eliciting an immune response which may comprise systemically administering to an animal in need thereof an effective amount of any one of the mRNAs encoding the non-naturally occurring protein(s) of the present invention, including mRNAs that may have at least 90% or 95% homology or identity thereto. Advantageously, the mRNA is formulated in lipid nanoparticles (LNPs). The animal may be a mammal, advantageously a human.

[0109] The invention pertains to the identification, design, synthesis and isolation of mutant trimers disclosed herein as well as nucleic acids encoding the same. The present invention also relates to homologues, derivatives and variants of the sequences of the mutant trimers and nucleic acids encoding the same, wherein it is preferred that the homologue, derivative or variant have at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99% homology or identity with the sequence of the mutant trimers and nucleic acids encoding the same. It is noted that within this specification, homology to sequences of the mutant proteins and nucleic acids encoding the same refers to the homology of the homologue, derivative or variant to the binding site of the mutant proteins and nucleic acids encoding the same.

[0110] The invention still further relates to nucleic acid sequences expressing the mutant immunogens disclosed herein, or homologues, variants or derivatives thereof. One of skill in the art will know, recognize and understand techniques used to create such. Additionally, one of skill in the art will be able to incorporate such a nucleic acid sequence into an appropriate vector, allowing for production of the amino acid sequence of mutant proteins and nucleic acids encoding the same or a homologue, variant or derivative thereof.

[0111] The invention still further relates to mRNA sequences expressing the mutant immunogens disclosed herein, or homologues, variants or derivatives thereof. One of skill in the art will know, recognize and understand techniques used to create such mRNAs.

[0112] Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

[0113] The term “isolated” or “non-naturally occurring” is used herein to indicate that the isolated moiety (e.g. peptide or compound) exists in a physical milieu distinct from that in which it occurs in nature. For example, the isolated peptide may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs. The absolute level of purity is not critical, and those skilled in the art may readily determine appropriate levels of purity according to the use to which the peptide is to be put. The term “isolating” when used a step in a process is to be interpreted accordingly.

[0114] In many circumstances, the isolated moiety will form part of a composition (for example a more or less crude extract containing many other molecules and substances), buffer system, matrix or excipient, which may for example contain other components (including proteins, such as albumin).

[0115] In other circumstances, the isolated moiety may be purified to essential homogeneity, for example as determined by PAGE or column chromatography (for example HPLC or mass spectrometry). In preferred embodiments, the isolated peptide or nucleic acid of the invention is essentially the sole peptide or nucleic acid in a given composition. In other preferred embodiments, the isolated mRNA of the invention is essentially the sole mRNA in a given composition.

[0116] In an advantageous embodiment, a tag may be utilized for purification or biotinylation. The tag for purification may be a his tag. In another embodiment, the tag for biotinylation may be an avi-tag. Other tags are contemplated for purification, however, purification may be accomplished without a tag. In another embodiment, antibody (such as, not limited to, a broadly neutralizing antibody) affinity columns are contemplated. In another embodiment, lectin columns are contemplated.

[0117] Native-like soluble trimers can be made by several methods that all involve stabilizing associations between envelope protein subunits. See, e.g., Steichen et al., Immunity. 2016 Sep 20;45(3):483-496. doi: 10.1016 / j.immuni.2016.08.016. Epub 2016 Sep 8.PMID: 27617678, Kulp et al . , Nat Commun. 2017 Nov 21 ;8(1): 1655. doi : 10.1038 / s41467-017-01549-6. PMID : 29162799 and R.W. Sanders et al., “HIV-1 neutralizing antibodies induced by native-like envelope trimers,” Science, doi: 10.1126 / science.aac4223, 2015.

[0118] The proteins and compounds of the invention need not be isolated in the sense defined above, however.

[0119] The term “pharmaceutical composition” is used herein to define a solid or liquid composition in a form, concentration and level of purity suitable for administration to a patient (e.g. a human patient) upon which administration it may elicit the desired physiological changes. The terms “immunogenic composition” and “immunological composition” and “immunogenic or immunological composition” cover any composition that elicits an immune response against the targeted pathogen, HIV. Terms such as “vaccinal composition” and “vaccine” and “vaccine composition” cover any composition that induces a protective immune response against the targeted pathogen or which efficaciously protects against the pathogen; for instance, after administration or injection, elicits a protective immune response against the targeted pathogen or provides efficacious protection against the pathogen. Accordingly, an immunogenic or immunological composition induces an immune response, which may, but need not, be a protective immune response. An immunogenic or immunological composition may be used in the treatment of individuals infected with the pathogen, e.g., to stimulate an immune response against the pathogen, such as by stimulating antibodies against the pathogen. Thus, an immunogenic or immunological composition may be a pharmaceutical composition. Furthermore, when the text speaks of ‘immunogen, antigen or epitope’, an immunogen may be an antigen or an epitope of an antigen. A diagnostic composition is a composition containing a compound or antibody, e.g., a labeled compound or antibody, that is used for detecting the presence in a sample, such as a biological sample, e.g., blood, semen, vaginal fluid, etc., of an antibody that binds to the compound or an immunogen, antigen or epitope that binds to the antibody; for instance, an anti-HIV antibody or an HIV immunogen, antigen or epitope.

[0120] A ‘conservative amino acid change’ is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine and histidine), acidic side chains (e.g. aspartic acid and glutamic acid), non-charged amino acids or polar side chains (e g. glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan), beta-branched side chains (e.g. threonine,valine and isoleucine), and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan and histidine).

[0121] The terms “protein”, “peptide”, “polypeptide”, and “amino acid sequence” are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.

[0122] As used herein, the terms “antigen” or “immunogen” are used interchangeably to refer to a substance, typically a protein, which is capable of inducing an immune response in a subject. The term also refers to proteins that are immunologically active in the sense that once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector that encodes the protein) is able to evoke an immune response of the humoral and / or cellular type directed against that protein.

[0123] The term “antibody” includes intact molecules as well as fragments thereof, such as Fab, F(ab’)2, Fv and scFv which are capable of binding the epitope determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and include, for example:(a) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule may be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;(b) Fab’, the fragment of an antibody molecule may be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab’ fragments are obtained per antibody molecule;(c) F(ab’)2, the fragment of the antibody that may be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab’)2 is a dimer of two Fab’ fragments held together by two disulfide bonds;(d) scFv, including a genetically engineered fragment containing the variable region of a heavy and a light chain as a fused single chain molecule.

[0124] General methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), which is incorporated herein by reference). Fabs, Fv and scFV may also be made recombinantly, i.e. expressed as Fab, Fv or scFV rather than cleaving an intact IgG.

[0125] A “neutralizing antibody“ may inhibit the entry of HIV-1 virus for example SF162 and / or JR-CSF with a neutralization index >1.5 or >2.0. Broad and potent neutralizing antibodies may neutralize greater than about 50% of HIV-1 viruses (from diverse clades and different strains within a clade) in a neutralization assay. The inhibitory concentration of the monoclonal antibody may be less than about 25 mg / ml to neutralize about 50% of the input virus in the neutralization assay.

[0126] An “isolated antibody“ or “non-naturally occurring antibody“ is one that has been separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody ’ s natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0127] The term “monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies which may comprise the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal“ is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.

[0128] An “antibody fragment" may comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, scFV and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10): 1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0129] It should be understood that the proteins of the invention may differ from the exact sequences illustrated and described herein. Thus, the invention contemplates deletions, additions and substitutions to the sequences shown, so long as the sequences function in accordance with the methods of the invention. In this regard, particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic— aspartate and glutamate; (2) basic— lysine, arginine, histidine; (3) non-polar— alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar— glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. It is reasonably predictable that an isolated or non-naturally occurring replacement of leucine with isoleucine or valine, or vice versa; an aspartate with a glutamate or vice versa; a threonine with a serine or vice versa; or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. Proteins having substantially the same amino acid sequence as the sequences illustrated and described but possessing minor amino acid substitutions that do not substantially affect the immunogenicity of the protein are, therefore, within the scope of the invention.

[0130] As used herein the terms “nucleotide sequences" and “nucleic acid sequences" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences, including, without limitation, messenger RNA (mRNA), DNA / RNA hybrids, or synthetic nucleic acids. The nucleic acid maybe single-stranded, or partially or completely double-stranded (duplex). Duplex nucleic acids may be homoduplex or heteroduplex.

[0131] As used herein the term “transgene“ may used to refer to “recombinant“ nucleotide sequences that may be derived from any of the nucleotide sequences encoding the proteins of the present invention. The term “recombinant“ means a nucleotide sequence that has been manipulated “by man“ and which does not occur in nature, or is linked to another nucleotide sequence or found in a different arrangement in nature. It is understood that manipulated “by man“ means manipulated by some artificial means, including by use of machines, codon optimization, restriction enzymes, etc.

[0132] For example, in one embodiment the nucleotide sequences may be mutated such that the activity of the encoded proteins in vivo is abrogated. In another embodiment the nucleotide sequences may be codon optimized, for example the codons may be optimized for human use. In preferred embodiments the nucleotide sequences of the invention are both mutated to abrogate the normal in vivo function of the encoded proteins, and codon optimized for human use. For example, each of the sequences of the invention, such as the mutant trimers, may be altered in these ways.

[0133] As regards codon optimization, the nucleic acid molecules of the invention have a nucleotide sequence that encodes the antigens of the invention and may be designed to employ codons that are used in the genes of the subject in which the antigen is to be produced. Many viruses, including HIV and other lentiviruses, use a large number of rare codons and, by altering these codons to correspond to codons commonly used in the desired subject, enhanced expression of the antigens may be achieved. In a preferred embodiment, the codons used are “humanized" codons, i.e., the codons are those that appear frequently in highly expressed human genes (Andre et al., J. Virol. 72: 1497-1503, 1998) instead of those codons that are frequently used by HIV. Such codon usage provides for efficient expression of the transgenic HIV proteins in human cells. Any suitable method of codon optimization may be used. Such methods, and the selection of such methods, are well known to those of skill in the art. In addition, there are several companies that will optimize codons of sequences, such as Geneart (geneart.com). Thus, the nucleotide sequences of the invention may readily be codon optimized.

[0134] The invention further encompasses nucleotide sequences encoding functionally and / or antigenically equivalent variants and derivatives of the antigens of the invention and functionally equivalent fragments thereof. These functionally equivalent variants, derivatives, and fragmentsdisplay the ability to retain antigenic activity. For instance, changes in a DNA sequence that do not change the encoded amino acid sequence, as well as those that result in conservative substitutions of amino acid residues, one or a few amino acid deletions or additions, and substitution of amino acid residues by amino acid analogs are those which will not significantly affect properties of the encoded polypeptide. Conservative amino acid substitutions are glycine / alanine; valine / isoleucine / leucine; asparagine / glutamine; aspartic acid / glutamic acid; serine / threonine / methionine; lysine / arginine; and phenylalanine / tyrosine / tryptophan. In one embodiment, the variants have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology or identity to the antigen, epitope, immunogen, peptide or polypeptide of interest.

[0135] For the purposes of the present invention, sequence identity or homology is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical algorithms. A nonlimiting example of a mathematical algorithm used for comparison of two sequences is the algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268, modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993;90: 5873-5877.

[0136] Another example of a mathematical algorithm used for comparison of sequences is the algorithm of Myers & Miller, CABIOS 1988;4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 may be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85: 2444-2448.

[0137] Advantageous for use according to the present invention is the WU-BLAST (Washington University BLAST) version 2.0 software. WU-BLAST version 2.0 executable programs for several UNIX platforms may be downloaded from ftp: / / blast.wustl.edu / blast / executables. This program is based on WU-BLAST version 1.4, which in turn is based on the public domain NCBLBLAST version 1.4 (Altschul & Gish, 1996, Localalignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschul et al., Journal of Molecular Biology 1990; 215: 403-410; Gish & States, 1993;Nature Genetics 3: 266-272; Karlin & Altschul, 1993;Proc. Natl. Acad. Sci. USA 90: 5873-5877; all of which are incorporated by reference herein).

[0138] The various recombinant nucleotide sequences and proteins of the invention are made using standard recombinant DNA and cloning techniques. Such techniques are well known to those of skill in the art. See for example, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al. 1989).

[0139] The nucleotide sequences of the present invention may be inserted into “vectors. “ The term “vector" is widely used and understood by those of skill in the art, and as used herein the term “vector" is used consistent with its meaning to those of skill in the art. For example, the term “vector“ is commonly used by those skilled in the art to refer to a vehicle that allows or facilitates the transfer of nucleic acid molecules from one environment to another or that allows or facilitates the manipulation of a nucleic acid molecule.

[0140] Any vector that allows expression of the proteins of the present invention may be used in accordance with the present invention. In certain embodiments, the proteins of the present invention may be used in vitro (such as using cell-free expression systems) and / or in cultured cells grown in vitro in order to produce the encoded HIV- proteins, which may then be used for various applications such as in the production of proteinaceous vaccines. For such applications, any vector that allows expression of the proteins in vitro and / or in cultured cells may be used.

[0141] For applications where it is desired that the proteins be expressed in vivo, for example when the transgenes of the invention are used in DNA or DNA-containing vaccines, any vector that allows for the expression of the proteins of the present invention and is safe for use in vivo may be used. In preferred embodiments the vectors used are safe for use in humans, mammals and / or laboratory animals.

[0142] For the proteins of the present invention to be expressed, the protein coding sequence should be “operably linked" to regulatory or nucleic acid control sequences that direct transcription and translation of the protein. As used herein, a coding sequence and a nucleic acid control sequence or promoter are said to be “operably linked" when they are covalently linked in such a way as to place the expression or transcription and / or translation of the coding sequence under the influence or control of the nucleic acid control sequence. The “nucleic acid control sequence" maybe any nucleic acid element, such as, but not limited to promoters, enhancers, IRES, introns, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence that is operably linked thereto. The term “promoted will be used herein to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II and that when operationally linked to the protein coding sequences of the invention lead to the expression of the encoded protein. The expression of the transgenes of the present invention may be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when exposed to some particular external stimulus, such as, without limitation, antibiotics such as tetracycline, hormones such as ecdysone, or heavy metals. The promoter may also be specific to a particular cell-type, tissue or organ. Many suitable promoters and enhancers are known in the art, and any such suitable promoter or enhancer may be used for expression of the transgenes of the invention. For example, suitable promoters and / or enhancers may be selected from the Eukaryotic Promoter Database (EPDB).

[0143] The vectors used in accordance with the present invention should typically be chosen such that they contain a suitable gene regulatory region, such as a promoter or enhancer, such that the proteins of the invention may be expressed.

[0144] Any suitable vector may be used depending on the application. For example, plasmids, viral vectors, bacterial vectors, protozoal vectors, insect vectors, baculovirus expression vectors, yeast vectors, mammalian cell vectors, and the like, may be used. Suitable vectors may be selected by the skilled artisan taking into consideration the characteristics of the vector and the requirements for expressing the proteins under the identified circumstances.

[0145] In an advantageous embodiment, IgGl and Fab expression vectors may be utilized to reconstitute heavy and light chain constant regions if heavy and light chain genes of the proteins of the present invention are cloned.

[0146] When the aim is to express the proteins of the invention in vivo in a subject, for example in order to generate an immune response against an HIV-1 antigen and / or protective immunity against HIV-1, expression vectors that are suitable for expression on that subject, and that are safe for use in vivo, should be chosen. For example, in some embodiments it may be desired to express the proteins of the invention in a laboratory animal, such as for pre-clinical testing of the HIV-1 immunogenic compositions and vaccines of the invention. In other embodiments, it will be desirable to express the proteins of the invention in human subjects, such as in clinical trials andfor actual clinical use of the immunogenic compositions and vaccine of the invention. Any vectors that are suitable for such uses may be employed, and it is well within the capabilities of the skilled artisan to select a suitable vector. In some embodiments it may be preferred that the vectors used for these in vivo applications are attenuated to vector from amplifying in the subject. For example, if plasmid vectors are used, preferably they will lack an origin of replication that functions in the subject so as to enhance safety for in vivo use in the subject. If viral vectors are used, preferably they are attenuated or replication-defective in the subject, again, so as to enhance safety for in vivo use in the subject.

[0147] In preferred embodiments of the present invention viral vectors are used. Viral expression vectors are well known to those skilled in the art and include, for example, viruses such as adenoviruses, adeno-associated viruses (AAV), alphaviruses, herpesviruses, retroviruses and poxviruses, including avipox viruses, attenuated poxviruses, vaccinia viruses, and particularly, the modified vaccinia Ankara virus (MVA; ATCC Accession No. VR-1566). Such viruses, when used as expression vectors are innately non-pathogenic in the selected subjects such as humans or have been modified to render them non-pathogenic in the selected subjects. For example, replicationdefective adenoviruses and alphaviruses are well known and may be used as gene delivery vectors.

[0148] The nucleotide sequences and vectors of the invention may be delivered to cells, for example if the aim is to express the HIV-1 antigens in cells in order to produce and isolate the expressed proteins, such as from cells grown in culture. For expressing the proteins in cells any suitable transfection, transformation, or gene delivery methods may be used. Such methods are well known by those skilled in the art, and one of skill in the art would readily be able to select a suitable method depending on the nature of the nucleotide sequences, vectors, and cell types used. For example, transfection, transformation, microinjection, infection, electroporation, lipofection, or liposome-mediated delivery could be used. Expression of the proteins may be carried out in any suitable type of host cells, such as bacterial cells, yeast, insect cells, and mammalian cells. The proteins of the invention may also be expressed using including in vitro transcription / translation systems. All of such methods are well known by those skilled in the art, and one of skill in the art would readily be able to select a suitable method depending on the nature of the nucleotide sequences, vectors, and cell types used.

[0149] A synthetic mutant trimer may be chemically synthesized in whole or part using techniques that are well-known in the art (see, e g., Kochendoerfer, G. G., 2001). Additionally, homologs and derivatives of the polypeptide may be also be synthesized.

[0150] Alternatively, methods which are well known to those skilled in the art may be used to construct expression vectors containing nucleic acid molecules that encode the polypeptide or homologs or derivatives thereof under appropriate transcriptional / translational control signals, for expression. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., 1989.

[0151] The HIV envelope protein (Env) is the target of broadly neutralizing antibodies (bnAbs) in natural infection. Env is a membrane protein composed of a trimer of gpl20 and gp41 subunits that contains a high degree of sequence diversity and a surface that is shielded by N- linked glycans. The bnAbs that target Env often have unusual features such as a long complementarity-determining region (CDR) H3, high levels of somatic hypermutation (SHM), and insertions and deletions (INDELS). Furthermore, most of the bnAbs recognize complex epitopes that are typically non-linear and have both protein and glycan components.

[0152] Applicants claim sequences of different types of immunogen sequences. The sequences provided below are exemplary examples, the stabilizing mutations, modifications, (such as, but not limited to, cleavage-independent modifications), and / or a membrane anchoring strategy (such as, but not limited to, linker plus platelet-derived growth factor receptor (PDGFR)) described herein are applicable to any HIV strain or clade, such as but not limited to, those described below.

[0153] The present invention relates to non-naturally occurring proteins, which may be involved in forming immunogenic proteins of the present invention.

[0154] The invention relates to a non-naturally occurring protein which may comprise any one of the following sequences in Tables 1-4.Table 1: Immunogens.Table 2: Immunogens inducing VRCOl-class bnAbsTable 3: Immunogens inducing BG18 bnAb precursor response.Table 4: Immunogens inducing 10E8-class bnAB precursor response.

[0155] The protein may have at least 90% or 95% homology or identity with the sequence of the non-naturally occurring protein(s) of the invention.

[0156] Each of the protein sequences of Tables 1-4 optionally has a leader sequence of MGILPSPGMPALLSLVSLLSVLLMGCVAETG (SEQ ID NO: 54).

[0157] Some of the protein sequences of Tables 1-4 may comprise a linkl4 sequence of SHSGSGGSGSGGHA (SEQ ID NO: 55). The linkl4 sequence is optional in each of the sequences comprising a link! 4 sequence.

[0158] The invention also encompasses trimers which may comprise any one of the non- naturally occurring protein(s) of the invention.

[0159] The proteins of the invention may comprise an additional cysteine and / or be fused to be a multimerization motif. The proteins of the invention may also comprise a tag for purification or biotinylation, such as a his tag or a avi-tag.

[0160] The invention also encompasses nucleic acids encoding the non-naturally occurring protein(s) of the present invention, including nucleic acids that may have at least 90% or 95% homology or identity with a nucleotide encoding the sequence of the non-naturally occurring protein(s) of the invention. In one embodiment, the nucleic acid may be a mRNA.

[0161] In one embodiment, the nucleic acids of the present invention may be delivered as a therapeutic mRNA.

[0162] The present invention contemplates expressing the herein disclosed immunogens as mRNAs, advantageously as mRNA vaccines. The disclosures of US Patent Nos. 9,675,668;9,464,124; 9,447,164; 9,428,535; 9,334,328; 9,303,079; 9,301,993; 9,295,689; 9,283,287;9,271,996; 9,255,129; 9,254,311; 9,233,141; 9,221,891; 9,220,792; 9,220,755; 9,216,205;9,192,651; 9,186,372; 9,181,319; 9,149,506; 9,114,113; 9,107,886; 9,095,552; 9,089,604;9,061,059; 9,050,297; 8,999,380; 8,980,864; 8,822,663; 8,754,062; 8,710,200; 8,680,069 and 8,664,194 and US Patent Publication Nos. 20220047518, 20200254086, 20200206362, 20180311336, 20180303929, 20170204152, 20160331828, 20160317647 and 20160194368 are herein incorporated by reference.

[0163] Exemplary aspects of the invention feature efficacious mRNA vaccines. Described herein are mRNA vaccines designed to achieve particular biologic effects. Exemplary vaccines of the invention feature mRNAs encoding a particular antigen of interest (or and mRNA or mRNAs encoding antigens of interest), optionally formulated with additional components designed tofacilitate efficacious delivery of mRNAs in vivo. In exemplary aspects, the vaccines of the invention feature and mRNA or mRNAs encoding antigen(s) of interest, complexed with polymeric or lipid components, or in certain aspects, encapsulated in liposomes, or alternatively, in lipid nanoparticles (LNPs). Chemical modification of mRNAs can facilitate certain desirable properties of vaccines on the invention, for example, influencing the type of immune response to the vaccine. For example, appropriate chemical modification of mRNAs can reduce unwanted innate immune responses against mRNA components and / or can facilitate desirable levels of protein expression of the antigen or antigens of interest. Further description of such features of the invention is provided infra.

[0164] Provided herein are isolated nucleic acids (e.g., modified mRNAs encoding a peptide described herein) which may comprise a translatable region and at least two different nucleoside modifications, wherein the nucleic acid exhibits reduced degradation in a cell into which the nucleic acid is introduced, relative to a corresponding unmodified nucleic acid. For example, the degradation rate of the nucleic acid is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to the degradation rate of the corresponding unmodified nucleic acid. In certain embodiments, the nucleic acid may comprise RNA, DNA, TNA, GNA, or a hybrid thereof. In certain embodiments, the nucleic acid comprises messenger RNA (mRNA). In certain embodiments, the mRNA does not substantially induce an innate immune response of the cell into which the mRNA is introduced. In certain embodiments, the mRNA may comprise at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2- thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2 -thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1 -methyl - pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1 -methyl- 1- deaza-pseudouridine, 2-thio-l -methyl- 1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2- methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In certain embodiments, the mRNA may comprise at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5- formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine,pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza- pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-l -methyl -pseudoisocytidine. In other embodiments, the mRNA may comprise at least one nucleoside selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2- aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyl adenine, 2-methylthio-adenine, and 2- methoxy-adenine. In yet other embodiments, the mRNA may comprise at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza- guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza- 8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy- guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo- guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0165] In some embodiments, the nucleic acids provided herein comprise a 5' untranslated region (UTR) and / or a 3'UTR, wherein each of the two different nucleoside modifications are independently present in the 5'UTR and / or 3'UTR. In some embodiments, nucleic acids are provided herein, wherein at least one of the two different nucleoside modifications are present in the translatable region. In some embodiments, nucleic acids provided herein are capable of binding to at least one polypeptide that prevents or reduces an innate immune response of a cell into which the nucleic acid is introduced.

[0166] Further provided herein are isolated nucleic acids (e.g., modified mRNAs described herein) which may comprise (i) a translatable region encoding a peptide described herein, (ii) at least one nucleoside modification, and (iii) at least one intronic nucleotide sequence capable of being excised from the nucleic acid.

[0167] Further provided herein are isolated nucleic acids (e.g., modified mRNAs described herein) which may comprise (i) a translatable region encoding a peptide described herein, (ii) at least two different nucleoside modifications, and (iii) a degradation domain.

[0168] Further provided herein are non-enzymatically synthesized nucleic acids (e.g., modified mRNAs described herein) which may comprise at least one nucleoside modification, and which may comprise a translatable region encoding a peptide described herein. In certain embodiments, the non-enzymatically synthesized mRNA may comprise at least two different nucleoside modifications.

[0169] Further provided herein are isolated nucleic acids (e.g., modified mRNAs described herein) which may comprise a noncoding region and at least one nucleoside modification that reduces an innate immune response of a cell into which the nucleic acid is introduced, wherein the nucleic acid sequesters one or more translational machinery components. In certain embodiments, the isolated nucleic acids which may comprise a noncoding region and at least one nucleoside modification described herein are provided in an amount effective to reduce protein expression in the cell. In certain embodiments, the translational machinery component is a ribosomal protein or a transfer RNA (tRNA). In certain embodiments, the nucleic acid may comprise a small nucleolar RNA (sno-RNA), microRNA (miRNA), small interfering RNA (siRNA) or Piwi -interacting RNA (piRNA).

[0170] Further provided herein are isolated nucleic acids (e.g., modified mRNAs described herein) which may comprise (i) a first translatable region, (ii) at least one nucleoside modification, and (iii) an internal ribosome entry site (IRES). In certain embodiments, the IRES is obtained from a picomavirus, a pest virus, a polio virus, an encephalomyocarditis virus, a foot-and-mouth disease virus, a hepatitis C virus, a classical swine fever virus, a murine leukemia virus, a simian immune deficiency virus or a cricket paralysis virus. In certain embodiments, the isolated nucleic acid further may comprise a second translatable region. In certain embodiments, the isolated nucleic acid further may comprise a Kozak sequence. In some embodiments, the first translatable region encodes a peptide described herein. In some embodiments, the second translatable region encodes peptide described herein. In some embodiments, the first and the second translatable regions encode peptides described herein.

[0171] Provided herein are pharmaceutical compositions which may comprise: (i) an effective amount of a synthetic messenger ribonucleic acid (mRNA) encoding peptide described herein; and(ii) a pharmaceutically acceptable carrier, wherein i) the mRNA may comprise pseudouridine, 5'methyl-cytidine, or a combination thereof, or ii) the mRNA does not comprise a substantial amount of a nucleotide or nucleotides selected from the group consisting of uridine, cytidine, and a combination of uridine and cytidine, and wherein the composition is suitable for repeated administration (e.g., intravenous administration) to a mammalian subject in need thereof. In some embodiments,

[0172] Further provided herein are pharmaceutical compositions which may comprise and / or consisting essentially of: (i) an effective amount of a synthetic messenger ribonucleic acid (mRNA) encoding peptide described herein; (ii) a cell penetration agent; and (iii) a pharmaceutically acceptable carrier, wherein i) the mRNA may comprise pseudouridine, 5'methyl- cytidine or a combination thereof, or ii) the mRNA does not comprise a substantial amount of a nucleotide or nucleotides selected from the group consisting of uridine, cytidine, and a combination of uridine and cytidine, and wherein the composition is suitable for repeated administration (e.g., intravenous administration) to an animal (e.g., mammalian) subject in need thereof.

[0173] This invention provides nucleic acids, including RNAs such as mRNAs that contain one or more modified nucleosides (termed “modified nucleic acids”), which have useful properties including the lack of a substantial induction of the innate immune response of a cell into which the mRNA is introduced. Because these modified nucleic acids enhance the efficiency of protein production, intracellular retention of nucleic acids, and viability of contacted cells, as well as possess reduced immunogenicity, these nucleic acids having these properties are termed “enhanced nucleic acids“ herein.

[0174] The term “nucleic acid,“ in its broadest sense, includes any compound and / or substance that is or can be incorporated into an oligonucleotide chain. Exemplary nucleic acids for use in accordance with the present invention include, but are not limited to, one or more of DNA, RNA, hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc., described in detail herein.

[0175] Provided are modified nucleic acids containing a translatable region encoding a peptide described herein, and one, two, or more than two different nucleoside modifications. In some embodiments, the modified nucleic acid exhibits reduced degradation in a cell into which thenucleic acid is introduced, relative to a corresponding unmodified nucleic acid. For example, the degradation rate of the nucleic acid is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to the degradation rate of the corresponding unmodified nucleic acid. Exemplary nucleic acids include ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or a hybrid thereof. In preferred embodiments, the modified nucleic acid includes messenger RNAs (mRNAs). As described herein, the nucleic acids of the invention do not substantially induce an innate immune response of a cell into which the mRNA is introduced.

[0176] In some embodiments, modified nucleosides include pyridin-4-one ribonucleoside, 5- aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1- methyl-l-deaza-pseudouridine, 2-thio-l -methyl- 1-deaza-pseudouri dine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2- methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

[0177] In some embodiments, modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l- methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2- thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy- pseudoisocytidine, and 4-methoxy-l-methyl-pseudoisocytidine.

[0178] In other embodiments, modified nucleosides include 2-aminopurine, 2,6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2- methoxy-adenine.

[0179] In certain embodiments it is desirable to intracellularly degrade a modified nucleic acid introduced into the cell, for example if precise timing of protein production is desired. Thus, the invention provides a modified nucleic acid containing a degradation domain, which is capable of being acted on in a directed manner within a cell.

[0180] In other embodiments, modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl- guanosine, 7-methylinosine, 6-methoxy -guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio- guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0181] Other components of nucleic acid are optional, and are beneficial in some embodiments. For example, a 5' untranslated region (UTR) and / or a 3'UTR are provided, wherein either or both may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the translatable region. Also provided are nucleic acids containing a Kozak sequence.

[0182] Further, nucleic acids encoding a peptide described herein, and containing an internal ribosome entry site (IRES) are provided herein. An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. An mRNA containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes (“multicistronic mRNA”). When nucleic acids are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the invention include without limitation, those from picornaviruses (e.g., FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).

[0183] The therapeutic mRNAs as described, for example, in US Patent Nos. 9,464,124; 9,447,164; 9,428,535; 9,334,328; 9,303,079; 9,301,993; 9,295,689; 9,283,287; 9,271,996; 9,255,129; 9,254,311; 9,233,141; 9,221,891; 9,220,792; 9,220,755; 9,216,205; 9,192,651;9,186,372; 9,181,319; 9,149,506; 9,114,113; 9,107,886; 9,095,552; 9,089,604; 9,061,059; 9,050,297; 8,999,380; 8,980,864; 8,822,663; 8,754,062; 8,710,200; 8,680,069 and 8,664,194 may be utilized for the present invention.

[0184] Advantageously, the mRNA of the present invention is formulated as a lipid nanoparticle (LNP) formulation, such as a PEG lipid, which are useful in pharmaceutical compositions, cosmetic compositions, and drug delivery systems, e.g., for use in LNP formulations. The LNPs described in US Patent Publication Nos. 20220047518 and 20200254086 are useful for the delivery of an agent (e.g., therapeutic agent such as a nucleic acid) to a subject.

[0185] In one set of embodiments, lipid nanoparticles (LNPs) are provided. In one embodiment, a lipid nanoparticle comprises lipids including an ionizable lipid (such as an ionizable cationic lipid), a structural lipid, a phospholipid, and mRNA. Each of the LNPs described herein may be used as a formulation for the mRNA described herein. In one embodiment, a lipid nanoparticle comprises an ionizable lipid, a structural lipid, a phospholipid, and mRNA. In some embodiments, the LNP comprises an ionizable lipid, a PEG-modified lipid, a phospholipid and a structural lipid. In some embodiments, the LNP has a molar ratio of about 20-60% ionizable lipid: about 5-25% phospholipid: about 25-55% structural lipid; and about 0.5-15% PEG-modified lipid. In some embodiments, the LNP comprises a molar ratio of about 50% ionizable lipid, about 1.5% PEG-modified lipid, about 38.5% structural lipid and about 10% phospholipid. In some embodiments, the LNP comprises a molar ratio of about 55% ionizable lipid, about 2.5% PEG lipid, about 32.5% structural lipid and about 10% phospholipid. In some embodiments, the ionizable lipid is an ionizable amino or cationic lipid and the phospholipid is a neutral lipid, and the structural lipid is a cholesterol. In some embodiments, the LNP has a molar ratio of 50:38.5: 10: 1.5 of ionizable lipid: cholesterokDSPC: PEG2000-DMG.

[0186] In other aspects the invention is a composition for or method of vaccinating a subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide wherein a dosage of between 10 pg / kg and 400 pg / kg of the nucleic acid vaccine is administered to the subject. In some embodiments the dosage of the RNA polynucleotide is 1-5 pg, 5-10 pg, 10-15 pg, 15-20 pg, 10-25 pg, 20-25 pg, 20-50 pg, 30-50 pg, 40-50 pg, 40-60 pg, 60-80 pg, 60-100 pg, 50-100 pg, 80-120 pg, 40-120 pg, 40-150 pg, 50-150 pg, 50-200 pg, 80-200 pg, 100-200 pg, 120- 250 pg, 150-250 pg, 180-280 pg, 200-300 pg, 50-300 pg, 80-300 pg, 100-300 pg, 40-300 pg, 50-350 pg, 100-350 pg, 200-350 pg, 300-350 pg, 320-400 gg, 40-380 gg, 40-100 gg, 100-400 gg, 200-400 gg, or 300-400 gg per dose. In some embodiments, the nucleic acid vaccine is administered to the subject by intradermal, intraperitoneal or intramuscular injection. Advantageously, the administration is an intramuscular injection. In some embodiments, the nucleic acid vaccine is administered to the subject on day zero. In some embodiments, a second dose of the nucleic acid vaccine is administered to the subject on day twenty one.

[0187] In some embodiments, a dosage of 25 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 100 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 50 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 75 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 150 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 400 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 200 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, the RNA polynucleotide accumulates at a 100 fold higher level in the local lymph node in comparison with the distal lymph node. In other embodiments the nucleic acid vaccine is chemically modified and in other embodiments the nucleic acid vaccine is not chemically modified.

[0188] In exemplary embodiments of the invention, an efficacious vaccine produces an antibody titer of greater than 1 :40, greater that 1 : 100, greater than 1 :400, greater than 1 : 1000, greater than 1 :2000, greater than 1 :3000, greater than 1 :4000, greater than 1 :500, greater than 1 :6000, greater than 1 :7500, greater than 1 :10000. In exemplary embodiments, the antibody titer is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination. In exemplary embodiments, the titer is produced or reached following a single dose of vaccine administered to the subject. In other embodiments, the titer is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose.) In exemplary aspects of the invention, antigen-specific antibodies are measured in units of pg / ml or are measured in units of IU / L (International Units per liter) or mIU / ml (milli International Unitsper ml). In exemplary embodiments of the invention, an efficacious vaccine produces >0.5 pg / ml, >0.1 pg / ml, >0.2 pg / ml, >0.35 pg / ml, >0.5 pg / ml, >1 pg / ml, >2 pg / ml, >5 pg / ml or >10 pg / ml. In exemplary embodiments of the invention, an efficacious vaccine produces >10 mIU / ml, >20 mIU / ml, >50 mIU / ml, >100 mIU / ml, >200 mIU / ml, >500 mIU / ml or >1000 mIU / ml. In exemplary embodiments, the antibody level or concentration is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination. In exemplary embodiments, the level or concentration is produced or reached following a single dose of vaccine administered to the subject. In other embodiments, the level or concentration is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose.) In exemplary embodiments, antibody level or concentration is determined or measured by enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments, antibody level or concentration is determined or measured by neutralization assay, e.g., by microneutralization assay.

[0189] Methods for the chemical conjugation of polypeptides, carbohydrates, and / or lipids are well known in the art (see, for example, Hermanson. Bioconjugate Techniques (Academic Press; 1992); Aslam and Dent, eds. Bioconjugation: Protein coupling Techniques for the Biomedical Sciences (MacMillan: 1998); and Wong Chemistry of Protein Conjugation and Cross-linking (CRC Press: 1991)). For instance, primary amino groups may be incorporated by reaction with ethylenediamine in the presence of sodium cyanoborohydride and sulfhydryls may be introduced by reaction of cysteamin dihydrochloride followed by reduction with a standard disulfide reducing agent. Heterobifunctional crosslinkers, such as, for example, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, which link the epsilon amino group on the D-lysine residues of copolymers of D- lysine and D-glutamate to a sulfhydryl side chain from an amino terminal cysteine residue on the peptide to be coupled, may be used as well. Chemical conjugation also includes anything covalently bonded directly via side chain bonds or via a linker or spacer group.

[0190] The nanoparticle formulations may be a carbohydrate nanoparticle which may comprise a carbohydrate carrier and a modified nucleic acid molecule (e.g., mmRNA). As a nonlimiting example, the carbohydrate carrier may include, but is not limited to, an anhydride- modified phytoglycogen or glycogen-type material, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g., International Publication No. W02012109121; herein incorporated by reference in its entirety).

[0191] Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP). Ionizable cationic lipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin- MC3-DMA, have been shown to accumulate in plasma and tissues over time and may be a potential source of toxicity. The rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles by an order of magnitude from a 1 mg / kg dose to a 10 mg / kg dose in rat. Inclusion of an enzymatically degraded ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation. The ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain. The internal ester linkage may replace any carbon in the lipid chain.

[0192] The average diameter of the nanoparticle employed in the compositions of the invention can be at least one member selected from the group consisting of about 20 nanometers, about 25 nanometers, about 30 nanometers, about 40 nanometers, about 50 nanometers, about 75 nanometers, about 100 nanometers, about 125 nanometers, about 150 nanometers, about 175 nanometers and about 200 nanometers. In another embodiment, the average diameter of the particle is at least one member selected from the group consisting of between about 10 to about 200 nanometers, between about 0.5 to about 5 microns and between about 5 to about 10 microns. In another embodiment, the average diameter of the microparticle is selected from the group consisting of about 0.1 pm, about 0.2 pm, about 0.4 pm, about 0.5 pm, about 1 pm and about 2 pm.

[0193] Nanoparticles for use in the compositions of the invention can be made from lipids or other fatty acids (see, for example, U.S. Pat. Nos. 5,709,879; 6,342,226; 6,090,406; Lian, et al., J. of Pharma. Sci. 90:667-680 (2001) and van Slooten, et al., Pharm Res. 17:42-48 (2000)) and nonlipid compositions (see, for example, Kreuter, J. Anat. 189:503-505 (1996), the teachings of all of which are hereby incorporated by reference in their entirety). The compositions can be bilayer or multilamellar liposomes and phospholipid based. Polymerized nanoparticles, as described, for example, in U.S. Pat. No. 7,285,289, the teachings of which are incorporated by reference in their entirety.

[0194] Metallic oxide nanoparticles for use in the compositions of the invention can be chemically substituted with at least one reactive moiety capable of forming a thioether bondemploying conventionally techniques as described herein and in U.S. Pat. No. 6,086,881, the teachings of which are hereby incorporated by reference in their entirety. The antigen described herein can be coupled in a single step onto the metallic oxide particles by the formation of at least one thioether bond or it may be synthesized or assembled stepwise onto the metallic oxide particles after the initial thioether bond formation. The chemical derivatization reagents for the metallic oxide particles can include organosilane reagents that provide thioalkane functionality or other groups that may readily be converted into thiols or thiol-reactive moieties. Organosilane reagents which may be utilized for this purpose may be, but are not limited to, 3- mercaptopropyltrimethoxysilane, 3 -aminopropyltri ethoxy silane, 3 -iodopropyltrimethoxy silane, 2-chloroethyltrichlorosilane, 3-glycidoxypropyltrimethoxysilane, vinyltrichlorosilane and 3- acryloxypropyltrimethoxysilane. Moieties that include one or more disulfide components may also be joined to the metallic oxide particle surface and thereby provide the corresponding reactive moiety able to enter into and form a thioether bond and juncture. Exemplary nanoparticles for use in the compositions of the invention include at least one member selected from the group consisting of poly (D,L-lactide-co-glycolide, also referred to as “poly(lactic-co-glycolic acid) and bi sacy 1 oxy propyl cy steine .

[0195] Nanoparticles for use in the compositions of the invention can be made of inorganic material. Nanoparticles for use in the compositions of the invention can be made of a polymer material, such as at least one member selected from the group consisting of polystyrene, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyamide, polyacrylamide, polyacrolein, polybutadiene, poly caprolactone, polycarbonate, polyester, polyethylene, polyethylene terephthalate, polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polymethylmethacrylate, polylactide, polyglycolide, poly(lactide- co-glycolide), polyanhydride, polyorthoester, polyphosphazene, polyphosophaze, a carbohydrate, carboxymethyl cellulose, hydroxyethyl cellulose, agar, gel, proteinaceous polymer, polypeptide, eukaryotic and prokaryotic cells, viruses, lipid, metal, resin, latex, rubber, silicone (e.g., polydimethyldiphenyl siloxane), glass, ceramic, charcoal, kaolinite and bentonite.

[0196] It is noted that these therapeutics may be a chemical compound, a composition which may comprise a polypeptide of the present invention and / or antibody elicited by such a chemical compound and / or portion thereof or a pharmaceutically acceptable salt or a composition whichmay comprise a polypeptide of the invention, and may be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, diluents, and vehicles, as well as other active ingredients.

[0197] The compounds or compositions may be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and infusion techniques.

[0198] It is noted that humans are treated generally longer than the mice or other experimental animals which treatment has a length proportional to the length of the disease process and drug effectiveness. The doses may be single doses or multiple doses over a period of several days, but single doses are preferred. Thus, one may scale up from animal experiments, e.g., rats, mice, and the like, to humans, by techniques from this disclosure and documents cited herein and the knowledge in the art, without undue experimentation.

[0199] In a particularly advantageous embodiment, the mRNAs of the present invention are administered in combinations of a prime dose followed by one or more boost doses over time. mRNA doses of about 100 pg are advantageous, however, dosages of about 10 pg to about 1000 pg, about 20 pg to about 900 pg, about 30 pg to about 800 pg, about 40 pg to about 700 pg, about 50 pg to about 600 pg, about 60 pg to about 500 pg, about about 70 pg to about 400 pg, about 80 pg to about 300 pg, or about 900 pg to about 200 pg, are contemplated. Varying combinations are presented below as non-limiting examples.

[0200] The treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient being treated.

[0201] When administering a therapeutic of the present invention parenterally, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion). The pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier may be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

[0202] Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, corn oil,sunflower oil, or peanut oil and esters, such as isopropyl myristate, may also be used as solvent systems for compound compositions.

[0203] Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, may be added. Prevention of the action of microorganisms may be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the compounds.

[0204] Sterile injectable solutions may be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired.

[0205] A pharmacological formulation of the present invention, e.g., which may comprise a therapeutic compound or polypeptide of the present invention, may be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicles, adjuvants, additives, and diluents; or the compounds utilized in the present invention may be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, iontophoretic, polymer matrices, liposomes, and microspheres.

[0206] A pharmacological formulation of the compound and composition which may comprise a polypeptide utilized in the present invention may be administered orally to the patient. Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable. Known techniques, which deliver the compound orally or intravenously and retain the biological activity, are preferred.

[0207] In one embodiment, a formulation of the present invention may be administered initially, and thereafter maintained by further administration. For instance, a formulation of the invention may be administered in one type of composition and thereafter further administered in a different or the same type of composition. For example, a formulation of the invention may be administered by intravenous injection to bring blood levels to a suitable level. The patient's levelsare then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition, may be used. In the instance of a vaccine composition, the vaccine may be administered as a single dose, or the vaccine may incorporate set booster doses. For example, booster doses may comprise variants in order to provide protection against multiple clades of HIV. For example, one or more boost immunogens may be from HIV pseudo viruses (PS Vs) or derivatives or mutations or a portion thereof.

[0208] The quantity to be administered will vary for the patient being treated and whether the administration is for treatment or prevention and will vary from a few micrograms to a few milligrams for an average 70 kg patient, e.g., 5 micrograms to 5 milligrams such as 500 micrograms, or about 100 ng / kg of body weight to 100 mg / kg of body weight per administration and preferably will be from 10 pg / kg to 10 mg / kg per administration. Typically, however, the antigen is present in an amount on, the order of micrograms to milligrams, or, about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and most preferably about 0.05 to about 5 wt %.

[0209] Of course, for any composition to be administered to an animal or human, including the components thereof, and for any particular method of administration, it is preferred to determine therefor: toxicity, such as by determining the lethal dose (LD) and LDso in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable immunological response, such as by titrations of sera and analysis thereof for antibodies or antigens, e.g., by ELISA and / or REFIT analysis. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations may be ascertained without undue experimentation. For instance, dosages may be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art. Thus, the skilled artisan may readily determine the amount of compound and optional additives, vehicles, and / or carrier in compositions and to be administered in methods of the invention. Typically, an adjuvant or additive is commonly used as 0.001 to 50 wt % solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, most preferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and most preferably about 0.05 to about 5 wt %. Such determinations do not require undue experimentation from the knowledge of the skilled artisan,this disclosure and the documents cited herein. And, the time for sequential administrations may be ascertained without undue experimentation.

[0210] Examples of compositions which may comprise a therapeutic of the invention include liquid preparations for orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric, mucosal (e.g., perlingual, alveolar, gingival, olfactory or respiratory mucosa) etc., administration such as suspensions, syrups or elixirs; and, preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The compositions may also be lyophilized. The compositions may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0211] Compositions of the invention, are conveniently provided as liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions or viscous compositions which may be buffered to a selected pH. If digestive tract absorption is preferred, compositions of the invention may be in the “solid“ form of pills, tablets, capsules, caplets and the like, including “solid“ preparations which are time-released or which have a liquid filling, e.g., gelatin covered liquid, whereby the gelatin is dissolved in the stomach for delivery to the gut. If nasal or respiratory (mucosal) administration is desired, compositions may be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser. Aerosols are usually under pressure by means of a hydrocarbon. Pump dispensers may preferably dispense a metered dose or, a dose having a particular particle size.

[0212] Compositions of the invention may contain pharmaceutically acceptable flavors and / or colors for rendering them more appealing, especially if they are administered orally. The viscous compositions may be in the form of gels, lotions, ointments, creams and the like (e.g., for transdermal administration) and will typically contain a sufficient amount of a thickening agent so that the viscosity is from about 2500 to 6500 cps, although more viscous compositions, even up to 10,000 cps may be employed. Viscous compositions have a viscosity preferably of 2500 to 5000cps, since above that range they become more difficult to administer. However, above that range, the compositions may approach solid or gelatin forms, which are then easily administered as a swallowed pill for oral ingestion.[002131 Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection or orally. Viscous compositions, on the other hand, may be formulated within the appropriate viscosity range to provide longer contact periods with mucosa, such as the lining of the stomach or nasal mucosa.

[0214] Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form), or solid dosage form (e.g., whether the composition is to be formulated into a pill, tablet, capsule, caplet, time release form or liquid-fdled form).

[0215] Solutions, suspensions and gels, normally contain a major amount of water (preferably purified water) in addition to the active compound. Minor amounts of other ingredients such as pH adjusters (e.g., a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents, jelling agents, (e.g., methylcellulose), colors and / or flavors may also be present. The compositions may be isotonic, i.e., it may have the same osmotic pressure as blood and lacrimal fluid.

[0216] The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.

[0217] Viscosity of the compositions may be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.

[0218] A pharmaceutically acceptable preservative may be employed to increase the shelf-life of the compositions. Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed. A suitable concentration of the preservative will be from 0.02% to 2% based on the total weight although there may be appreciable variation depending upon the agent selected.

[0219] Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert with respect to the active compound. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems may be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

[0220] It is generally envisaged that compounds and compositions of the invention will be administered by injection, as such compounds are to elicit anti-HIV antibodies, and the skilled artisan may, from this disclosure and the knowledge in the art, formulate compounds and compositions identified by herein methods for administration by injection and administer such compounds and compositions by injection.

[0221] The inventive compositions of this invention are prepared by mixing the ingredients following generally accepted procedures. For example, the selected components may be simply mixed in a blender, or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity. Generally, the pH may be from about 3 to 7.5. Compositions may be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the composition form used for administration (e.g., solid vs. liquid). Dosages for humans or other mammals may be determined without undue experimentation by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art.

[0222] Suitable regimes for initial administration and further doses or for sequential administrations also are variable, may include an initial administration followed by subsequent administrations; but nonetheless, may be ascertained by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art.

[0223] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

[0224] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.ExamplesExample 1 - HIV broadly neutralizing antibodiesAn HIV vaccine first-boost candidate immunogen promoted maturation of VRC01 -class antibodies in a humanized mouse model

[0225] Heterologous prime-boost vaccination has been shown to drive early maturation of HIV broadly neutralizing antibody precursors in humanized mice.

[0226] A protective human immunodeficiency virus (HIV) vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRCOl-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, applicants designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost following eOD-GT8 60mer priming. Applicants found, using a humanized mouse model approximating human conditions of VRCOl-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRCOl-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. Applicants further showed that VRCOl-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRCOl-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.

[0227] An effective prophylactic vaccine against human immunodeficiency virus (HIV) is needed to help prevent the 1.3 million new infections occurring each year (A. S. Fauci et al., 2017; UNAIDS, 2023). HIV broadly neutralizing antibodies (bnAbs), which bind partially conserved surface patches (epitopes) on the highly variable HIV envelope glycoprotein (Env), can provide sterilizing protection in non-human primate models (A. Pegu et al., 2019) and can protect humans against infection (Y. Huang et al., 2022; L. Corey et al., 2021). Hence, bnAb elicitation is widely considered essential for an effective HIV vaccine. However, bnAb elicitation faces at least two major challenges. First, bnAb germline precursors typically have no detectable affinity for wildtype Env (X. Xiao et al., Viruses 2009; X. Xiao et al., Biochem Biophys Res Commun 390 2009;D. S. Dimitrov et al., 2010; M. Pancera et al., 2010; T. Zhou et al., 2010; M. Bonsignori et al., 201 1 ; J. F. Scheid et al., 201 1 ; S. Hoot et al., 2013; J. Jardine et al., 2013; A. T. McGuire et al., 2013; J. G. Jardine et al., 2016), which implies that wild-type Env proteins are unlikely to serve as effective priming immunogens to initiate bnAb induction. Second, most bnAbs are highly mutated from germline, with multiple mutations enabling high affinity binding to the cognate epitope, which indicates that repeated vaccination with a single antigen is unlikely to induce sufficient maturation to produce bnAbs.

[0228] Germline-targeting vaccine design (J. Jardine et al., 2013; A. T. McGuire et al., 2013; J. G. Jardine et al., 2016; J. M. Steichen et al., 2016; A. Escolano et al., 2016; M. Medina- Ramirez et al., 2017; J. M. Steichen et al., 2019; B. Briney et al., 2016; M. Tian et al., 2016; K. R. Parks et al., 2019; X. Chen et al., 2021) offers a potential solution to these challenges. In this vaccine strategy, a priming immunogen is designed to induce responses from diverse bnAb precursors for any single bnAb class, in order to prime the desired responses in all or most vaccine recipients. A series of booster immunogens are then designed to be successively closer in structure to the native glycoprotein, such that each boost should engage B cells produced by the prior immunization and select for additional mutation toward bnAb development. The IAVI G001 phase 1 trial provided clinical proof-of-principle for the priming step in the germline-targeting strategy: immunization with the germline-targeting priming immunogen eOD-GT8 60mer as protein with AS01B adjuvant was found to induce bnAb precursors of the VRC01 class in 97% of vaccine recipients (A. C. deCamp et al., 2023; D. J. Leggat et al., 2022; K. W. Cohen et al., 2023 ).

[0229] VRC01 -class bnAbs bind the Env CD4-binding site (CD4bs) and prevent HIV Env from binding its primary receptor on human CD4+ T cells (J. F. Scheid et al., 2011; X. Wu et al., 2010; X. Wu et al., 2011; T. Zhou et al., 2015; J. Huang et al., 2016; M. M. Sajadi et al., 2018; J. Umotoy et al., 2019). Prior work has shown that passively administered VRC01 IgG can protect humans from infection with diverse VRC01 -sensitive viruses (Y. Huang et al., 2022; L. Corey et al., 2021), providing evidence that similar VRCOl-class bnAbs elicited by vaccination can protect humans from HIV infection. The eOD-GT8 60mer is a self-assembling nanoparticle designed to bind to diverse VRCOl-class naive human precursors with substantial affinity and avidity (J. G. Jardine et al., 2015) and in pre-clinical studies was shown to prime VRCOl-class B cell responses in multiple knock-in and adoptive transfer mouse models (B. Briney et al., 2016; M. Tian et al., 2016; J. G. Jardine et al., 2015; P. Dosenovic et al., 2015; R. K. Abbott et al., 2018; H. Duan etal., 2018; X. Chen et al., 2021; X. Wang et al., 2021; D. Huang et al., 2020; S. Luo et al., 2023; D. Sok et al., 2016).

[0230] The next critical test of the germline-targeting strategy is to determine whether a suitably designed first-boost immunogen can advance maturation toward bnAb development. To support use of mRNA delivery to accelerate clinical testing, pre-clinical studies with mRNA immunogens must be performed. Here, applicants used VRC01 -class and CD4bs-specific non- VRC01 -class monoclonal antibodies (mAbs) from the IAVI G001 study to aid in the design of first-boost candidates to follow eOD-GT8 60mer priming, and applicants evaluated different prime-boost regimens based on mRNA or adjuvanted protein in a mouse model with VRC01 -class naive precursor frequencies and affinities approximately similar to those in humans. The results supported the initiation of the IAVI G002 human clinical trial (NCT05001373) testing eOD-GT8 60mer priming and core-g28v2 60mer boosting delivered by mRNA lipid nanoparticles (LNPs).

[0231] Materials and Methods

[0232] Study Design

[0233] The main objectives of this study were to evaluate first-boost immunogens following eOD-GT8 60mer priming for their capacity to induce increases in VRC01 -class B cell frequency, SHM, key mutations, and affinity and to compare adjuvanted protein versus mRNA immunization for delivery of self-assembling nanoparticle immunogens for prime and boost. The number of mice in each group was limited by mouse availability and the costs of analysis; however, the number of mice used was judged to be sufficient to detect clear differences between groups. Experiments were conducted once with group sizes ranging from 5 to 15 mice. Mice were randomly assigned to groups. Blinding was not used. Samples were excluded from analysis if fewer than 50,000 CD 19+ B cells were detected during fluorescence activated cell sorting (FACS), indicating poor viability.

[0234] Statistical analysis

[0235] Individual-level data for experiments where n < 20 are presented in data file S 1. Except in figure S9, significant differences between groups were calculated with Kruskal-Wallis test followed by Dunn’s test for multiple comparisons with an alpha of 0.05 and shown as: ns, no significant difference, P>0.05; *P<0.05; **P<0.01; ***P<0.001; and ****P<0.0001. In figure S9, statistical comparisons were made by Mann-Whitney test and shown as *p<0.05, **p<0.01, ns, not significant. Correlation analysis was performed with simple linear regression of the loglOtransform of the core-g28v2 KD versus VH %SHM, VK %SHM, or key VRC01 -class HC residues. All statistical analyses were performed using GraphPad Prism v9.5.1 .

[0236] Immunogen Design

[0237] The goal was to develop boost immunogens to follow eOD-GT8 60mer priming after realizing that eOD-GT8 60mer could successfully prime VRC01 -class responses in humans.

[0238] Boost candidates were developed and tested to follow eOD-GT8 60mer priming using a relatively permissive knockin mouse model (VRCOlgH, (J. G. Jardine et al., 2015)). Boost candidates were BG505-GT3-core 60mer and BG505-GT3-SOSIP (B. Briney et al., 2016) and also HxB2 core-e-2cc N276D 60mer. However, based on concerned that the permissive nature of this knockin mouse model might be misleading (reduced competition due to about 85% usage of VRCOlgH heavy chains, and potentially easier and more consistent SHM because all bnAb precursors had the same heavy chain that included the mature bnAb HCDR3); specifically concern that boost candidates selected in the VRCOlgH mouse might not perform well in more stringent mousemodels or in humans.

[0239] The binding of The applicants’ boost candidates to eOD-GT8-induced VRC01 -class antibodies from the more stringent Vnl-2 mouse model (M. Tian et al., 2016) was tested and no detectable binding was found (Fig. 24A), suggesting that these boost candidates had little promise in the more stringent mouse or in humans. eOD-GT6 (J. Jardine et al., 2013) and the more engineered eOD-GT7 (J. G. Jardine et al., 2016) both had affinity for post-GT8 VRCOl-class Abs from the Vnl-2 mouse (Fig. 24A), suggesting that eOD-GT6 60mer or eOD-GT7 60mer might serve as a boost. However, the aim was to develop boost candidates that would be more different from the eODGT8 prime and at least somewhat more native-like in structure. Therefore, development of modified eODs and cores, aiming to identify a boost that succeeded in the Vnl-2 mouse (or at least had affinity for post-GT8 VRCOl-class Abs from that mouse) was initiated, postulating that this would be the best candidate for human testing. Stabilized, N276-lacking versions of native-like trimers from isolates highly sensitive to VRC01 -classneutralization, such as the isolate 191084 (Fig. 9, (B. Briney et al., 2016)) were developed as even more native-like potential boostimmunogens. An eOD-GT8 60mer immunization study in Kymab mice was carried out. Kymab mice have extremely low frequencies of VRCOl-class precursors and therefore produce very low frequencies of VRCOl-class responses to eOD-GT8 60mer (D. Sok et al., 2016), so it was hypothesized that the Kymab model was not well-suited for evaluating boosters forVRC01 -class responses. However, it was also hypothesized that the Kymab model might give insights into how the human antibody repertoire might respond to eOD-GT8 60mer in terms of off-target non-VRCOl -class responses and their cross-reactivity to boost candidates. In the study, two shots of eOD-GT8 60mer were delivered six weeks apart and, two weeks after the second shot, similar amounts of serum antibody binding to HxB2 core-e-2cc N276D and a VRC01 epitope-knockout version of HxB2 core-e-2cc N276D (KOI lb) were detected, indicating that human immunoglobulin non-VRCO l -class responses primed by eOD-GT8 were binding to HxB2 coree-2cc N276D. It was hypothesized that such non-VRCOl -class serum Ab binding might impede boosting of of VRCOl-class responses in general. Thus, the aim was to minimize non- CD4bs cross-reactivity between the boost immunogens and eOD-GT8.

[0240] Development of the new eOD-based boost candidates started from eOD-GT6 (J. Jardine et al., 2013). The design goals were to make the CD4bs more native-like by removing selected germline-targeting mutations present in GT6 and to minimize reactivity to GT8-induced, non-VRCOl -class, CD4bs-specific and non-CD4bs-specific Abs. Removing GT mutations was expected to reduce affinity for GT8-primed Abs, so a stepwise approach was taken to identify constructs that retained as few GT mutations as possible while also retaining at least low detectable affinity for GT8-primed Abs from the VH1-2 mouse. This resulted in a series of eOD-GT6 variants termed eOD-GT6v2, eOD-GT6v3, and eOD-GT6v4 (D. J. Leggat et al., 2022). These GT6 variants eliminated 2, 3, and 4 GT mutations, respectively. It was hypothesized that these changes to the VRC01 epitope would reduce reactivity to GT8-induced, non-VRCOl -class, CD4bs-specific Abs. eOD-GT6v2 was noted to have better affinities for GT8-induced VRCOl-class Abs from the Vnl- 2 mouse. To reduce reactivity to GT8-induced non-CD4bs Abs, Rosetta was used to computationally resurface eOD-GT6 outside the CD4bs and outside the existing glycosylation sites. This resulted in resurfaced variants of the above-mentioned GT6 variants, including resurfaced eOD-GT6v2, termed eOD-GT6v2-cRSF.

[0241] Development of the new core-based boost candidates started from HxB2 core-e-2cc N276D (J. G. Jardine et al., 2015). The design goals were (i) to maintain or improve affinity for eOD-GT8 60mer-induced VRCOl-class Abs from the VH1-2 mouse model; (ii) to maintain a strong affinity gradient in which mature VRCOl-class bnAbs bind more strongly than post-GT8 VRCOl-class mAbs; (iii) to minimize boosting of GT8-induced, non-VRCOl -class, CD4bs- specific and non-CD4bs-specific Abs (iv); to minimize priming of non-CD4bs (off-target)responses; (v) to ensure robust shared CD4 T help with eOD-GT8 60mer, by utilizing the same lumazine synthase nanoparticle as employed for eOD-GT8 60mer; (vi) to ensure good thermal stability and nanoparticle formation; and (vii) to increase the content of shared sequence with HIV Envelope (Env) trimers so as to provide potential T-helper priming prior to a subsequent trimer boost immunization. To achieve these goals, the aim was to develop a core with the CD4bs as native-like as possible that would still allow binding to GT8-induced VRC01 -class Abs from the VH1-2 mouse; with the non-CD4bs surface differing as much as possible from eOD-GT8 (by resurfacing) and as non-immunogenic as possible (by V3-loop minimization and as much glycan- masking as possible); and with good thermal stability and nanoparticle expression. Starting from HxB2 core-e-2cc N276D, multiple iterations of V3-loop truncation were carried out (to minimize potential immunogenicity of the already reduced V3 loop present on HxB2 core-e-2cc N276D), computational resurfacing (to reduce antigenic similarity to eOD-GT8 outside the CD4bs), and glycan-masking (to minimize immunogenicity outside the CD4bs). Analogous modifications were made in the context of an extended version of HxB2 core-e-2cc N276D (TH6 version), in which the N- and C-termini were extended and two internal segments were adjusted to increase the content of sequence shared with largely conserved, non-glycosylated regions of HIV Env trimers, as an attempt to provide for potential shared T-help between the applicants’ core booster and any subsequent trimer boost. Modified versions of HxB2 core-e-2cc N276D led to core-g5, and modified versions including the TH6 extensions and internal changes led to core-g28 and core- g28v2.

[0242] Modifications were generally designed using Rosetta, starting from an initial homology model of HxB2 core-e-2cc N276D based on the structure of mature VRC01 bound to a core-e gp!20 (PDB: 3ngb). The different types of modifications were tested alone and in combinations. In the early iterations, only monomers were produced and tested, but in the later stages both monomers and 60mers were produced and tested. Readouts included: (i) fundamental biophysical readouts of expression amount, solution multimeric state by SECMALS, and thermal stability; (ii) surface plasmon resonance (SPR) affinities for mature VRC01 -class antibodies; and (iii) SPR affinities for GT8-induced VRC01 -class antibodies from the VH1-2 mouse (antibodies were from Tian et al. 2016 (M. Tian et al., 2016) and from a 2018 in-house experiment in which a single immunization of eOD-GT8 60mer was delivered with Sigma adjuvant system and day 35 GT8 / KO7IgG memory an germinal center B cells were sorted into 96-well plates and their BCRsequences were obtainedby reverse transcription polymerase chain reaction and Sanger sequencing.) Late-stage core boost candidates for binding to GT8-induced VRC01 -class and non- VRC01 -class antibodies from G001 were tested once those antibodies became available starting in 2020.

[0243] For V3-loop truncation, Rosetta KIC (D. J. Mandell et al., 2009) was employed to model loops of lengths 2 to 6 aa, with the aim of introducing a glycosylation site within the V3. One thousand models were generated for each loop length. The final candidates, selected based on loop closure score and total energy, were produced in vitro and evaluated for expression amount, stability and binding to a panel of VRCOl-class bnAbs (VRC01, 12a21, 12al2, CHA31, PGV04, 3BNC60, 3BNC117, VRC07, PG19, PGV20) and the non-nAb B6. In the final selected V3-loop minimization design, the starting v3 loop sequence “RPNNGGSGSGGNMRQ” (SEQ ID NO: 56) was substituted with a 6-residue loop sequence “AGNGTA” (SEQ ID NO: 57), which incorporated an engineered glycosylation site, referred to as position “300*”, with the star signifying that this position is present only on an artificial truncated loop. This modified V3-loop was incorporated into the final core-g5, core-g28, and core-g28v2 designs.

[0244] For glycan masking, two glycans (N206 and N246) from a previously reported hyperglycosylated core called 6G (J. Ingale et al., 2014) were included. To identify additional sites for glycosylation, the focus was on surface residues located at least 5 A away from the CD4bs (HxB2 numbering 92-97, 276-282 and 455-476) and at least 5 A away from the asparagine residues within existing glycosylation sites. Using Rosetta, the energy difference resulting from the introduction of the Nx(S / T) sequon were evaluated, where x is any amino acid except proline. Mutations that did not destabilize the protein were deemed suitable for experimental evaluation. For the initial design round, only single glycan mutations were incorporated. Each glycan variant was screened for expression and antigenicity against a panel of mature VRC01 antibodies. Promising glycan candidates were chosen and combined for the subsequent rounds of optimization, and glycosylation designs were also combined with V3-loop minimization and resurfacing designs as those modifications were being developed iteratively. In the design of glycosylation sites, it was also attempted to utilize NxT sequons whenever possible, as they have been shown to be glycosylated more efficiently than NxS sequons (H. A. Kaplan et al., 1987; L. Kasturi et al., 1995; R. Derking et al., 2021). In core-g5, which includes the final V3-loop minimization design and resurfacing, there are 8 engineered glycosylation sites (at positions 63,82, 113, 202, 206, 246, 300, and 441 in HxB2 numbering) and 16 native glycosylation sites (at positions 88, 230, 234, 241, 262, 289, 295, 332, 339, 356, 386, 392, 397, 406, 448 and 463 in HxB2 numbering). In core-g28v2, which includes the final V3-loop minimization design and resurfacing in the context of the TH6 extended core to increase potential shared T-helper epitopes with HIV Env trimers, there are a total of 28 glycosylation sites, with 12 engineered sites (at positions 113, 120, 300*, 344, 402, 409, 413, 419, 423, 434, 439, and 442 in HxB2 numbering) and 16 native sites (at positions 88, 230, 234, 241, 262, 289, 295, 332, 339, 356, 386, 392, 397, 406, 448, and 463 in HxB2 numbering). Core-g28 was the same as core-g28v2, except that core- g28 contained an engineered glycosylation site at 399 instead of the native glycosylation site at 397. It was found that core-g28v2 60mer demonstrated superior nanoparticle formation (Fig. 10), with a higher fraction of nanoparticles and greater homogeneity, compared to core-g28 60mer, which was the primary reason that the core-g28v2 was selected over core-g28 for clinical testing. Subsequently, it was also found that core-g28v2 monomer exhibited superior binding to GT8- elicited antibodies from the G001 study.

[0245] For core resurfacing, surface positions with identical or similar amino acids in both

[0246] eOD-GT8 and HxB2 core-e-2cc N276D (419, 462, 264, 267, 268, 270, 275, 278, 279, 298, 334, 335, 340, 344, 347, 351, 360, 362, 363, 381, 382, 444, and 457) were identified, and RosettaScripts (S. J. Fleishman et al., 2011) was used to design new amino acids at those positions. Minimal backbone movement was allowed. To guide the sequence design process, a positionspecific scoring matrix (PSSM) was generated by aligning HIV Env trimer sequences from the LANL database (www.hiv.lanl.gov); only amino acids present in native Env trimers were permitted, and an energy bonus was given to the native amino acid to reduce the likelihood of introducing destabilizing mutations. Most resurfaced designs were tested as fully resurfaced proteins, but for selected positions within the edge or near the CD4bs, individual point mutations were tested. Resurfacing modifications were found to affect thermal stability and nanoparticle formation propensity, with some designs reducing stability by as much as 10° and correspondingly lacking the ability to form nanoparticles, and other designs improving both thermal stability and nanoparticle formation. Although this resurfacing effort was almost entirely focused on making mutations at non-CD4bs positions, Rosetta was permitted to make substitutions at other positions, including position 278 within loop D at the edge of the CD4bs, because an additional study of non- VRCOl-class responses to eODGT8 60mer in Kymab mice (T. Schiffner et al., 2024) hadidentified loop D as an important target of CD4bs-specific non-VRCOl -class responses. Computational resurfacing identified the mutation R278M as the most favorable mutation at that position. This mutation had previously and independently been selected by a yeast display library screening method of developing a resurfaced eOD-GT8 (T. Schiffner et al., 2024) and it was found that the T278M mutation was effective at reducing binding to GT8 of CD4bs-specific non-VRCOl - class antibodies induced by GT8 in Kymab mice (T. Schiffner et al., 2024), hence it was hypothesized that including T278M in The applicants’ core booster might reduce binding of such antibodies to core. Although it was found experimentally that the T278M mutation was slightly destabilizing on core proteins (reducing thermal stability by about 1°C), it was also found that the T278M mutation improved binding to mature VRC01 -class bnAbs and to GT8-induced VRC01- class antibodies from IAVI G001 (Fig. 24B). Given the beneficial VRC01 -class antigenicity and the potential for reducing boosting of CD4bs-specific non-VRCOl -class responses primed by GT8, this mutation was retained in The applicants’ final designs. The core-g5 design included a total of 13 resurfacing mutations, and the core-g28 and core-g28v2 designs each included a total of 8 resurfacing mutations.

[0247] It was also sought to make in vivo comparisons between core-g28v2 and C13.G4.2 (X. Chen et al., 2021), which had previously been reported as a ferritin nanoparticle-based immunogen. To enable direct comparisons, C13.G4.2 60mers were produced using the same lumazine synthase platform used for core-g28v2, and core-g28v2 ferritin nanoparticles were produced, using the same ferritin platform previously described for C13.G4.2 (X. Chen et al., 2021).

[0248] Lumazine synthase stabilization

[0249] All lumazine synthase nanoparticles tested in this study utilized the previously reported d41m3 version of lumazine synthase. This version includes two engineered disulfides and three mutations to disable the enzymatic active site (D. Sok et al., 2016). When eOD-GT8 60mer was evaluated previously for delivery by an RNA replicon platform, the d41m3 version was found to perform better than the original version (M. Melo et al., 2019).

[0250] VRC01 -class epitope knockout mutants

[0251] The eOD-GT8 KO used here was eOD-GT8 KO11 (with mutations 280R, 365L, and 371R) (D. Sok et al., 2016). The core-g28v2 KO used here was core-g28v2 KOI lb. This version has the same mutations as KOI 1 but also included D368R.

[0252] Molecular modeling

[0253] A de novo model for core-g28v2 was generated using AlphaFold2. Man9 glycans were added and relaxed using Rosetta (J. Jumper et al., 2021; J. Adolf-Bryfogle et al., 2021; J. W. Labonte et al., 2017). Figures were made using UCSF Chimera (E. F. Pettersen et al., 2004).

[0254] Protein production

[0255] His-tagged and His-Avi-tagged monomeric and trimeric antigens were produced by transient transfection of HEK-293F cells (Thermo Fisher) and purified by immobilized metal ion affinity chromatography (IMAC) using HisTrap excel columns (Cytiva) followed be sizeexclusion chromatography (SEC) using either Superdex 75 10 / 300 GL or Superdex 200 Increase 10 / 300 GL columns (Cytiva). The molecular weight and the homogeneity of antigens were confirmed by size exclusion chromatography-multi-angle light scattering (SEC-MALS) in phosphate-buffered saline (PBS) using Superdex 75 10 / 300 GL or Superdex 200 Increase 10 / 300 GL columns (Cytiva) columns operating with an isocratic flow of 0.5 mL / minute followed by DAWN HELEOS II and Optilab T-rEX detectors (Wyatt Technology). His-Avi-tagged antigens were biotinylated using BirA (Avidity) and purified again to remove excess biotin using SEC with either Superdex 75 10 / 300 GL or Superdex 200 Increase 10 / 300 GL columns (Cytiva).

[0256] Nanoparticle 60mer immunogens were produced by transient transfection of HEK- 293F cells (Thermo Fisher). Immunogens were then purified by Galanthus nivalis lectin affinity chromatography (Vectorlabs) followed by SEC using a Superose 6 16 / 600 PG column (Cytiva). Immunogen preps confirmed to contain < 5 EU / mg of endotoxin using an Endosafe instrument (Charles River).

[0257] Genes encoding the antibody variable fragment (Fv) regions were synthesized by GenScript and cloned into antibody expression vectors pCW-CHIg-hGl and pCW-CLIg-hk. Monoclonal antibodies were produced using transient transfection of HEK-293F cells (Thermo Fisher). They were then purified using rProtein A Sepharose Fast Flow resin (Cytiva).

[0258] Site specific glycan profiling of core-g28v2 60mer

[0259] Engineered N-linked glycosylation sites are frequently under-occupied. To assess the glycosylation profile of the core-g28v2 60mer immunogen, site specific glycan profiling was conducted as previously described (S. Baboo et al., 2021). The degree of glycan occupancy and proportion of glycans that were complex and oligomannose / hybrid type were determined.

[0260] Immunizations in SE09 Mice

[0261] All work followed IACUC guidelines associated with animal protocol number 20- 0001. Mice were injected with l Opg (50pl total volume) of mRNA intramuscularly (I.M.) under anesthesia (5% isoflurane induction) in the left quadriceps muscle. All primes and subsequent boosts were done in the same location. Protein injections (20pg, 200pl total volume) were performed intraperitoneally (I.P.), utilizing 100 pL of the Sigma Adjuvant System (Cat#S6322). I.P. injections consisted of two 100 pL injections given on either side of the abdomen. Insulin syringes were used for all injections (BD #328440).

[0262] Tissue harvest and serum isolation

[0263] Mice were euthanized with compressed CO2 (100%) in a clear chamber to allow for visualization of respiration and subsequent death through respiratory cessation. Blood was collected from the chest cavity prior to the removal of the spleen and lymph nodes (mesenteric, inguinal, and popliteal (RNA injections only, left leg only). Tissues were placed in 3 mb resuspension buffer (lx PBS Ca / Mg++ free, ImM EDTA, 25mM HEPES, pH 7.0, 1% heat- inactivated fetal bovine serum [FBS]) in a 15 m polypropylene tube on ice. Tissues were disassociated using the rough ends of two sandblasted microscope slides in a 5mL petri dish, then returned to the same 15 mb polypropylene tube for centrifugation (460xg for 5 minutes at 4°C). Red blood cell lysis was performed using 1 mb of ACK buffer (Quality Biological, Cat#l 18-156- 721) for 2 minutes on ice in a 15 mb polypropylene tube. Lysis was halted by adding 14 mb resuspension buffer per sample. Post lysis and centrifugation (460xg for 5 minutes), cells were resuspended in 3 mb Bambanker freezing medium (Bulldog Bio, Cat# BB01) prior to filtration through a cottonplugged, borosilicate Pasteur pipette into a borosilicate glass test tube. 1 mb filtered-cell solution was subsequently divided into three cryovials per mouse, which were precooled in a Styrofoam rack on dry ice. Cells were stored at -80°C for 2 to 7 days prior to longterm storage in liquid nitrogen. Serum samples were collected by spinning the blood at 14,000 RPM for 30 minutes. Serum samples were stored at -20°C.

[0264] Sample preparation and B cell sorting

[0265] For immunized samples, frozen splenocytes and lymphocytes were thawed in 10 mb 50:50 heat inactivated FBS (Omega Scientific, cat# FB-02):RPMI-1640 (Gibco, cat# 61870-036) pre-warmed to 37°C. Unimmunized splenocytes for naive B cell sorting were used fresh after processing. Cells were centrifuged at 400xg for 5 minutes. After the supernatant was removed, cells were resuspended in 3 mb FACS buffer (1% v / v heat inactivated FBS, 1 mM EDTA(Invitrogen, cat# 15575-038), 1 mM HEPES (Gibco, cat# 15630-080) in Dulbecco’s PBS (DPBS; Corning, cat # 21-031 -CV)), and enumerated. Because cells in SE09 mice constitutively express green fluorescent protein (GFP), in some cases a small amount of cells were set aside for GFP compensation. After counting, cells were subjected to B cell isolation using the StemCell) Easy Sep Mouse Pan-B Cell Isolation Kit (StemCell), cat #19844 A) according to manufacturer-provided instructions.

[0266] Streptavidin (SA) conjugated-baits were prepared by combining biotinylated monomeric baits or Env trimer baits with fluorescent SA at room temperature for at least 1 hour in the dark. Wild-type baits were complexed with SA- Alexa Fluor 647 (Invitrogen, cat# S21374) and SA-brilliant violet (BV) 421 (BioLegend, cat# 405225). Knockout (KO) baits, if used, were conjugated with SAphycoerythrin (PE)-cyanine (Cy) 7 (BioLegend, cat# 405206). Monomeric baits were conjugated with SA at a 4: 1 (bait: SA) ratio and used at a final bait concentration of 200 nM for staining. Env trimer baits were conjugated with SA at a 2: 1 (bait: SA) ratio and used at 100 nM.

[0267] Isolated B cells were transferred over to 15 mL conical tubes, washed once with FACS buffer, and stained with 100 pL antibody cocktail mix consisting of PE anti-CD19 (BD Biosciences, cat# 553786), BV786 anti-IgM (BD Biosciences, cat#743328), Peridinin chlorophyll protein (PerCP)-Cy5.5 anti-IgD (BD Biosciences, cat# 564273), allophycocyanin (APC)-Cy7 anti- F4 / 80 (BioLegend, cat # 123118), APC-Cy7 anti-CDl lc (BD Biosciences, cat# 561241), APC- Cy7 anti-Ly-6C (BD Biosciences, cat# 557661), APC-H7 anti-CD8a (BD Biosciences, cat# 560182), and APC-H7 anti-CD4 (BD Biosciences, cat# 560181). All antibodies were used at 1 : 100 dilution. When only wild type (WT) baits were used, the prepared fluorescent SA-baits were added along with the antibody master mix for 30 minutes at 4°C in the dark. When using KO baits, fluorescent SA-KO baits were first added to cells with the antibody master mix for 15 minutes, followed by the addition of WT baits for an extra 30 minutes. During the addition of antibody master mix, a unique TotalSeq-C anti-mouse hashtag antibody (BioLegend) was added to each sample at a concentration of 2.5 pL I up to 20 million cells. Following antibody staining, 1 :300 1 mL live / dead stain (LIVE / DEAD Fixable Aqua, Invitrogen, cat# L34966) was added to each sample and incubated for an additional 15 minutes at 4°C. At the end of staining, cells were washed with 10 mL of FACS buffer, and resuspended in 500 pL FACS buffer.

[0268] All samples were sorted on a BD FACSMelody. Single color compensations were performed using C57BL / 6 splenocytes with matched antibodies. For channels used for bait detection, cells were stained with biotinylated anti-CD19 (BioLegend, cat# 115503) followed by secondary staining with the appropriate fluorescent SA. Samples were fdtered through a 35 pm mesh-cap FACS tube (Falcon, cat# 352235) prior to being loaded on the sorter. A maximum of 15,000 cells were sorted using purity mode into a PCR plate well containing 20 pL of 0.2 pm filtered FBS. Event rates were typically maintained at about 1000 events per second and no more than 1500 events per second to ensure high sorting efficiencies.

[0269] BCR sequencing using lOx Genomics

[0270] Sorted samples were prepared for BCR sequencing by the 10X Genomics Single Cell Immune Profiling platform. After cell sorting, DPBS was added up to near top of the sample collection well (about 100 pL) and gently mixed to dilute the FBS catch buffer. The plate was sealed and cells were centrifuged for 2 minutes at 2000 rpm, after which the excess buffer was removed except for about 38 pL required for the 10X Genomics GEM reaction. Samples were processed according to manufacturer’s user guide for Chromium Next GEM Single Cell 5’ Reagent Kits v2 (Dual Index) with Feature Barcoding, with two main modifications. The number of PCR cycles in the cDNA amplification step were determined by assuming that only 20% of the total number of cells sorted would be recovered. This modification was made based on the observation that on average, the number of unique paired-BCR sequences recovered from the 10X Genomics platform was typically about 20% of the total number of cells sorted. In the second modification, the number of PCR cycles for each of the V(D)J amplification steps were increased to 10 cycles if the number of cells sorted (according to the sorter) was fewer than 1000 cells. Details regarding the modified protocol can also be found in Hurtado et al. (J. Hurtado et al., 2022). Pooled libraries were sequenced on an Illumina NextSeq 2000 using a 100-cycle P3 reagent kit (Illumina, cat# 20040559) with a target depth of 5000 paired-end reads for both the V(D)J and Feature Barcode Libraries and run using read parameters indicated in the lOx Genomics user guide.

[0271] Sequence analysis

[0272] Raw sequencing data were demultiplexed, processed into assembled VDJ contigs and counts matrix files, and assigned to specific animal IDs based on TotalSeq-C antibody hashtag counts using Cell Ranger (v6.1) and scab as previously described (J. Hurtado et al., 2022). Gene assignment, annotation, and formatting into Adaptive Immune Receptor Repertoire (AIRR) format(F. Breden et al. 2017 et al., 2017) for paired heavy and light chain antibody sequences was performed using Sequencing Analysis and Data library for Immunoinformatics Exploration (SADIE) with a custom SE09 mouse germline reference database (D. J. Leggat et al., 2022). VRC01 -class identification and mutational analysis was performed using the SADIE renumbering module by numbering each sequence using Kabat numbering and checking for the following key VRCOl-class residues: (i) germline: 47W, 50W, 55G, 71R; (ii) nonparatope: K19R, G31D / A, Y33I / V / T, M34I / L, S76E / D, S82aK / R; and (iii) paratope: N52K / R, N53 / R / Q / K / L / M / V / E, S54Y / G / H / F / R, G56A, T57V, Q61R / H / G, K62Q / G, T73V / I, S74Y, Trpio3-5.

[0273] The frequency of VRCOl-class memory B cells (MBCs) was calculated by multiplying the frequency of antigen-specific MBCs among all MBCs processed by FACS and the frequency of VRCOl-class BCRs among all sequenced heavy / light pairs. Similarly, the frequency of VRCOl- class with human VK1-33 light chains was calculated by multiplying the frequency of antigenspecific MBCs among all MBCs processed by FACS and the frequency of VRC01- classVK1'33BCRs among all sequenced heavy / light pairs.

[0274] ELISA

[0275] ELISA plates (Corning 96-Well Half-Area Plates, Catalog # 3690) were directly coated with ELISA antigens at 2 pg / mL on Day 1. Plates were incubated overnight at 4°C. Plates were washed three times with PBST (PBS + 0.2% tween 20) and blocked with PBST containing 5%

[0276] skim milk (BD Difco Skim Milk Catalog # 232100) and 1% FBS (Thermo Fisher, Catalog # 16000044) for 1 hour at room temperature on Day 2. Plates were then washed three times and 25pL of serum serially diluted (1 : 1000) in blocking buffer (PBST, 1% [w / v] FBS) was added for 1 hour at 37°C and 80% humidity. Plates were washed five times and 25 pL of AntiMouse IgG (H+L) (Jackson ImmunoResearch Catalog # 115-035 166) was added at 1 :5,000 dilution in PBST + 1% FBS. After a 1 hour incubation at room temperature, plates were washed three times and TMB Chromogen Solution (Thermo Fisher Catalog # 002023) substrate was added. To stop the reaction, 25 pl 0.5 M H2SO4 were added after 5 minutes. Absorption was read at 450 and 570 nm on a Molecular devices VersaMax plate reader (VersaMax). Background subtraction was performed by subtracting the 570 nm value from the corresponding 450 nm value. Data were subsequently analyzed in GraphPad Prism v9.5.1. using the Agonist vs response - variable slope equation to calculate the 50% effective concentrations (ECso).

[0277] SPR

[0278] The kinetics and affinity of antibody-antigen interactions were measured on Carterra LSA using HC30M or CMDP Sensor Chip (Carterra) and lx HBS-EP+ pH 7.4 running buffer (20x stock from Teknova, Cat. No H8022) supplemented with bovine serum albumin (BSA) at 1 mg / mL. The chip surface was prepared for ligand capture according Carterra software instructions. In a typical experiment, about 2500 to 2700 resonance units (RU) of capture antibody (SouthemBiotech Cat no 2047-01) in 10 mM Sodium Acetate pH 4.5 was amine coupled. Phosphoric Acid 1.7% was the applicants’ regeneration solution with 60 seconds contact time and injected three times per each cycle. The ligand solution concentrations were 1 to 5 pg / mL for the standard method used to assess monovalent analytes and 0.1 to 0.2 pg / mL for the low-capture IgG method used to assess binding to trimeric analytes. In both methods, the contact time was 3 to 5 minutes. Raw sensograms were analyzed using Kinetics software (Carterra), interspot and blank double referencing, Langmuir model. Analyte concentrations were quantified on NanoDrop 2000c Spectrophotometer using Absorption signal at 280 nm. Analyte samples were buffer-exchanged into the running buffer using dialysis. A broad range of affinities in runs was covered and the best referencing practices are different depending on how fast the off-rate is for particular ligand. For fast off-rate (faster than le-2 1 / s) automated batch referencing was used that includes overlay y- aline and higher analyte concentrations. For slow off-rates (9e-3 1 / s or less) a manual process referencing that includes serial y-align and lower analyte concentrations was used. After automated data analysis by Kinetics software, additional filtering was performed to remove datasets with highest response signals smaller than signals from negative controls. This additional filtering was performed automatically using a R-script.

[0279] Serum IgG Isolation

[0280] Polyclonal IgG was isolated from mouse serum samples using Protein G Sepharose 4 Fast Flow resin (Cytiva, 17-0618-05). Resin was washed 3 times in lx PBS and resuspended at 1 : 1 ratio resin to PBS. 50 pL of resin / PBS mixture was added to 200 pL heat-inactivated mouse serum and incubated with agitation overnight at room temperature. Samples were loaded into empty spin columns (Pierce, 89868) and centrifuged at 5000xg for 2 minutes. Resin was washed 3 times with lx PBS. IgGs were eluted into 50 pL IM Tris pH 8 using 50 pL 0. IM glycine pH 2.7. IgGs were concentrated and buffer exchanged into IX TBS using 30 kDa Amicon Ultra 0.5 mL centrifugal filters (Millipore, UFC503096).

[0281] Competition biolayer interferometry (BLI)

[0282] A competition BLI experiment was performed to assess epitope specificity of elicited VRC01 -class mAbs. Biotinylated core-g28v2 monomer was loaded onto streptavidin OCTET biosensors (Sartorius, 18-5019) at 5 pg / mL in kinetics buffer (IxPBS, pH 7.4, 0.01% [w / v] BSA, and 0.002% [v / v] Tween 20) until a response of 1 nanometer shift was reached. Loaded biosensors were dipped into kinetics buffer for 1 minute to acquire a baseline and then moved to wells containing VRC01 Fab at 2 pM in kinetics buffer. The VRC01 Fab was allowed to saturate the core-g28v2 load biosensors for 2 minutes. The biosensors were then moved to wells containing competitor IgGs at 500 nM in kinetics buffer for 2 minutes. Control binding experiments were conducted in which no VRC01 Fab was used. Competition was apparent by a lack of signal for IgG binding when VRC01 Fab was used compared to the signal acquired when no VRC01 Fab was used. All BLI experiments were conducted at 25°C.

[0283] Neutralization

[0284] Pseudovirus neutralization assays were performed as previously described (M. Li et al., 2005 et al., 2005) with minor modifications. Briefly, single cycle infectious pseudoviruses were generated by co-transfecting HEK293T cells with Envs of interest with an Env deficient HIV-1 backbone plasmid pSG3DEnv using Fugene 6 (Promega, E2692), or PEI MAX (Polysciences, Inc, 24765-1). Viruses were harvested 72 hours post-transfection and kept frozen at -80°C until use. 50 pL of target TZM-bl cells at 100,000 cells / mL were distributed into half area 96 well plates (Corning, 3688) the day before the assay. On the day of the neutralization assay, mAbs were serially diluted in D10 media (Dulbecco's Modified Eagle Medium (Gibco, 10313-021), 10% FBS (Omega Scientific, FB-02), lx PenStrep (Gibco, 15070-063), and lx GlutaMAX (Gibco, 35050- 061)). Viruses where prepared by thawing at 37°C, and concentrated or diluted with D10 according to previously defined titers. DEAE-dextran (Spectrum Chemical Mfg. Corp., DE132) was added to the viruses at 10 pg / mL. The prepared mAb dilutions and viruses were distributed into 96 well round bottom plates (Corning, 3788) at a 1 : 1 v / v ratio and incubated at 37°C for 1 hour. Following the 1 hour incubation, the supernatant was carefully aspirated from the prepared TZM-bl plates, then 25 pL of appropriate mAb:virus mixtures were added to the target cells and returned to the incubator. 24 hours later, 75 pL of D10 were added to each well and further incubated for an additional 48 hours. The supernatant was removed and TZM-bl cells were lysed in 45 pL / well of IX Cell Lysis Buffer (Promega, cat# E4550) for 15 minutes, after which 30 pL / well of substrate was added (Promega, cat# E4550) and luminescence was read on a BioTek Synergy Hl PlateReader. Half-maximal inhibitory concentration (IC50) values were interpolated from the One site Fit logIC50 model in Prism 9 (GraphPad). All curve fits were constrained between 0 to 100% neutralization. Both TZMbl cells and HEK293T cells were cultured and maintained in DIO media in a 37°C humidity and CO2 controlled incubator.

[0285] Results

[0286] VH1-2 / VK1-33 rearranging mice harbor VRC01 -class precursors with frequencies and affinities approximating those in humans.

[0287] VRC01 -class antibodies are defined by their use of the human immunoglobulin heavy chain (HC) V gene alleles VH1-2*02 or *04 and any light chain (LC) complementarity determining region (LCDR) 3 with a length of five amino acids (aa) (J. Jardine et al., 2013, A. P. West et al., 2012). Most of the interactions between VRCOl-class bnAbs and HIV Env come from the HC complementarity determining region (HCDR) 2, with the HCDR3 providing a minor supporting role (T. Zhou et al., 2010, T. Zhou et al., 2015, J. Huang et al., 2016), allowing VRCOl-class bnAbs to have diverse HCDR3 lengths and sequences.

[0288] The Vnl-2JH2 / VKl-33hTdTmouse (referred to as SE09 from this point forward) has the human IGHV1-2*O2 and human IGHJ2*01 alleles knocked into the mouse IGH locus and the human IGKVl-33*01 allele knocked into the mouse IGK locus (Fig. 7A) (S. Luo et al., 2023). Additionally, the SE09 mouse has a knockout of the intergenic control region 1 (IGCR1), resulting in dominant usage of the human IGHV1-2*O2 allele, and a human terminal deoxynucleotidyl transferase (TdT) knock-in that increases the percentage of LCs with 5 aa LCDR3s (Fig. 7A) (S. Luo et al., 2023). The knock-in genes participate in normal V(D)J recombination, resulting in a diverse repertoire of CDR3 lengths and sequences, and the human VH1-2 HCS and VK1-33 LCS are expressed in approximately 45% and 2% of naive B cells, respectively (S. Luo et al., 2023). Applicants evaluated the VRCOl-class precursor frequency in the SE09 mouse model by sorting CD19+ / IgM+IgD+ / eOD-GT8++splenocytes from 10 unimmunized mice and subjecting those cells to paired single-cell B cell receptor (BCR) sequencing using lOx Genomics. The frequency of VRCOl-class naive B cells was higher in the SE09 mouse (1 in 13,600) compared to humans (1 in 228,000) (Fig. 7B) (J. G. Jardine et al., 2016, J. H. Lee et al., 2021, C. Havenar-Daughton et al., 2018). The frequency of VRCOl-class naive B cells with human VK1-33 LCS (VRC01-class^K1‘33B cells) was 1 in 583,000 in SE09 mice, which was approximately 3 times higher than the frequency of VRC01-classVK1'33naive B cells in humans (1 in 1.78 million) (Fig. 7C) (J. G. JardineI l let al., 2016, J. H. Lee et al., 2021, C. Havenar-Daughton et al., 2018). The LCDR3s from VRC01- classVK1'33naive B cells for humans and SE09 mice were similar, with slightly less diversity seen in the sequences from SE09 mice (Fig. 7D). Applicants expressed BCRs from VRC01 -class naive B cells as IgG mAbs and assessed them for binding to eOD-GT8 using surface plasmon resonance (SPR). VRCOl-class naive mAbs from SE09 mice had nearly identical geomean dissociation constant (KD) for eOD-GT8 as previously isolated human naive VRCOl-class mAbs (J. G. Jardine et al., 2016, J. H. Lee et al., 2021, C. Havenar-Daughton et al., 2018) (3.5 pM for SE09 [N=42] vs. 3.4 pM for humans [N=73]) (Fig. 7E). Restricting the analysis to VRC01-classVK1'33naive BCRs, the geomean KD for binding to eOD-GT8 was also nearly identical for antibodies from SE09 mice or humans (1.5 pM for SE09 [N=l 1] vs. 1.6 pM for humans [N=14]) (Fig. 7F). Overall, the SE09 mouse provided a model system with a diverse B cell repertoire containing VRCOl-class naive precursors with comparable affinities to human VRCOl-class naive precursors that were present at physiologically relevant frequencies.

[0289] First-boost candidates were designed to boost maturation of VRCOl-class BCRs primed by eOD-GT8 60mer.

[0290] An ideal first-boost immunogen to follow eOD-GT8 60mer priming would be the most native-like protein that can bind to eOD-GT8-induced VRCOl-class BCRs and have no measurable affinity for eOD-GT8-induced non-VRCOl -class BCRs specific for the CD4bs. In humans, eOD- GT6 and more native variants of eOD-GT6 have been shown to bind to eOD-GT8-primed VRCOl- class BCRs but to have limited binding to eOD-GT8-primed non-VRCOl -class BCRs (D. J. Leggat et al., 2022). Although eOD-GT6 variants could potentially function as a first boost, immunogens that are more native-like in structure are preferred because they have the potential to shepherd B cells further down the path towards producing bnAbs. Structural platforms that would be more native-like than eOD-GT6 variants would need to include both core-gpl20- and trimer-based immunogens, in which trimer-based immunogens would be the closest in structure to the native glycoprotein.

[0291] Applicants set out to design a gpl20 core-based nanoparticle immunogen that would boost and drive further maturation of eOD-GT8-primed VRCOl-class B cells, but not boost eOD- GT8-primed non-VRCOl -class B cells. Starting from a conformationally stabilized HxB2 gpl20 core lacking the N276 glycan (HxB2 core-e-2cc N276D) (J. G. Jardine et al., 2015), applicants carried out iterative rounds of remodeling (minimization of the already trimmed V3 to reducepotential immunogenicity of this region), resurfacing (to minimize cross-reactivity to eOD-GT8 outside the CD4bs and to improve affinity for eOD-GT8-induced VRC01 -class BCRs) and glycan masking (to reduce responses outside the CD4bs) (Fig. 1A and B). New constructs were screened for protein expression and stability, antigenicity for VRCOl-class bnAbs and eOD-GT8-induced VRCOl-class Abs from a Vnl-2 mouse model (M. Tian et al., 2016) and from IAVI G001, and nanoparticle formation (Fig. 1 A). For some core designs, applicants extended the N- and C-termini and adjusted two internal segments to increase the content of sequence shared with largely conserved, non-glycosylated regions of HIV Env trimers, as an attempt to provide for potential shared T cell help between The applicants’ core booster and any subsequent trimer boost. The design path without added potential CD4 helper T cell epitopes resulted in the immunogen core- g5 (Fig. 1A and Fig. 8A). The design path with added potential CD4 helper T cell epitopes was subjected to additional rounds of protein resurfacing and glycan masking followed by screening for protein expression and stability, antigenicity, and nanoparticle formation, ultimately resulting in two immunogens, core-g28 and core-g28v2 (Fig. 1 A and Fig. 8A). All three core designs (core- g5, core-g28, and core-g28v2) were more native-like within the CD4bs than eOD-GT8 (Fig. 8B) and assembled into well-formed 60mer nanoparticles (Fig. 9A to E). Among these three designs, core-g28v2 60mer was prioritized due to its improved potential for priming CD4 T helper responses that might be engaged by subsequent trimer boosts, superior nanoparticle assembly compared to core-g28 (Fig. 9A), lack of affinity for CD4bs-specific non-VRCOl -class mAbs induced by eOD-GT8 60mer in G001 (Fig. 9F), and favorable affinity gradient, with considerably higher affinity for VRCOl-class bnAbs than for eOD-GT8 60mer-induced VRCOl-class mAbs (Fig. 9G), which should help guide maturation toward bnAb development. Additionally, core- g28v2 lacked detectable affinity for VRC01-classVK1-33precursors isolated from the SE09 mouse (Fig. 9H) and is therefore unlikely to prime VRCOl-class responses. Glycan occupancy analysis for core-g28v2 60mer (Fig. 91) showed that only 4 of 12 engineered N-linked glycosylation sites had > 50% occupancy, whereas 14 of 16 native N-linked glycosylation sites had > 50% occupancy.

[0292] Several additional booster immunogens have been suggested or used in previous studies, including but not limited to: a stabilized native HxB2 gpl20 core lacking the N276 glycan (J. G. Jardine et al., 2016, D. J. Leggat et al., 2022), a chimeric gpl20 core immunogen termed cl3 lacking the N276 glycan (M. Medina-Ramirez et al., 2017, M. Tian et al., 2016), and a BG505 gpl20 core lacking the N276 glycan with additional germline-targeting mutations (A. Escolano etal., 2016). Finally, a native HIV Env trimer lacking the N276 glycan could theoretically be used as a first-boost immunogen, provided it had appreciable affinity to eOD-GT8-primed VRC01 -class BCRs. Therefore, applicants used SPR to assess affinities of mAbs isolated from humans after one (G001 wk4+wk8) or two (G001 wkl0+wkl6) immunizations with eOD-GT8 60mer protein (D. J. Leggat et al., 2022) and from SE09 mice primed with one dose of eOD-GT8 60mer protein to the following first-boost candidates: eOD-GT6v2-cRSF (a resurfaced version of eOD-GT6v2 (D. J. Leggat et al., 2022), core-g28v2, 191084-N276D stabilized Env trimer (from isolate 191084_B7_19, hereafter abbreviated as 191084), C13.G4.2 core (X. Chen et al., 2021), and BG505-core-VRC01-GT3.3 (B. Briney et al., 2016) (Fig. 1C, Fig. 8 and 11). VRCOl-class mAbs isolated in the IAVI G001 trial after eOD-GT8 60mer immunization had affinity for boost immunogen candidates that decreased as the immunogens became more native (Fig. 1C, Fig. 8 and 11). Overall, core-g28v2 showed moderate affinity for eOD-GT8 elicited mAbs (Fig. 1C), could assemble into a high avidity 60mer nanoparticle (Fig. 9), and contained a more native CD4bs than eOD-GT6v2-cRSF and the other gpl20 core immunogens (Fig. 8).

[0293] VRCOl-class B cells were boosted with protein immunogens.

[0294] Applicants evaluated core-g28v2 60mer and six other candidate first-boost immunogens as adjuvanted proteins in SE09 mice. SE09 mice were primed with eOD-GT8 60mer protein, received first-boost immunogens at week 6, and responses were evaluated at weeks 6 and 12. The primary readouts were the frequencies, degrees of mutation, and affinities of VRCOl-class responses. Splenocytes and draining lymph node cells from week 6 (post-prime) or week 12 (postboost) were sorted for antigen-specific CD19+ / IgMTgD‘ B cells and subjected to paired single-cell BCR sequencing using lOx Genomics followed by bioinformatic analyses (Fig. 11). Samples from each group were sorted with antigens matched to the immunogen received; placebo samples were sorted with core-g28v2. Applicants refer to the sorted cells as antigen-specific memory B cells (MBCs), because they were expected to be enriched in memory-phenotype cells, although other types of cells such as germinal center B cells could also be present.

[0295] In the primary set of experiments, mice were primed with eOD-GT860mer protein and then boosted with either core-g28v2 60mer, eOD-GT6v2-cRSF 60mer, 191084-N276D soluble native Env trimer, or phosphate buffered saline (PBS) as a placebo (Fig. 2A). Antigen-specific MBCs were detected in all immunization groups, although frequencies were very low for both the placebo and 191084-N276D groups (Fig. 2B). Substantial fractions of the antigen-specific MBCswere VRCOl-class for both the core-g28v2 and eOD-GT6v2-cRSF boost groups (Fig. 2C), and median frequencies of VRCOl-class MBCs were 0.5% to 1% for those two boost groups (Fig. 2D). In contrast, VRCOl-class MBCs were detected in only two of the five animals boosted with the 191084-N276D trimer (Fig. 2C), and an appreciable frequency of VRCOl-class MBCs was detected in only one such animal (Fig. 2D), from which applicants concluded that this particular N276-lacking native-like soluble trimer would not be an effective first boost.

[0296] Consideration of VRC01-classK1’33responses offered the most direct comparison to human responses. Priming with eOD-GT8 60mer protein led to a 712-fold expansion of VRC01- classVK1'33MBCs relative to the naive IgD+IgM+VRC01-classVK1'33precursor pool in SE09 mice (Fig. 2E and Fig. 7C). For comparison, in the G001 clinical trial, the frequency of VRC01-classVK1'33IgG+B cells was 45-fold or 317-fold higher than the naive VRC01-classXK1'33precursor frequency at eight weeks after the first or second dose of eOD-GT8 60mers, respectively (D. J. Leggat et al., 2022). Thus, a single immunization with adjuvanted eOD-GT8 60mer protein in SE09 mice was approximately as effective as two immunizations in humans for inducing substantial frequencies of VRC01-classK1‘33MBCs.

[0297] Boosting with core-g28v2 60mer after one immunization of eOD-GT8 60mer in SE09 mice led to an additional 3-fold expansion of VRC01-classVK1'33MBCs, resulting in a VRC01- class^K1‘33frequency of 1 in 258 MBCs at week 12 (Fig. 2E). Compared to a placebo boost, the core-g28v2 60mer boost elicited higher frequencies of antigen-specific MBCs (median 4.7% vs. 0.3%, p=0.009; Fig. 2B) and VRCOl-class MBCs (median 0.59% vs. 0.14%, p=0.032; Fig. 2D). Furthermore, core-g28v2 lacked detectable affinity for VRC01-classK1'33precursors in the SE09 mouse (Fig. 9H), hence it was not expected to elicit de novo VRCOl-class responses. The increased VRCOl-class frequencies detected after the core-g28v2 60mer boost were therefore most likely due to boost-induced expansion of eOD-GT8 60mer-primed B cells and not to either de novo priming of VRCOl-class responses by the boost immunogen or to evolution of the eOD-GT8 60mer-primed cells over time independent of the boost.

[0298] Boosting with eOD-GT6v2-cRSF 60mer elicited similar frequencies of VRCOl-class MBCs and VRC01-classVK1'33MBCs compared to core-g28v2 60mer (Fig. 2D and E). However, VH and VK / VL somatic hypermutation (SHM) among VRCOl-class MBCs did not increase after boosting with eOD-GT6v2-cRSF 60mer relative to the post-prime response (Fig. 2F and G). In contrast, boosting with core-g28v2 60mer produced VRCOl-class MBCs with higher VH andVK / VL SHM compared to the post-prime response and compared to boosting with eOD-GT6v2- cRSF 60mer (Fig. 2F and G). As the core-g28v260mer boost generated similar VRCOl -class MBC frequencies but higher SHM in VRC01 -class MBCs (and similar SHM in non-VRCOl -class MBCs) compared to the eOD-GT6v2-cRSF 60mer boost, and as core-g28v2 had a more nativelike structure than eOD-GT6v2-cRSF (Fig. 8), applicants concluded that core-g28v2 60mer was superior to eOD-GT6v2-cRSF 60mer as a first-boost immunogen candidate.

[0299] BCR sequences from the eOD-GT8 60mer prime and core-g28v2 60mer boost immunization groups were expressed as mAbs and assessed for affinity to eOD-GT8, core-g28v2, and core-g28v2 with theN276 glycan (Fig. 3A). The geomean affinity of post-prime VRCOl-class mAbs for eOD-GT8 increased by over 1000-fold relative to the affinity of the VRCOl-class naive precursors from SE09 mice (Fig. 3 A). Approximately half of the post-prime VRCOl-class mAbs assayed had measurable affinity for core-g28v2, with geomean affinity among binders of approximately 12 pM (Fig. 3 A). Boosting with core-g28v2 60mer led to a 5900-fold increase in geomean affinity for core-g28v2 relative to the post-prime VRCOl-class mAbs and produced VRCOl-class mAbs that were able to bind a variant of core-g28v2 containing the N276 glycan (Fig. 3A).

[0300] Applicants used serum enzyme-linked immunosorbent assay (ELISA) analyses to understand the degree to which serum antibodies primed by eOD-GT8 60mer might interfere with core-g28v2 60mer boosting. Applicants found that serum antibody responses post eOD-GT8 60mer prime bound to both eOD-GT8 and the epitope knockout version of eOD-GT8, but showed no detectable binding to core-g28v2 or the epitope knockout version of core-g28v2, suggesting that post-prime serum antibodies would not contribute to antibody feedback during the boosting immunization (Fig. 3B). Boosting with core-g28v2 60mer elicited serum antibodies with higher 50% effective dilution (ED50) values for eOD-GT8 and core-g28v2 relative to the corresponding epitope-knockout versions, indicating that the core-g28v2 boost induced a strong CD4bs-specific response (Fig. 3B), as intended by the core-g28v2 design.

[0301] To evaluate other alternatives to core-g28v2 60mer, applicants also tested boosting with core-g28v2 as a 24mer ferritin nanoparticle, C13.G4.2 as a 24mer ferritin nanoparticle (X. Chen et al., 2021) or a 60mer nanoparticle, and BG505-core-VRC01-GT3.3 60mer (B. Briney et al., 2016) (Fig. 12). None of the alternative booster immunogens were superior to core-g28v2 60mer in terms of the magnitude of the VRCOl-class MBC response or the amount of SHMinduced (Fig. 12). In addition, the amounts of SHM among boost-antigen-specific, non-VRCOl- class BCRs were similar for the different boosters (Fig. 12H and I) and not different from the SHM following priming with eOD-GT8 60mer (Fig. 2H and I), suggesting that each first-boost immunogen elicited a de novo non-VRCOl -class response, but none caused substantial boosting of eOD-GT8 60mer-primed non-VRCOl -cl ass B cells. From these analyses, applicants selected core-g28v260mer as the applicants’ lead first-boost candidate to follow eOD-GT8 60mer priming.

[0302] The number of key VRCOl-class residues was increased by protein and adjuvant boosting.

[0303] Applicants selected a representative panel of 19 VRC01 -class bnAbs with minimal (< 3 aa) indels as aspirational goals for vaccine elicitation (D. J. Leggat et al., 2022) and performed sequence analysis to identify key residues commonly utilized by VRC01 -class bnAbs. As described previously (D. J. Leggat et al., 2022), applicants identified 20 positions (19 within Vul- 2, plus Trpl03-5) as key VRCOl-class residues, four of which are germline-encoded in the Vnl- 2*02 and *04 alleles (47W, 50W, 55G, and 71R). Applicants counted the key VRCOl-class residues on a scale ranging from -4 to +16, to allow for all possibilities from losing all germline- encoded key residues, to gaining key residues at all 16 non-germline-encoded positions (D. J. Leggat et al., 2022). Here, structural analysis of the 16 non-germline-encoded key VRCOl-class residues revealed that 10 residues were involved in the VRCOl-class paratope (Fig. 4A) and made contact with the CD4bs and adjacent regions on HIV Env (Fig. 4B). The six non-paratope key VRCOl-class residues likely stabilize the HCDR2 of VRCOl-class bnAbs or potentially make contact with distal N-linked glycans.

[0304] To assess the ability of different prime-boost regimens to drive maturation toward bnAb development, applicants counted the number of key VRCOl-class residues in each VRCOl- class BCR isolated for each animal, then computed the 90th percentile number of key VRCOl- class residues per animal (as representative of the best 20% of BCRs per animal), and finally computed the median 90th percentile number of key VRCOl-class residues for each immunization group (D. J. Leggat et al., 2022). Applicants carried out this analysis for different groups primed with eOD-GT8 60mer and boosted with core-g28v2 60mer, eOD-GT6v2-cRSF 60mer, 191084- N276D trimer, or PBS placebo (Fig. 4C). The median 90th percentile number of key VRCOl-class residues increased significantly from approximately +2 after priming with eOD-GT8 60mer to approximately +5 after boosting with core-g28v2 60mer (p=0.002) (Fig. 4C). None of the otherboosts resulted in an increase in key VRCOl-class residues relative to post-prime (Fig. 4C). For comparison, the representative VRCOl-class bnAbs had a median of 13 key residues, and a range of +8 to + 16 (Fig. 4C and Fig. 13 A), illustrating that additional maturation will be required to elicit bnAbs. Paratope key VRCOl-class residues at positions within the HCDR2 (L. Mesin et al., 2020; T. T. Wu et al., 1970; D. J. Mandell et al., 2009) were detected after boosting with core-g28v2 60mer, but not after priming with eOD-GT8 60mer, whether followed by a placebo boost (Fig. 4D) or not (Fig. 13B), illustrating a direct effect of the boost. Key paratope VRCOl-class residues at positions within the frame work region 3 (FWR3; 73 and 74) make contact with residues on HIV Env that are either absent on core-g28v2 or are present in a different conformation (Fig. 4B). Accordingly, key VRCOl-class residues at positions 73 and 74 were not detected after boosting with core-g28v2 60mer and would likely need to be elicited using a native HIV Env trimer (Fig. 4D). The number of key VRCOl-class residues and the Vn amino acid (aa) SHM both correlated with higher affinity binding to core-g28v2 (Fig. 4E and F), indicating that in vivo selection for higher affinity to core-g28v2 served to guide immune responses toward bnAb development. eOD- GT8 and core-g28v2 make little contacts with the light chains of VRCOl-class antibodies, except for the LCDR3. Hence it was not surprising that the VK / L aa SHM was not correlated with higher affinity binding to core-g28v2 (Fig. 4G). Applicants concluded that the core-g28v2 60mer boost immunization selected for desirable maturation of VRCOl-class responses.

[0305] eOD-GT8 60mer mRNA primes VRCOl-class responses.

[0306] mRNA / LNP vaccine platforms can provide excellent immunogenicity and safety with rapid timelines for entering clinical trials, as illustrated by the SARS-CoV-2 mRNA vaccines (F. P. Polack et al., 2020; L. R. Baden et al., 2021). Using Moderna mRNA / LNP immunogens in the SE09 mouse model, applicants evaluated eOD-GT8 60mer priming (one or two immunizations) and core-g28v2 60mer boosting (after one or two eOD-GT8 60mer priming immunizations), and as controls applicants tested a placebo boost and a core-g28v260mer priming group (Fig. 5A). B cell analyses were carried out as for the protein experiments, except that replicate samples from placebo-boosted animals were sorted twice, once with eOD-GT8 probes and once with core-g28v2 probes. Similar frequencies of antigen-specific (eOD-GT8-specific) MBCs were induced by one or two immunizations with eOD-GT8 60mer mRNA or by eOD-GT8 60mer mRNA followed by placebo (Fig. 5B), suggesting that the second immunization of eOD- GT8 60mer mRNA had little effect. Furthermore, the percentage of eOD-GT8-specific MBCs thatwere VRC01 -class was not significantly different for one eOD-GT860mer immunization followed by placebo compared to two immunizations with eOD-GT8 60mer (Fig. 5C), indicating that the second eOD-GT8 60mer mRNA immunization did not cause substantial additional priming of VRC01 -class precursors. One or two eOD-GT8 60mer mRNA immunizations induced similar frequencies of VRC01 -class and VRC01-classVK1'33MBCs (Fig. 5D and E), and the VRC01 -class MBCs in both cases had minimal VH and VK / L SHM (Fig. 14A and B). Comparing mRNA to adjuvanted protein for one eOD-GT8 60mer immunization, applicants found that mRNA vaccination induced higher frequencies of VRCOl-class MBCs (median values of 0.8% vs. 0.24%, P=0.0067, Fig. 15A), similar frequencies of VRC01-classVK1’33MBCs (median values of 0.07% vs. 0.03%, P=0.76, Fig. 15B), low SHM in both cases (e.g. median VH SHM values of 0% vs. 1%, P=0.07, Fig. 15C), and similar numbers of key VRCOl-class residues (median 90th percentile values of 1.9 vs 2, P=0.23, Fig. 15D). In terms of VRCOl-class mAbs affinities, the results for mRNA were also similar to the case of protein immunization: priming with eOD-GT8 60mer mRNA resulted in a >500-fold increase in the geomean affinity of VRCOl-class mAbs for eOD- GT8 relative to the affinity of the VRCOl-class naive precursors from SE09 mice (Fig. 6 A), with similar geomean KD values of 6 nM and 2 nM from mRNA and protein immunization, respectively. Serum antibody responses post eOD-GT8 60mer mRNA prime bound to both eOD- GT8 and the epitope KO version of eOD-GT8, but showed no detectable binding to core-g28v2 or the epitope KO of core-g28v2 (Fig. 6B), analogous to what applicants found with the protein immunizations (Fig. 3B). Applicants concluded that mRNA performed as well or better than adjuvanted protein for eOD-GT8 60mer priming in the SE09 mouse, and that two immunizations was not better than one.

[0307] Core-g28v2 60mer mRNA / LNP boosting enhances VRCOl-class responses.

[0308] Having dissected the frequency and SHM effects of eOD-GT8 60mer mRNA priming, applicants then turned attention to analysis of core-g28v260mer mRNA boosting experiments and controls. After one eOD-GT8 60mer mRNA priming immunization, boosting with core-g28v2 60mer mRNA elicited higher frequencies of core-g28v2-specific MBCs, VRCOl-class MBCs, and VRC01-classVK1'33MBCs compared to placebo boost (Fig. 5B, D, and E). Furthermore, priming with core-g28v2 60mer mRNA elicited antigen-specific MBCs, but no detectable VRCOl-class MBCs (Fig. 5B and C), confirming that the increased frequency of VRCOl-class MBCs detected after core-g28v2 60mer mRNA boosting was due to expansion of eOD-GT8 60mer-primedVRC01 -class B cells and not to de novo VRC01 -class priming by core-g28v2 60mer. In concert with that expansion, heterologous boosting induced BCR maturation: boosting with core-g28v2 60mer mRNA after a single eOD-GT8 60mer mRNA prime elicited higher amounts of VH SHM among VRC01 -class MBCs compared to boosting with placebo (median SHM values of 8.7% vs. 6.1% for VH [P=0.02]) (Fig. 14A and B, with core-g28v2 and placebo boost groups sorted at week 12 with core-g28v2 probes). Boosting with core-g28v2 60mer mRNA after a single eOD-GT8 60mer mRNA prime also elicited higher amounts of key VRC01 -class HC residues among VRCOl-class MBCs compared to boosting with placebo (median of 90th percentile values of 5 vs. 2 for core-g28v2 and placebo boost groups sorted at week 12 with core-g28v2 probes, respectively, P=0.02; Fig. 6C). After eOD-GT8 60mer mRNA priming, VRC01-classVK1’33BCRs were enriched for LCDR3s with Glu at position 96, the most prevalent residue at that position in VRC01-classVK1'33bnAbs (Fig. 17A versus Fig. 7D), and this enrichment increased after core-g28v2 60mer mRNA boosting (Fig. 17B). Comparing the output of the core-g28v260mer mRNA boost after one or two priming immunizations with eOD-GT8 60mer mRNA, applicants detected no improvements in frequencies of antigen-specific MBCs, VRCOl-class MBCs, or VRC01-classVK1’33MBCs associated with double priming (Fig. 5B to E), and applicants found that the double priming regimen induced significantly lower amounts of VH and VK / L SHM (p<0.001 and p<0.05 respectively) (Fig. 14A and B) and key VRCOl-class residues (p<0.001) (Fig. 6C). Hence, in the SE09 mouse, the core-g28v2 60mer mRNA boost was effective for expanding and maturing VRCOl-class responses after a single eOD-GT8 60mer mRNA prime, and there was no benefit to using a double eOD-GT8 60mer mRNA prime.

[0309] Applicants used multiple metrics to compare the performance mRNA to adjuvanted protein for one eOD-GT8 60mer immunization followed by a core-g28v2 60mer boost. mRNA induced higher frequencies of VRCOl-class MBCs (median values of 1.5%% vs. 0.6%, P=0.003; Fig. 18A), similar frequencies of VRC01-classVK1'33MBCs (median values of 0.33% vs. 0.54%; Fig. 18B), similar amounts of SHM (e.g. median VH SHM values of 8.7% vs. 7.2%; Fig. 18C), and similar numbers of key VRCOl-class HC residues (median 90th percentile values of 5 vs. 4.6; Fig. 18D). In terms of mAb affinity analysis, mRNA performed similarly as protein. Approximately one third of the post-prime VRCOl-class mAbs assayed had measurable affinity for core-g28v2, with a geomean KD among binders of about 7.6 pM (Fig. 6A), values similar to those obtained with protein immunization (Fig. 3A). Boosting with core-g28v2 60mer mRNA ledto a 900-fold increase in geomean affinity for core-g28v2 relative to the post-prime VRC01 -class mAbs and produced VRC01 -class mAbs that were able to bind a more native-like variant of core- g28v2 containing the N276 glycan (Fig. 6A), with a geomean KD among binders of 720 nM similar to the value of 870 nM obtained for protein immunization (Fig. 3A). Boosting with core-g28v2 60mer mRNA after a single eOD-GT8 60mer mRNA prime induced more key VRC01 -class HCDR2 residues compared to priming alone (Fig. 19A) or priming followed by a placebo boost (Fig. 6D and Fig. 19B), as was true for protein immunization. Applicants also found that boosting with core-g28v2 60mer mRNA after a single eOD-GT8 60mer mRNA prime induced more key VRC01 -class HCDR2 residues compared to two immunizations with eOD-GT8 60mer mRNA (Fig. 19C and D). The number of key VRC01 -class residues, percent VH SHM, and percent VK / VI. SHM were all correlated with VRCOl-class mAb KD for core-g28v2, for mAbs derived from MBCs isolated after core-g28v2 60mer mRNA boosting (Fig. 6E to G), similar to the applicants’ correlation findings for protein immunization (Fig. 4E to G). To confirm the epitope specificity of the VRCOl-class mAbs derived from MBCs isolated after core-g28v2 60mer mRNA boosting, applicants measured binding to the epitope KO version of core-g28v2 with SPR and assessed competition with VRC01 using biolayer interferometry (Fig. 20A and B). Only one VRCOl-class mAb had measurable affinity for the epitope KO version of core-g28v2 and it had >8000-fold reduction in affinity relative to the non-epitope KO version of core-g28v2 (Fig. 20A) indicating that all of the VRCOl-class mAbs targeted the CD4bs epitope. Competition biolayer interferometry (BLI) with VRC01 Fab showed similar results in that mature VRC01 Fab fully blocked binding to all of the VRCOl-class mAbs (Fig. 20B). In serum antibody binding analyses, the core-g28v2 60mer mRNA boost induced a similar pattern of responses as protein, with significantly higher ED50 values for eOD-GT8 and core-g28v2 compared to the epitope KO versions (p<0.001), indicating that the majority of the response elicited after boosting with mRNA was CD4bs-specific (Fig. 6B), as intended by the core-g28v2 design. Furthermore, there were significantly higher ED50 values for eOD-GT8 after boosting with core-g28v2 60mer mRNA compared to post eOD- GT8 60mer prime (p<0.001) but no increase in ED50 values for eOD-GT8 KO (Fig. 6B), which also suggested that boosting with core-g28v2 60mer preferentially boosted CD4bs-specific responses over non-CD4bs off-target responses. Applicants concluded that mRNA delivery of eOD-GT8 60mer followed by core-g28v2 60mer was effective for priming VRCOl-class naive B cells and driving their expansion and early maturation in the SE09 mouse. Similar findings weremade in a different stringent mouse model (X. Wang et al., 2024), demonstrating the robustness of the applicants’ conclusions.

[0310] Non-VRCOl -class BCR affinity and specificity

[0311] Given that the applicants’ B cell sorting strategy isolated antigen-specific, rather than epitope-specific B cells, it was of interest to determine what proportion of the antigen-specific non-VRCOl -class MBCs were CD4bs-specific and therefore direct epitope competitors for the VRC01 -class responses. Applicants employed SPR analyses to interrogate the specificity of the non-VRCOl -class mAbs recovered from MBCs after eOD-GT8 60mer mRNA priming and core- g28v2 60mer mRNA boosting. Twelve randomly selected non-VRCOl -class, eOD-GT8-binding BCRs isolated after priming with eOD-GT8 60mer mRNA were expressed as mAbs and assessed for affinity to eOD-GT8, eOD-GT8-KO, and core-g28v2 (Fig. 21A). Half of the mAbs had >50- fold reduction in affinity for eOD-GT8-KO compared to eOD-GT8, indicating that they were CD4bs-specific competitors. None of the mAbs had measurable affinity to core-g28v2 (Fig. 21A), suggesting that eOD-GT8 60mer-primed non-VRCOl -class MBCs were not readily boosted by core-g28v2. Seventeen randomly selected non-VRCOl -class, core-g28v2-binding BCRs isolated after boosting with core-g28v2 60mer mRNA were expressed as mAbs and assessed for affinity to core-g28v2 and core-g28v2 KO (Fig. 2 IB). More than fifty percent of post core-g28v2 non- VRCOl -class mAbs had >50-fold reduction in affinity for g28v2-KO compared to core-g28v2, indicating that they too were CD4bs-specific competitors (Fig. 21B). Applicants concluded that the expansion and favorable maturation of VRC01 -class responses induced by priming with eOD- GT8 60mer mRNA and boosting by core-g28v2 60mer mRNA were achieved in the presence of CD4bs-specific non-VRCOl -class competition.

[0312] With the design of core-g28v2 aiming to avoid boosting non-VRCOl -class responses primed by eOD-GT8, applicants sought to determine if that goal was achieved. The strongest line of evidence came from SHM analyses in protein vaccination: SHM in VRC01 -class MBCs was higher after the core-g28v2 boost compared to post-prime (Fig. 2F and G), but SHM in non- VRCOl-class MBCs was similar after the core-g28v2 boost and after the eOD-GT8 prime (Fig. 2H and I). Additionally, the quantities of SHM (VH and VK / VL) among non-VRCOl -class MBCs were comparable between animals primed with core-g28v2 mRNA and those primed with eOD- GT8 mRNA and boosted with core-g28v2 mRNA (Fig. 22A and B). These SHM analyses suggested that the core-g28v2-specific, non-VRCOl -class responses detected after the core-g28v260mer boost were likely de novo primed by the core immunization. In further support of that hypothesis, applicants found that none of the 12 post-prime non-VRCOl -class mAbs tested had detectable affinity for core-g28v2 (Fig. 21A).

[0313] Identifying potential boost-2 immunogens

[0314] Applicants hypothesized that the next step in sequential boosting after core-g28v2 60mer would be to immunize with a prefusion conformation-stabilized HIV Env trimer lacking the N276 glycan (J. G. Jardine et al., 2016, B. Briney et al., 2016; M. Tian et al., 2016; K. R. Parks et al., 2019, X. Chen et al., 2021, J. G. Jardine et al., 2016). Therefore, determining if post-core- g28v2 VRC01 -class antibodies can bind to such trimers would further support the use of core- g28v2 60mer as a first boost and would identify potential second-boost candidates. Applicants tested VRC01 -class mAbs isolated after core-g28v2 60mer boosting (protein or mRNA) for binding to a panel of MD39-stabilized (J. M. Steichen et al., 2016) HIV Env trimers that were highly sensitive to neutralization by VRC01 -class bnAbs, according to available data on CATNAP (H. Yoon et al., 2015; S. Gnanakaran et al., 2010; M. Caskey et al., 2017; S. S. Kulkarni et al., 2009). The N276 glycan was removed from these trimers by introducing N276D, N276Q, or T278M mutations. VRC01 -class mAbs from both protein- and mRNA-immunized animals bound to N276-lacking trimers with similar affinities (Fig. 7A and B). Higher percent binders and higher affinity was noted for the N276D version of 191084 over the N276Q version and for the T278M version of HIV_001428_2 (hereafter referred to as 001428) over N276Q, indicating preferences for D276 and M278 (Fig. 7A and B). Both D276 and M278 are present in core-g28v2, and D276 is also present in eOD-GT8, likely contributing to the mutation preference. Fifteen of the VRC01- class mAbs elicited after boosting with core-g28v2 60mer mRNA were assessed for neutralization against a panel of N276-lacking and corresponding wildtype pseudoviruses (Fig. 7C). Neutralization was detected against N276D, T278M, and N276Q pseudoviruses, but not against wildtype pseudoviruses (Fig. 7C). Although neutralization activity was detected for mAbs, neutralization activity in polyclonal IgGs isolated from serum after boosting with core-g28v2 60mer was detected in only two of six animals tested (Fig. 23). Thus, at least a subset of VRC01- class responses elicited by core-g28v2 60mer mRNA boosting showed detectable affinity for heterologous native-like trimers lacking the N276 glycan and had the capacity to neutralize corresponding pseudoviruses.

[0315] Discussion

[0316] Learning how to induce bnAbs against HIV by vaccination represents both a critical goal for global public health and a major challenge for immunology and vaccinology. Germlinetargeting vaccine design provides a promising strategy for priming bnAb naive precursors and shepherding them into acquiring the somatic mutations required for neutralization breadth and potency. The IAVI G001 clinical trial provided proof-of-principle that a germline-targeting priming immunogen could activate diverse bnAb naive precursors (D. J. Leggat et al., 2022). Applicants used mAbs isolated from IAVI G001 to guide the selection of a first-boost immunogen, core-g28v2 60mer. Applicants then showed, in a mouse model approximating human conditions of precursor frequency, affinity, and diversity, that VRC01 -class B cells primed by eOD-GT8 60mer and boosted by core-g28v2 60mer acquired additional key VRC01 -class residues and gained affinity for heterologous HIV Env trimers lacking the N276 glycan and also for a core- g28v2 variant containing the N276 glycan. The key VRC01 -class residues within the HCDR2 induced by core-g28v2 60mer boosting are known to be important for neutralization breadth and potency (J. G. Jardine et al., 2016). Furthermore, the correlations between affinity for core-g28v2 and both key mutations and SHM demonstrated that the structure of core-g28v2 selected for favorable directional maturation. Thus, applicants have demonstrated that a suitably designed boost immunogen that is closer in structure to the native glycoprotein than the prior immunogen and that possesses an affinity gradient in which bnAbs have higher affinity than precursors, can drive maturation toward bnAb development in an animal model. These findings provide important support for the germline-targeting vaccine strategy and represent important steps toward the goal of bnAb induction.

[0317] Applicants note that core-g28v2 60mer was an effective boost after a single priming immunization by eOD-GT8 60mer in this mouse model even though the applicants’ SPR analysis showed that only 56% or 28% of post-GT8 mAbs at week 6 (the timepoint for the core-g28v2 60mer boost) had detectable affinity for core-g28v2, for protein or mRNA priming, respectively, and among those binders, the geomean affinities for core-g28v2 were 12 pM and 8 pM, respectively. These data showed that successful boosting of bnAb precursors could be achieved even when only a subset of the previously primed precursors had measurable monovalent affinity for the booster immunogen and even if that affinity was relatively low. Whether this proves to be a general rule for booster design remains to be determined through additional studies, includingfor core-g28v2 60mer mRNA boosting in humans in IAVI G002, and also for other boost candidates for various bnAb classes in different animal models or humans.

[0318] There are several limitations to The applicants’ study. First, none of the vaccine regimens elicited bnAbs that would be necessary for an effective vaccine. However, the goal of germline-targeting priming followed by sequential boosting is to initiate B cell responses that can mature into bnAbs and guide those B cells to acquire the appropriate mutations. Priming with eOD-GT8 60mer followed by boosting with core-g28v260mer successfully initiated VRC01 -class B cell responses and guided the responses to acquire additional key mutations that allowed binding to more native-like antigens. Secondly, the VRC01 -class precursor frequency in the SE09 mouse was 17 times higher than in humans; however, the VRC01 -class precursors were still rare (1 in 13,600 naive B cells), highly diverse with different CDR3s and light chains, and had affinities for eOD-GT8 that were comparable to human VRC01 -class precursors. Boosting MBCs in mice to re-enter the germinal center is highly inefficient (L. Mesin et al., 2020) and likely requires a higher precursor frequency present at priming compared to humans to generate sufficient MBCs as targets for boosting. Finally, the elevated frequencies of B cells with human Vnl-2 and VK1-33 in the SE09 mouse reduced the genetic diversity of potential competitor B cells. However, applicants identified ample CD4bs epitope-specific, high-affinity, non-VRCOl antibodies among randomly sampled antigen-specific non-VRCOl antibodies elicited after eOD-GT8 60mer priming or core- g28v2 60mer boosting. This indicated that epitope-specific competition was present in the SE09 mouse model.

[0319] mRNA vaccine technology will likely prove essential for HIV vaccine development, as the favorable immunogenicity combined with increased speed and lower cost of producing clinical material should improve the feasibility and timelines of clinical trials testing multiple immunogens in series. For that reason, applicants compared adjuvanted protein immunization, as in IAVI G001, to mRNA immunization, and applicants found that mRNA performed at least as well, if not slightly better than, protein. This not only provided preclinical support for the IAVI G002 clinical trial evaluating eOD-GT8 60mer mRNA priming followed by core-g28v2 60mer mRNA boosting, but also demonstrated the feasibility of using mRNA to deliver self-assembling nanoparticle immunogens in vivo. Germline-targeting priming and sequential heterologous boosting with protein immunogens in highly permissive mouse models has been shown to induce increased SHM (B. Briney et al., 2016; M. Tian et al., 2016; K. R. Parks et al., 2019, K. O. Saunderset al., 2019) and to produce bnAbs (J. M. Steichen et al., 2016; A. Escolano et al., 2016; X. Chen et al., 2021). The critical differences in The applicants’ study are that applicants have demonstrated maturation under more stringent, human-like conditions of precursor frequency, affinity, and diversity; applicants have validated mRNA as a viable delivery platform for germline-targeting priming and boosting; and The applicants’ results build directly on the IAVI G001 trial leading directly to the IAVI G002 trial. Overall, the applicants’ results predict favorable outcomes in IAVI G002 and suggest that the germline-targeting strategy combined with mRNA vaccination have promise for HIV vaccine development. These endeavors to induce VRC01 -class bnAbs also provide guidance for application of the germline-targeting strategy to induce other bnAb classes to HIV and to other antigenically diverse pathogens.Sequential heterologous boosting drives development of VRCOl-class mAbs

[0320] Humanized Vnl-2 / VKl-33hTdTmice were initially primed with eOD-GT8 60mer delivered by mRNA / LNPs and boosted with core-g28v2 60mer mRNA / LNP (Figure 71). Subsequentially, these mice were boosted at week 12 with one of four mRNA / LNP immunogens encoding for stabilized membrane-bound HIV Env trimers lacking the N276 glycan (1HD2- N276Q-gpl51, lHD2-T278M-gpl51, 001428-N276Q-gpl51, or 001428-T278M-gpl51). The frequencies of VRCOl-class memory B cells were determined by antigen-specific cell sorting (Figure 72) and single cell lOx Genomics VDJ sequencing conducted six weeks after boosting. A subset of mice was boosted again at week 18 with corresponding wildtype membrane-bound trimers delivered by mRNA / LNPs matched to those received at week 12.

[0321] Boosting with stabilized membrane-bound HIV trimers lacking the N276 glycan at week 12 elicited VRCOl-class memory B cells (Figure 73). Subsequent boosting with wildtype membrane-bound HIV Env trimers elicited VRCOl-class memory B cells that bind wildtype trimers (Figures 74 and 75) and have BCRs capable of neutralizing the autologous wildtype HIV pseudovirus and heterologous pseudoviruses lacking the N276 glycan when expressed as recombinant IgGs (Figure 76). These IgGs also bind to wildtype heterologous trimers with moderate affinity (Figure 77).Example 2 - Vaccine priming by targeting BG18Vaccine priming of rare HIV broadly neutralizing antibody precursors in non-human primates

[0322] Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to human immunodeficiency virus (HIV) and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3 -dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. Here, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3 -dominant bnAb BG18, primed bnAb-precursor B cells in 8 of 8 rhesus macaques to substantial frequencies and with diverse lineages, in germinal center and memory B cells. Applicants confirmed bnAb-mimicking, HCDR3 -dominant, trimer-binding interactions with cryoelectron microscopy. The results demonstrate proof of principle for HCDR3 -dominant bnAb- precursor priming in outbred animals and suggest that N332-GT5 has promise to induce similar responses in humans.

[0323] Humanity could benefit substantially from the design and development of vaccines that induce broadly neutralizing antibodies (bnAbs) to protect against major human pathogens (D. R. Burton et al., 2017). A leading strategy for developing such vaccines is germline-targeting vaccine design (J. Jardine et al. et al., 2013; A. T. McGuire et al. et al., 2013; J. G. Jardine et al. et al., 2016; J. M. Steichen et al. et al., 2016; J. M. Steichen et al. et al., 2019; M. Medina-Ramirez et al. et al., 2017). This strategy aims to induce bnAbs by first priming rare bnAb-precursor B cells and then guiding B cell affinity maturation with a series of rationally designed boosting immunogens (A. Escolano et al. et al., 2016; B. Briney et al. et al., 2016; M. Tian et al. et al., 2016; J. G. Jardine et al. et al., 2016). Clinical proof of principle for germline-targeting vaccine priming was recently demonstrated in the IAVI G001 Phase 1 trial, in which the priming immunogen eOD-GT8 60mer was found to induce responses from diverse VRC01 -class bnAb-precursor B cells in 97% of vaccine recipients and to generate substantial frequencies of bnAb-precursor-derived germinal center (GC) and memory IgG B cells (D. J. Leggat et al. et al., 2022). However, that trial tested a special case of antibody-antigen interaction in which the antibody HCDR3 plays a minor role. Most antibodies, including most HIV bnAbs, interact with antigen in an HCDR3 -dominant manner. Hence, if the germline-targeting strategy is to be employed for induction of other bnAbs to HIV or other pathogens, it must work with HCDR3-dominant antibodies. This requires designing a priming immunogen that can engage a broad pool of bnAb precursors sharing keyHCDR3 features but otherwise containing diverse sequences. In the case of HIV, vaccines will need to induce several different classes of bnAbs targeting different epitopes in order to achieve optimal neutralization coverage, increasing the need for effective HCDR3 -dominant germlinetargeting.

[0324] Applicants previously described a generalized method for the design of HCDR3- dominant germline-targeting priming immunogens, and applicants illustrated the design and testing of HIV envelope (Env) trimer-based N332-GT priming immunogens for the HIV V3- glycan / N332 supersite bnAb BG18 (J. M. Steichen et al. et al., 2019). These immunogens induced responses from rare (~1 in 150,000) bnAb-precursor B cells in a BG18 germline heavy chain knockin mouse model and bound two types of potential bnAb-precursor human naive B cells that share key features with BG18 in ex vivo screens (types I and II). However, the BG18 type I precursors, which show greater HCDR3 similarity to BG18 than type II precursors, occurred at a very low frequency in humans, approximately 1 in 50 million human naive B cells. This frequency was too low to be tested in mouse models and more than 150-fold lower than the VRCOl-class precursor frequency in humans (1 in 300,000) for which consistent bnAb-precursor priming was observed in the IAVI G001 trial. Furthermore, consistent priming in humans will likely require activation of BG18 precursors with diverse HCDR3s, heavy chain V genes and light chains, whereas the mouse experiments validated priming of precursors with a single BG18 inferred- germline heavy chain bearing exact HCDR3 junctions from the bnAb itself. The very low BG18 type I human precursor frequency and the need to prime diverse BG18 precursors led us to question the plausibility of consistent priming of BG18 precursors in humans. Reasoning that similar challenges will confront most or all other attempts at HCDR3 -dominant bnAb-precursor priming in humans, and that these challenges would be reproduced in non-human primates, applicants sought to test the concept of HCDR3 -dominant germline-targeting in rhesus macaques (RMs). Hence, applicants evaluated the capacity of the N332-GT5 trimer to prime BG18 type I responses in RMs.

[0325] Materials and Methods

[0326] BG18 frequency analysis in naive B cells from rhesus macaques and humans

[0327] Consolidated naive BCR NGS database from 70 Rhesus macaques

[0328] Rhesus macaque BCR NGS datasets from 70 animals were downloaded from the NCBI sequence read archive (M. M. Corcoran et al. et al., 2016; N. Vazquez Bernat et al. et al., 2021; C.A. Cottrell et al. et al., 2020; W. Zhang et al. et al., 2019; K. Guo et al. et al., 2015; G. E. Phad et al. et al., 2020) or obtained directly from the study authors (V. Vigdorovich et al. et al., 2016). For datasets contained in Guo et al. (K. Guo et al. et al., 2015), preinfection VHI, VH3, and VH4 IgM libraries for each animal were concatenated into single datasets for each animal and processed along with datasets from Vigdorovich et al. (V. Vigdorovich et al. et al., 2016) using Immcantation (J. A. Vander Heiden et al. et al., 2014; N. T. Gupta et al. et al., 2015). To reduce sequencing artifacts, sequences were filtered to include only reads that were observed more than twice. The resulting filtered fastq files served as inputs for the next processing step. All 164 datasets from 70 animals were processed through IgDiscover without germline inference to produce a standardized output for construction of a comprehensive database (M. M. Corcoran et al. et al., 2016). IGHV expression analysis on the IgDiscover outputs was used to cluster datasets from Zhang et al. (W. Zhang et al. et al., 2019) and assign animal IDs (zRhl to zRhl5) to each dataset.

[0329] Sequencing of naive B cells from four additional rhesus macaques

[0330] 40x106 frozen PBMC samples from 4 RMs were thawed and recovered in 10% FBS inRPMI. Recovered cells were counted and stained with a B cell staining panel (eBioscience Fixable Viability Dye eFluor 506 (Invitrogen), mouse anti-human CD3 APC-Cy7 (SP34-2, BD Biosciences, mouse anti -human CD 14 APC-Cy7 (M5E2, BioLegend), mouse anti-human CD 16 APC-eFluor780 (eBioCB16, Thermo Fisher Scientific), mouse anti -human CD20 PerCP-Cy5.5 (2H7, BioLegend), mouse anti-human CD27 PE-Cy7 (0323, BioLegend), goat anti-human IgD FITC (polyclonal, Southern Biotech), mouse anti-human IgG BV786 (G18-145, BD Biosciences), mouse anti-human IgM BV605 (G20-127, BD Biosciences)). Approximately 1.5 million CD20+IgG’ B cells were sorted for each animal into RPMI containing 50% FBS using a FACSymphony S6 (BD Biosciences). Immediately after sorting, cells were centrifuged at 500 x g for 10 minutes and resuspended in 350ul of buffer RET (Qiagen, 79216) by vortexing. Lysed cells were immediately frozen at -20C then shipped to the Emory National Primate Research center for repertoire sequencing.

[0331] RNA was isolated using QIAGEN RNeasy kits (Valencia, CA) with an input of 1.5M cells for REtl8, RGpl8, RPbl8, and RPzl8. Reverse transcription (RT) was performed using Clontech SMART er cDNA template switching which involves 5' CDS oligo(dT) (12 pM) being added to RNA and incubated at 72°C for 3 minutes and 4°C for at least 1 minute. The RT mastermix was made using 5x RT Buffer (250 mM Tris-HCl (pH 8.3), 375 mM KC1, 30 mMMgC12), Dithiothreitol, DTT (20 mM), dNTP Mix (10 mM), RNase Out (40 U / pL), SMARTer II A Oligo (12 pM), Superscript II RT (200 U / pL) and was added to the reaction and incubated at 42°C for 90 minutes and 70°C for 10 minutes. First-strand cDNA was purified using AMPure XP beads (Beckman Coulter). Following RT and purification, two PCR rounds were carried out to generate immunoglobulin amplicon libraries that were compatible with Illumina sequencing. All oligos were ordered from Integrated DNA Technologies. The first PCR amplification was carried out using KAPA Real-Time Library Amplification Kit (Roche Diagnostics). cDNA was combined with master mix containing 2X KAPA HiFi HS RT PCR Master Mix, 12 pM pL 5PIIA and 5 pL RhlgM Constant Primer (2 pM). The amplification was monitored using real-time PCR and was stopped at 19 cycles during the exponential phase. The amplified products were again purified using AMPure XP beads. A second round of PCR amplification was carried out for addition of barcodes and Illumina adaptor sequences. Each sample contained 2X KAPA HiFi HS RT PCR Master Mix 2x, Nuclease-free water, 10 pM of P5_Seq BC_XX 5PIIA oligo, 10 pM of P7_ i7_XX RhlgM oligo and were combined with amplified Immunoglobulin from the first round PCR and amplified for 7 cycles using real-time PCR monitoring. The P5_Seq BC XX 5PIIA primers contain a randomized stretch of four to eight random nucleotides followed by a barcode sequence and this step was followed by purification with AMPure XP beads. A final PCR step was performed for addition of remaining Illumina adaptors by mixing master mix (2X KAPA PCR Master Mix, 10 pM P5_Graft P5_seq, Nuclease-free water), 10 pM of P7_ i7_XX RhlgM oligo and amplified products from the previous PCR step followed by purification with AMPure XP beads. The quality of the library was assessed using Agilent Bioanalyzer 2100 and quantified on a Qubit 4 Fluorometer with IX dsDNA HS Assay Kit. The amplicon libraries were pooled and sequenced across two Illumina MiSeq v3 runs as a 309 paired-end to obtain a sequencing depth of 10M reads / sample. Sequencing was conducted at the Emory National Primate Research Center Genomics Core Laboratory (www.yerkes.emory.edu / nhp_genomics_core).

[0332] List of Oligonucleotides from IDT

[0333] CDS Oligo (dT):TTTTTTTTTTTTTTTTTTTTTTTTTVN(SEQID NQ.58)

[0334] SMARTer II A Oligo: AAGCAGTGGTATCAACGCAGAGTACATrGrGrG (SEQ ID NO: 59)

[0335] 5PIIA: AAGCAGTGGTATCAACGCAGAGT (SEQ ID NO: 60)

[0336] RhlgM Constant Discover: GGGGCATTCTCACAGGAGACGAGGGGGAAAAG (SEQ ID NO: 61)

[0337] P5 Seq BC XX 5PIIA: CACGACGCTCTTCCGATCT 4-8xN AACCACTA AAGCAGTGGTATCAACGCAGAGT (SEQ ID NO: 62)

[0338] P7_i7_XX_RhIgM_Discover: CAAGCAGAAGACGGCATACGAGAT CGATCGAA GGGGCATTCTCACAGGAGACGAGGGGGAAAAG (SEQ ID NO: 63)

[0339] P5_Graft P5_seq: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC T (SEQ ID NO: 64)

[0340] Annotating BCR sequences from 74 rhesus macaques

[0341] Datasets from all 74 animals were then annotated to AIRR format (J. A. Vander Heiden et al. et al., 2018) using the AIRR module from the SADIE library vO.5.4 (github.com / jwillis0720 / sadie) using the “macaque” option and as the input annotation species and the adaptable penalty set to true. Results were filtered for only productive reads and converted to parquet format using snappy compression. Only animals that had >100,000 IgM sequences were included in the precursor frequency analysis (60 animals in total). Spark facilitated by AWS EMR service as described in Steichen et al. (J. M. Steichen et al. et al., 2019) was used for querying.

[0342] BG18 precursor frequency estimates in rhesus macaques and humans

[0343] Applicants analyzed NGS datasets of 1.1 billion human BCR heavy chain sequences from 14 human donors that were previously described (J. M. Steichen et al. et al., 2019; B. Briney et al., 2019; I. R. Willis et al. et al., 2022), as well as 95.4 million macaque BCRs from 60 macaques, using the Spark analytics engine on the AWS EMR platform (EMR 6.4.0). Applicants utilized the precursor definitions provided in Fig. 29E and performed the analysis using PySpark scripts. Each node was configured with Spark, lupyterEnterpriseGateway, Hadoop, and JupyterHub via the EMR node configuration interface. Applicants then used the EMR notebook interface to run PySpark scripts and analyze precursor frequencies. A precursor frequency was estimated by taking the number of BCR sequences that met a specific query definition and dividing it by the total number of BCRs for each donor. Applicants then multiplied these numbers by 1,000,000 and plotted them as frequencies per million. Median per species was shown on log scale graphs. All plots were generated using GraphPad Prism.

[0344] Animals and immunizations

[0345] Indian rhesus macaques (Macaca mulatta) were housed at AlphaGenesis Inc. and maintained in accordance with NIH guidelines. This study was approved by the Alpha Genesis Inc. Institutional Animal Care and Use Committee (IACUC). All animals were between 2-3 years old at the time of the priming immunization. For the N332-GT5 plus SMNP escalating-dose immunization group, 8 RMs (4 females and 4 males) were immunized with 50 pg N332-GT5 and 375 pg SMNP each side. For the MD39 plus SMNP escalating-dose immunization group, 4 RMs (2 females and 2 males) were immunized with 50 pg MD39 and 375 pg SMNP each side. All immunizations were given subcutaneously (s.c.) in the left and right mid-thighs. For priming, a 7- dose 12-day escalating dose strategy was used (K. M. Cirelli et al. et al., 2019) and a bolus boost immunization was given at week 10. Data from the MD39 group have been previously published (J. H. Lee et al. et al., 2022).

[0346] Genotyping rhesus macaques

[0347] Targeted long-read Pacific Biosciences single molecule real-time (SMRT) sequencing data generated for each animal in study cohort (n=8) to genotype IGHD3-41. Sequencing data was generated by adapting applicants’ published human immunoglobulin (IG) loci targeted enrichment protocol (O. L. Rodriguez et al. et al., 2020, W. S. Gibson et al. et al., 2023). Briefly, a custom oligo probe panel was designed (“HyperExplore”, Roche) using IG heavy chain (IGH), kappa (IGK), and lambda (IGL) genomic region sequences from the RM genome reference build (RheMaclO) and alternative haplotype assemblies from Cirelli et al. (K. M. Cirelli et al. et al., 2019) as sequence targets.

[0348] High molecular weight genomic DNA was isolated from peripheral blood mononuclear cells (PBMCs) collected from each animal using the DNeasy kit (Qiagen). DNA (1-2 ug) was then sheared using g-tubes (Covaris) and size selected using the YYYY Kbp Marker SI -Improved Recovery cassette definition on the Blue Pippin (Sage Science). Size-selected DNA was End Repaired and A-tailed using the standard KAPA library protocol (Roche), followed by the ligation of sample-specific sequence barcodes and universal primers. PCR amplification was performed for 8-9 cycles using PrimeSTAR GXL polymerase (Takara), and the resulting products were further size-selected and purified using 0.7X AMPure PB beads (Pacific Biosciences). Targetenrichment hybridization was performed using IGH / K / L-specific oligonucleotide probes (Roche). Target fragments were recovered using streptavidin beads (Life Technologies), followed by a second round of PCR amplification for 16-18 cycles using PrimeSTAR GXL (Takara). Long-readsequencing libraries were prepared using the SMRTbell Express Template Preparation Kit 2.0 (Pacific Biosciences), including Damage Repair and End Repair mix to repair nicked DNA, followed by the addition of an A-tail and overhang ligation with SMRTbell adapters. Libraries were then treated with a nuclease cocktail to remove unligated input material and purified with 0.45X AMPure PB beads (Pacific Biosciences). The resulting libraries were pooled (n=y samples) and sequenced on the Sequel lie system (2.0 chemistry; 30h movies) to generate “HiFi” (high fidelity) reads, with average read quality >QX.

[0349] HiFi reads for each animal were mapped to the RheMaclO genome reference. To genotype IGHD3-41, phased single nucleotide variants representing two distinct alleles were resolved from HiFi reads spanning the IGHD3-41 gene. At least 10 representative HiFi reads were required to include a given allele in the genotype of an animal.

[0350] Analysis of plasma by ELISA

[0351] ELISA plates (Corning™ 96-Well Half-Area Plates, Catalog # 3690) were precoated with anti-His antibody (Ipg / ml; Genscript) or PGT128 Fab (Ipg / ml) on Dayl. The V3-peptide (Ipg / ml) was directly coated on plates on Dayl. Plates were incubated overnight at 4°C. Plates were washed with PBST (PBS + 0.2% tween 20) and HIV trimers were captured for 2h at room temperature on Day 2. Plasma serially diluted in blocking buffer (PBST, 1% (w / v) FBS) was added for Ih at at 37°C and 80% humidity. Plates were washed three times and 25 pl of Peroxidase AffiniPure Donkey Anti-Human IgG (H+L) (Jackson ImmunoResearch Catalog # 709-035-149) was added to each well at 1:5,000 dilution in PBST + 1% FBS. After Ih incubation at RT, plates were washed 3X and TMB Chromogen Solution (Thermo Fisher Catalog # 002023) substrate was added. To stop the reaction, 25 pl 0.5 M H2SO4 were added after 5 minutes. Absorption was read at 450 and 570 nm on a Molecular devices VersaMax plate reader (Versamax, USA). Background subtraction was performed by subtracting the 570 nm value from the corresponding 450 nm value. Area Under Curve (AUC) was calculated in Prism (GraphPad Software, La Jolla, USA) by the trapezoidal method, which is based on connection of a straight line between every set of adjacent points defining the curve, and on a sum of the areas beneath these areas.

[0352] Lymph node fine needle aspiration

[0353] Lymph node fine needle aspirates (LN FNAs) were used to sample the left and right draining inguinal LNs (iLNs) which were identified by palpation and were performed by a veterinarian. A 22-gauge needle attached to a 3-mL syringe was passed into the LN up to 5 times.Samples were transferred into RPMI containing 10% fetal bovine serum (FBS) and lx penicillin / streptomycin (pen / strep). Ammonium-Chloride-Potassium (ACK) lysing buffer was used if the sample was contaminated with red blood cells. LN FNA samples were frozen down and stored in liquid nitrogen until analysis.

[0354] Flow cytometry and sorting

[0355] Frozen FNA or PBMC samples were thawed and recovered in 10% FBS in RPMI. The recovered cells were counted and stained with the appropriate staining panel. Fluorescent antigen probes were prepared by mixing fluorophore-conjugated streptavidin (SA) with incremental amounts of either biotinylated N332-GT5 or N332-GT5-KO in lx PBS at room temperature (RT) over the course of 45 min. The KO probe, N332-GT5-KO, was first added to the cells for 20 minutes, followed by the addition of WT N332-GT5 for 30 minutes, and then with the surface antibodies for 30 minutes at 4°C, similar to previously described protocols (K. M. Cirelli et al. et al., 2019; J. H. Lee et al. et al., 2022). For samples being sorted, anti-human TotalSeq-C hashtag antibodies (BioLegend) were added to each individual sample at a concentration of 2 pg per 5 million cells at the time of addition with the antibody master mix. 10% FBS in RPMI (R10) supplemented with lx pen / strep and lx GlutaMAX was used as the FACS buffer. Preimmunization samples were acquired on either a FACSFusion (BD Biosciences) or a FACSymphony S6 (BD Biosciences), while post-immunization samples were sorted on a FACSymphony S6 (BD Biosciences). Depending on the timepoint, some LN FNA samples were sorted for both Env+ (N332-GT5-BV421+N332-GT5-BV650+) and Env+KO' (N332-GT5- BV421+N332-GT5-BV650+ / N332-GT5-KO-PE-) GC B cells. The indexed V(D)J, Feature Barcode and Gene Expression libraries of sorted LN FNA or PBMC samples were prepared following the protocol for Single Indexed 10X Genomics V(D)J 5' v.1.1, with Feature Barcoding kit (10X Genomics). Custom primers designed to target RM BCR constant regions were used at concentrations previously described (J. H. Lee et al. et al., 2022).

[0356] For LN FNA data inclusion in GC B cell gating, a threshold of 250 total B cells in the sample was used. For Env-specific GC B cell gating, a threshold of 75 total GC B cells was used. The limit of detection (LOD) was calculated as the median of [3 / (number of B cells recorded)] from the LN FNA samples at the pre-immunization timepoint. Samples DHHW left iLN and DHIC left iLN at week 10 were excluded from antigen-specific GC B cell analysis. Week 3 and week 4 samples were gated on all live cells.

[0357] The following reagents were used during staining: Alexa Fluor 647 streptavidin (Invitrogen), BV421 streptavidin (BioLegend), BV650 streptavidin (BioLegend), eBioscience Fixable Viability Dye eFluor 506 (Invitrogen), mouse anti-human CD3 BV786, APC-Cy7 (SP34- 2, BD Biosciences), mouse anti-human CD4 BV711 (OKT4, BioLegend), mouse anti-human CD8a APC-eFluor780 (RPA-T8, Thermo Fisher Scientific), mouse anti-human CD14 APC-Cy7 (M5E2, BioLegend), mouse anti -human CD 16 APC-eFluor780 (eBioCB16, Thermo Fisher Scientific), mouse anti-human CD20 Alexa Fluor 488, PerCP-Cy5.5 (2H7, BioLegend), mouse anti-human CD27 PE-Cy7 (0323, BioLegend), mouse anti-human CD38 APC (OKTIO, NHP Reagents), mouse anti-human CD71 PE-CF594 (L01.1, BD Biosciences), mouse anti-human PD- 1 BV605 (EH12.2H7, BioLegend), mouse anti-human CXCR5 PE-Cy7 (MU5UBEE, Thermo Fisher Scientific), goat anti-human IgD FITC (polyclonal, Southern Biotech), mouse anti-human IgG Alexa Fluor 700, BV786 (G18-145, BD Biosciences), mouse anti-human IgM PerCP-Cy5.5, BV605 (G20-127, BD Biosciences), TotalSeq-C anti-human Hashtag antibody 1-10 (LNH-94 and 2M2, BioLegend), and TotalSeq-C0953 PE streptavidin (BioLegend).

[0358] 10X BCR sequencing

[0359] Cell Ranger v.3.0.2 was used for full-length VDJ read assembly. A custom RM germline VDJ reference was generated using databases published previously (K. M. Cirelli et al. et al., 2019; N. Vazquez Bemat et al. et al., 2021; C. A. Cottrell et al. et al., 2020). The constants. py fde in the Cell Ranger python library was modified to increase the maximum CDR3 length to 110 nucleotides. N332-GT5-KO-binding in GC B cells and memory B cells (MBCs) were determined by the PE-hashtag read counts, which were assessed by flow cytometry for each timepoint. Depending on the sorting strategy and the ratio of sorted N332-GT5+and N332-GT5+K0‘ GC B cells in each catch tube, PE-hashtag thresholds were defined per tissue and timepoint based on the flow cytometry analyses. For GC B cells from timepoints of week 3 to 7, a threshold of 100 PE- hashtag read counts was used. A sequence with a read count equal to or less than 100 PE-hashtag read counts was considered as epitope-specific (N332-GT5+KO ). For week 10 and 13, a threshold of 200 PE-hashtag read counts was used. For MBCs, a threshold of 300 PE-hashtag read count was used. Filtered VDJ contigs from the VDJ pipeline were used in further analysis.

[0360] Longitudinal lineage and somatic hypermutation analysis of BCR sequences

[0361] The following analysis was used to generate Fig. 34. For lineage analysis and SHM calculations used in Figs. 41-42, the analysis was carried out as described in “Bioinformaticanalysis of BCR sequences”. The V(D)J filtered contig output from Cell Ranger was further analyzed using packages from the Immcantation Portal (J. A. Vander Heiden et al. et al., 2014; N. T. Gupta et al. et al., 2015). An IgBLAST database was built from the custom RM germline VDJ reference. The Change-0 pipeline was used to parse the 10X V(D)J sequence output from Cell Ranger into an AIRR community standardized format, to allow for more downstream analysis using the Immcantation Portal. Clonal lineages were calculated for each animal using DefineClones.py with the appropriate clustering threshold value as determined by the disToNearest command from the SHazaM package. Inferred germline V and I sequences from the RM reference were added with CreateGermline.py. The germline D gene sequences and N nucleotide additions were masked from analysis since these cannot be accurately predicted. The total number of mutations (within V- and J-genes) for each heavy chain (HC) was determined by counting the number of nucleotide changes between the observed sequence and the predicted germline sequence with the observedMutations command within SHazaM. For analysis of total HC mutations, all productive HC contigs were analyzed. Sequences in which the VH call aligned to alleles IGHV3-100*01, IGHV3-100*01_S4205, IGHV3-100*01_S4375, IGHV336*01_S5206, IGHV3-36*01_S6650, IGHV3-NL_1 l*01_S5714, IGHV4-79-a, IGHV4-NL_l*01_S0419 were found to have a tremendously high number of substituted nucleotides at all timepoints compared to their inferred germline sequences, observed previously (J. H. Lee et al. et al., 2022). This observation was likely due to poor V-gene assignment in an incomplete RM V(D)J reference library and these sequences were excluded from further analysis. Clones with paired HC-LC BCR sequences were used when building clonal trees. Maximum-likelihood lineage trees were built for clonal families using Dowser (K. B. Hoehn et al., 2022) with the pml method within the GetTrees function. For the lineage trees, the branch length indicates the estimated number of total HC mutations and its most recent common ancestor in lineage.

[0362] Cell Lines

[0363] MS40L-low (D. R. Burton et al., 2017) or irradiated 3T3msCD40L feeder cells (J. Jardine et al. et al., 2013) were used in single B cell culture assays. MS40L-low cells were maintained in Iscove's Modified Dulbecco's medium with GlutaMAX (IMDM) (Gibco), supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (Omega Scientific), 100 Units / ml Penicillin, 100 pg / ml Streptomycin (1% Pen-Strep) (Gibco), and 55 pM 2- Mercaptoethanol (2-ME) (Gibco) before sorting. For single B cell sorting, MS40L-low cells weremaintained in B cell activation media: RPMI-1640 with GlutaMAX supplemented with 10% FBS, 55 pM 2 -ME, 1% Pen-Strep, 10 mM HEPES (Gibco), 1 mM Sodium Pyruvate (Gibco), 1% MEM NEAA (Gibco), while irradiated 3T3msCD40L cells were thawed on day of sorting and maintained in IMDM supplemented with 10% FBS, IX MycoZap Plus-PR (Lonza). HEK293T cells (ATCC) were used to produce viruses and maintained in complete Dulbecco's modified Eagle's medium (DMEM) (Gibco), supplemented with 10% FBS, 2 mM L-glutamine (Gibco), and 1% Pen-Strep. TZM-bl cells (NIH AIDS Reagents Program) (RRID:CVCL_B478) were maintained in complete DMEM and used as target cells in pseudovirus neutralization assays. All cell lines were maintained at 37°C in a humidified atmosphere of 5% CO2.

[0364] Isolation of N332-GT5 epitope-specific memory B cells by flow cytometry for culture

[0365] Fluorescently labeled antigens used for sorting were generated on the day of the sort by incubating 4 pM biotinylated N332-GT5 WT with streptavidin Alexa Fluor 647 (N332-GT5- AF647) (Invitrogen, cat# S21374) and streptavidin Alexa Fluor 488 (N332-GT5-AF488) (Invitrogen, cat# SI 1223) separately; and 4 pM biotinylated N332-GT5 KO with BV421 streptavidin (N332-GT5 KO-BV421) (BD Biosciences, cat# 563259) at a 2:1 molar ratio at room temperature for 1 hour in the dark. Washes, staining master mix, and sample preparation were carried out in cold sterile FACS buffer composed of 2% FBS in DPBS (Gibco). Cryopreserved peripheral blood mononuclear cells (PBMCs) from N332-GT5-immunized rhesus macaques were thawed, washed, and stained with antibody master mix of CD3 (clone SP34-2, BD Biosciences, cat# 557757), CD4 (clone OKT4, Biolegend, cat# 317418), CD8 (clone RPA-T8, BD Biosciences, cat#557760), CD14 (clone M5E2, BD Biosciences, cat# 561384), CD20 (clone 2H7, Biolegend, cat# 302326), IgM (clone MHM-88, Biolegend, cat#314508), IgG (clone G18-145, BD Biosciences, cat# 564230), and N332-GT5 KO-BV421 at 4°C for 15 min in the dark. Next, N332- GT5-AF647 and N332-GT5-AF488 WT antigens were added and incubated for an additional 30 min at 4°C in the dark. All antibodies were added at a 1 : 100 dilution in lOOpL and antigens at 100 nM final. Finally, 1 :300 LIVE / DEAD fixable cell dye (Invitrogen, cat# L34957) was added and incubated for 15 min at 4°C in the dark, washed, and resuspended to the desired volume. Double positive N332-GT5 WT, KO negative epitope-specific memory B cells (N332-GT5++ / N332-GT5- KO’ IgMlgG+B cells) were single-cell sorted into 96-well plates pre-seeded with appropriate feeder cells using a BD FACSMelody Cell Sorter. Post-sort analyses were done with FlowJo 10.7.2 (FlowJo, LLC).

[0366] Single memory B cell culture and Activation

[0367] Single B cells were cultured and expanded as previously described with some modifications (X. M. Luo et al. et al., 2009; J. Huang et al. et al., 2013). Briefly, MS40L-low feeder cells (MS40L-low cultures) were pre-seeded 24 hours before in 96-well plates at a density of 3 x 103 cells / well in 100 pL B cell activation media and supplemented with 100 pL 2X cytokines on the day of sort: 20 ng / mL IL-4 (Peprotech, cat# 200-04), 20 ng / mL IL-21 (Peprotech, cat# 200-21), 200 ng / mL IL-2 (Peprotech, cat# 200-02), 200 ng / mL BAFF (Peprotech, cat# 310- 13). Media was replaced with 100-200 pL B cell activation media with IX cytokines at day 4, 8, 12, 15, and 18 (X. M. Luo etal. et al., 2009). For animals K397 andK916, irradiated 3T3msCD40L (3T3 cultures) feeder cells which can be seeded on the day of sorting and have similar activation efficiency to MS40L-low cells were used to complete sample sorting. Irradiated 3T3msCD40L feeder cells were seeded the day of sorting at a density of 4 x 104 cells / well in IMDM complete medium and supplemented with 50 ng / mL each of IL-4, IL-21, IL-2, 100 ng / mL Anti -rhesus IgG (H+L) (Bio-Rad, cat# AAI42) and cultured for 14 days (J. Huang et al. et al., 2013). B cell culture supernatants from days 12, 14, 15, and 18 were harvested and used for IgG and antigen ELISA. After media removal, B cells in 96-well plates were frozen at -80°C without any additional buffer and used for B cell receptor sequence analysis.

[0368] ELISA for B cell activation screening

[0369] B cell culture supernatants from days 12, 14, 15, and 18 were used to screen for B cell activation (IgG expression) and antigen binding. Briefly, 96-well half area high binding plates (Corning cat# 3690) were coated overnight at 4°C with AffiniPure F(ab’)2 Fragment Goat Antihuman IgG (H+L) (Jackson Immunoresearch, cat# 109-006-088) diluted 1 :500 in PBS, or 6x- His tag monoclonal antibody diluted 1 :500 in PBS (Invitrogen, cat# MAI-21315). Plates were washed 3x with wash buffer composed of 0.01% or 0.05% Tween-20 in PBS for antigen and IgG ELISA, respectively, and blocked with 3% BSA / PBS for 1 hour at RT. For antigen ELISA, His- tagged N332-GT5 WT, N332-GT5 KO, and B23 were separately added to anti-His coated plates at 1 pg / mL in 1% BSA / PBS and incubated for 1 hour at RT and washed 3x. 25 pL control antibodies (BG18, DEN3) serially diluted in 1% BSA / PBS, and B cell culture supernatants were added onto plates and incubated for 1 hour at RT. Plates were washed 3x, and detected with alkaline phosphatase (AP)-conjugated anti-Human IgG Fc fragment specific secondary (Jackson Immunoresearch, cat# 109-055-098) diluted 1 : 1000 in 1% BSA / PBS and incubated for 1 hour atRT. Plates were developed with phosphatase substrate (Sigma-Aldrich, cat# S0942), and absorbance was measured at 405 nm.

[0370] B cell receptor amplification, cloning, and sequencing

[0371] Antigen-specific activated B cells were selected for B cell receptor sequence analysis. mRNA extraction, cDNA, and nested PCR reactions were done as previously described (F. Zhao et al. et al., 2020). Briefly, frozen cells were thawed, lysed, and mRNA extracted using TurboCapture 96 mRNA kit (QIAGEN, cat# 72251) according to the manufacturer’s protocol. mRNA was reverse transcribed, and cDNA was subjected to nested PCR reactions for Ig heavy and light chain variable regions. Amplified PCR products were analyzed with 2% 96 E-gel (Thermofisher, cat# G720802), and wells with PCR products corresponding to Ig heavy and light chain were cleaned with SPRI beads (Beckman Coulter, cat# B23319). Cleaned PCR products were sequenced directly and / or selected for Gibson cloning (NEB, cat# E2621X) prior to Sanger sequencing.

[0372] Bioinformatic analysis of BCR sequence

[0373] Sequence Processing

[0374] The output from single-cell sorting Sanger sequences and 10X VDJ contigs were reannotated with the SADIE library (github.com / jwillis0720 / sadie. git) resulting in a paired AIRR compliant dataframe (www.frontiersin.or / articles / 10.3389 / fimmu.2018.02206 / full). The dataframe was split into IGH, IGL and IGK assigned locus and paired on 10X hashtag and animal ID if they had exactly one heavy IGH and one IGL or IGK call. Applicants also assigned the closest human ortholog to every rhesus germline V and J sequence. Somatic hypermutation was calculated by taking the number of mutations of the VH or VK / VL gene and divided by the total length of the VH or VK / VL gene.

[0375] BG18 Type I Definitions

[0376] The paired dataframe was assigned BG18 type I definitions as >= 22 HCDR3 amino acids long using the regular expression “ITIFG[LV]VI[IT]”. Each sequence was scored based on how close it was to a perfect regular expression match and the “best” regular expression was recorded. A BG18 type I precursor was defined if it had less than four mutations from perfect regular expression match and was found in index position 4,5 or 6 in the HCDR3 sequence. In addition, applicants recorded if a glutamate followed +2 positions from the end of the matching regular expression.

[0377] BG18 Type I Alternate Definitions

[0378] The paired dataframe sequences were assigned a BG18 type I alternate classification if the sequences were >= 22 HCDR3 and had the following regular expression [WFY]GVLQFLEWLLY (SEQ ID NO: 65) where up to four somatic mutations were tolerated in only VLQFLEWLLY (SEQ ID NO: 66) requiring a strict regular expression match to the [WFY]G (SEQ ID NO: 67).

[0379] Clustering of BG18 Type I and Type 1 Alternate BCR Sequences

[0380] The clustering module of SADIE library (github.com / jwillis0720 / Sadie. git) was used to cluster both the BG18 Type I and Type I alternate using the following criteria. The sequences were only clustered on the heavy chain and were first grouped by animal and HCDR3 length. Within each group, a distance was computed for all antibodies. The distance was calculated as a Levenshtein distance between the HCDRls + HCDR2s + HCDR3s. In addition, the somatic pad option was used in SADIE which a distance of 1 was subtracted for every common somatic amino acid mutation (D. J. Leggat et al. et al., 2022). The final distance matrix was used for agglomerative clustering using average linkage and a distance cutoff of 3. The final clusters were annotated in the dataframe.

[0381] Clustering of off-target Non-BGl 8 Sequences

[0382] The clustering module of SADIE was also used in the clustering of off-target sequences defined as those sequences with an N332-GT5 KO antigen count of <100 (D. J. Leggat et al. et al., 2022). These sequences had distance matrix constructed such that both heavy and light chain were considered where the distance between every antibody was computed across all six CDR chains. Single-linkage agglomerative clustering was used with a cutoff of 5 to get final cluster assignments. Large clusters that were found in multiple animals and multiple weeks were prioritized for synthesis and testing with SPR.

[0383] TZM-bl pseudovirus neutralization assay

[0384] Pseudoviruses were produced in HEK293T cells (RRID:CVCL_0063) co-transfected using FuGENE 6 (Promega, cat# E2691) with pseudovirus Env-expressing plasmid and Env- deficient backbone plasmid (PSG3AEnv). Pseudoviruses were harvested 72 hours posttransfection, sterile filtered (0.45 pm), and concentrated (EMD Millipore, cat# UFC905024). Equal volumes of serially diluted monoclonal antibodies at appropriate concentrations were incubated with HIV pseudovirus in half-area 96-well plates (Greiner, cat# 675083) at 37°C for 1hour. Next, 50 pL of TZM-bl cells at 200,000 cells / mL with or without DEAE-dextran (5 pg / mL final concentration) were added to each well containing the antibody-virus mixture and incubated at 37°C for 72 hours in a humidified atmosphere of 5% CO2. After incubation, culture media was removed, and cells were lysed with 45 pL / well lx Luciferase Culture Lysis buffer (Promega, cat# El 531) for 20 min at RT. Neutralization was measured by adding 30 pL luciferase reagent / well (Promega, cat# El 500) and measuring luminescence. IC50 was calculated using a nonlinear regression curve fit, sigmoidal, 4PL equation constrained from 0-100% in GraphPad Prism 9.3.1. IC50 is reported as the mean IC50 ± SD of two biological replicates.

[0385] Protein expression and purification

[0386] The N332-GT5 trimer immunogen contained two modifications compared to what has been described previously (J. M. Steichen et al. et al., 2019) (Fig. 46). First, the trimer was stabilized with a set of mutations called MD65, which is defined as the MD39 stabilizing mutations (J. M. Steichen et al. et al., 2016) plus four additional stabilizing mutations (V505T, V513A, V518S, L520D). Second, glycosylation sequons were added to fill the 241 and 289 glycan holes as described previously (D. W. Kulp et al. et al., 2017). Trimers in the pHLsec vector were cotransfected with furin in a 2:1 ratio into 293F cells (RRID:CVCL_D603) cultured in FreeStyle media using either 293Fectin or PEI as a transfection reagent. Proteins were harvested from the supernatant after 7 days incubation at 37°C and untagged trimers were purified by 2G12 antibody affinity chromatography using a HiTrap NHS-activated HP column (Cytiva, Cat#17-0717-01) run on an AKTA Pure 25L HPLC (Cytiva, Cat# 29-0182-24). C-terminal His-tagged trimers were purified using a HIS-TRAP column, starting with a wash buffer (20mM Imidazole, 500 mM NaCl, 20mM Na2HPO4) and mixing in elution buffer (500 mM Imidazole, 500 mM NaCl, 20 mM Na2HPO4) using a linear gradient. Trimers were polished by size exclusion chromatography (SEC) using a Superdex 200 16 / 600 size exclusion chromatography column (Cytiva, Cat 28-9893- 35) run on an AKTA Pure 25L HPLC. Final proteins were diluted in lx TBS and stored at -80°C. For biotinylated probes, proteins were expressed with a His-tag and avi-tag (GTKHHHHHHGGSGGSGLNDIFEAQKIEWHE) (SEQ ID NO: 68), purified using a HIS- TRAP column followed by SEC, and biotinylated using a BirA biotin-protein ligase reaction kit (Avidity, Cat# BirA500) according to the manufacturer instructions. The N332-GT5 and N332- GT5-KO sorting probes did not contain the 241 / 289 glycosylation sequons.

[0387] Fab and antibody purification

[0388] Paired HC and LC Fab variable region sequences from select NHP affinity matured mAbs were gene synthesized and inserted into human Fab HC constant region expressing vector pFabCW and human lambda or kappa expressing vectors pCW-CLig-hL2 or pCW-CLig-hk, respectively. Fabs were expressed in 500 mL FreeStyle™ 293F cell cultures or 30 mb ExpiCHO™ cell cultures (Thermo Fisher Scientific, Cat# A29133). For 293F cell transfection, 300 pg of HC and 150 pg of LC plasmids were mixed with 225 pg polyethylenimine (PEI; 1:3 DNA:PEI ratio) in 5 mL of Opti-MEM™ reduced serum medium (Thermo Fisher Scientific, Cat# 31985070) for 30 min, then added to 293F cells. Supernatant was collected after 5-6 days. ExpiCHO™ cell cultures were transfected according to manufacturer instructions, using 12.5 pg HC and 31.2 pg LC plasmids. Supernatant was collected 8 days post transfection. Harvested supernatants were filtered through 0.45 or 0.25 pm membrane filters and batch bound to CaptureSelect CH1-XL Affinity resin (Thermo Fisher Scientific, Cat# 1943462005). Resin was washed with PBS, and captured Fabs were eluted with 50 mM NaOAc pH 4.0, buffer exchanged into l x PBS, and concentrated using a 30k MWCO concentrator. For expression of IgG the HC variable region was cloned into the pCW-CHIg-hGl vector. Transfection was carried out as described above but batch binding occurred overnight at 4°C to Protein A resin (Thermo Fisher Scientific, Cat# 20334) while on a rocker. Unbound supernatant was allowed to flowthrough, and the resin was washed with PBS until protein A280 reading of the flowthrough measured by a nanodrop reached background levels. Protein A bound IgG was eluted with 0.1 M Glycine pH 2.7. Eluted mAbs were buffer exchanged into 1xPBS and concentrated using a 50k MWCO concentrator (Millipore).

[0389] SPR

[0390] Kinetics and affinities of antibody-antigen interactions were measured on a ProteOn XPR36 (Bio-Rad) using HC30M XanTec chips and 1XHBS-EP+ pH 7.4 running buffer (20xstock from Teknova, Cat. No. H8022) supplemented with BSA at 1 mg / ml. Two different capture antibodies were used: Anti-Human IgG (Fc) antibody (Cat. No. BR-1008-39, GE) for capturing IgG (ligand) at low density and flowing trimer as analyte, and His-tag Antibody (pAb, Rabbit, Cat. No. A00174, GenScript) for capturing His-tagged trimer (ligand) and flowing Fab as analyte (Figs. 42E-F and 43B). About 7,000 response units of capture antibody were covalently immobilized on the sensor surface via EDC / NHS. In case of IgG-antigen interaction studies about 50 to 100 RUs of IgGs at O.lug / mL were captured onto each flow cell. In case of Fab-antigen interaction studies about 300 to 400 RUs of antigen at 1 pg / mL were captured onto each flow cell. Analytes werepassed over the flow cell at 30 pl / min for 3 min followed by a 5-min dissociation time. Regeneration was accomplished using phosphoric acid 1.7% or 0.85% with a 180-s contact time and injected four times per cycle. ProteOn Manager software (Bio-Rad) was used to analyze raw sensograms, including interspot and column double referencing, and to perform either Equilibrium fits or Kinetic fits with Langmuir model, or both, when applicable.

[0391] Cryo-electron microscopy

[0392] For each complex, 0.2 mg of N332-GT5 were incubated with 0.3 mg of BG18-like Fab (from this study) and 0.3 mg of base-directed RM20A3 Fab (to increase angular sampling in cryo- EM). A total of 5 complexes were produced (including RM_N332_03, RM_N332_36, RM_N332_32, RM_N332_07, or RM_N332_08), incubated overnight at room temperature, and purified the following morning using a HiLoad 16 / 600 Superdex 200 pg (Cytiva) gel filtration column. The complexes were then concentrated to between 5-6 mg / mL for application onto cryoEM grids. From a separate study, N332-GT5 was incubated with mouse polyclonal Fabs, purified as above and concentrated to 2.6 mg / ml. Later data processing (below) revealed that a majority of trimers were unliganded. Cryo grids were prepared using a Vitrobot Mark IV (Thermo Fisher Scientific). The temperature was set to 4°C and humidity was maintained at 100% during the freezing process. The blotting force was set to 1 and wait time was set to 10 s. Blotting time was varied from 5 to 6 s. Detergents lauryl maltose neopentyl glycol (LMNG; Anatrace) or n- Dodecyl-P-D-Maltoside (DDM; Anatrace) at final concentrations of 0.005 or 0.06 mM, respectively, were used for freezing. Quantifoil R 1.2 / 1.3 (Cu, 300-mesh; Quantifoil Micro Tools GmbH) or UltrAuFoil 1.2 / 1.3 (Au, 300-mesh; Quantifoil Micro Tools GmbH) grids were used and treated with Ar / O2 plasma (Solarus plasma cleaner, Gatan) for 8 sec before sample application. 0.5 pL of detergent was mixed with 3.5 pL of samples and 3 pL of the mixture was immediately loaded onto the grid. Following blotting, the grids were plunge-frozen into liquid nitrogen-cooled liquid ethane.

[0393] Samples containing RM_N332_03, RM_N332_36 or RM_N332_32 were loaded into a Thermo Fisher Scientific Talos Arctica operating at 200 kV. Exposure magnification was set to 36,000x with a pixel size at the specimen plane of 1.15 A. Leginon software (C. Suloway et al. et al., 2005) was used for automated data collection. Micrograph movie frames were motion corrected and dose weighted using MotionCor2 (S. Q. Zheng et al. et al., 2017) and imported intocryoSPARC (A. Punjani et al., 2017) for the remainder of data processing. CTF correction was performed using cryoSPARC Patch CTF.

[0394] Samples containing RM_N332_07, or RM_N332_08 were loaded into a Thermo Fisher Scientific Glacios electron microscope operating at 200 kV. Exposure magnification was set to 190,000x with a pixel size at the specimen plane of 0.725 A. EPU software (Thermo Fisher Scientific) was used for automated data collection. Micrograph movie frames were motion and CTF corrected using cryoSPARC Live, including dose weighting, followed by micrograph import into cryoSPARC.

[0395] N332-GT5 (partially complexed with mouse polyclonal antibodies) data collection occurred at the Pacific Northwest Center for Cryo-EM (PNCC) using a Thermo Fisher Scientific Krios and a Gatan K3 direct electron detector (300 keV, 0.40075 A / pixel super-resolution mode). EPU (Thermo Fisher) was used for automated data collection, and Relion 3.1 (J. Zivanov et al., 2020) for motion correction Micrographs were binned during motion correction, with a resulting pixel size of 0.8015 A / pixel and imported in cryoSPARC. CTF correction was performed using cryoSPARC Patch CTF.

[0396] For all datasets, particle picking was performed using blob picker initially followed by template picker. For Glacios datasets, particles were downscaled to 1.044 A / pix during extraction to reduce box size and increase speed of downstream jobs. Arctica datatsets were processed at the native 1.15 A / pix size, and the Krios dataset was processed at the binned 0.8015 A / pix size. Multiple rounds of 2D classification and 3D ab-initio reconstruction were performed prior to 3D non-uniform refinement with global CTF refinement. For the unligandedN332-GT5 dataset, many rounds of 2D classification were performed to remove the subpopulation of particles with mouse polyclonal Fab. Final refinements were performed with C3 symmetry and global resolution estimated by FSC 0.143.

[0397] Fab Fv homology models were generated using SAbPred ABodyBuilder-ML (B. Abanades et al., 2022). Model building was performing by docking homology models of trimer and Fab Fv in UCSF Chimera (E. F. Pettersen et al. et al., 2004), manually building and refinement in Coot 0.9.8 (A. Casanal et al., 2020) and real space refinement using Rosetta (P. Conway et al., 2014) and Phenix (P. V. Afonine et al. et al., 2018). Final models were validated using MolProbity and EMRinger in the Phenix suite.

[0398] Angle of approach measurements

[0399] The trimer 3-fold axis was aligned on the z-axis with the center of mass of the CA residues of the N332 epitope on the x-axis. The center of mass for the CA residues in the N332 epitope was defined by residues G324, D325, V326, R327, M328, A329, H330, 1415, L416, and P417 and was aligned on the x-axis to coordinates (40.091, 0, 0). The center of mass for three N332 epitopes at the trimer 3-fold axis was aligned to coordinates (0, 0, 0) and a third point (the center of mass for the CA of L587 in all three protomers) was aligned to coordinates (0, 0, -50.88). The latitudinal angle was the angle formed by the z-axis and a vector from the N332 epitope center of mass to the HC center of mass (CA atoms for 6 beta strands, residues 21-24, 34-39, 46-52, 67- 71, 77-82, 89-92 for BG18_iGL0) in the x-z plane. The longitudinal angle was the angle formed by the x-axis and the same vector connecting the N332 epitope to the HC in the x-y plane. The HC-LC twist angle was the angle between the x-axis and a vector connecting the HC center of mass to the LC center of mass (CA atoms for 6 beta strands, residues 18-24, 34-37, 45-48, 62-66, 70-76, 84-88 for BG18_iGL0) in the x-y plane.

[0400] Design of trimers with improved VI loop glycan occupancy

[0401] BG505_B23 was found to have low glycan occupancy by mass spectrometry analysis and therefore three new trimers were designed using three different approaches. In the first approach, BG505 B38 was designed by reverting four VI loop amino acids from BG505 B23 back to the WT BG505 amino acid. BG505 SOSIP has been shown to have better glycan occupancy in the VI loop than what the applicants observed with BG505_B23, which indicated that the germline targeting mutations in the VI loop were causing reduced glycan occupancy (L. Cao et al. et al., 2017). Therefore, reverting more GT mutations back to the WT amino acid should potentially improve the glycosylation. In the second approach, BG505_B46 was designed using the NetNGlyc server (R. Gupta et al., 2002) to optimize the sequence adjacent to the N133 and N137 glycosylation sites. The N-terminal portion of the BG505_B23 sequence was submitted along with mutated versions containing all 20 AA at each of the following VI loop positions: 132- 136, 138, 140-143. Five VI loop sequences were designed based on the output glycosylation potential and tested by incorporating those VI loop sequences into the BG505 B23 trimer. They were screened for acceptable expression levels in 293F cells, SECMALS profiles to determine percent trimer, and binding affinities to BG18 type I Fabs from wks 7 and 10 post prime. Two of the trimers passed those filters and were then subjected to site-specific glycan analysis to determine glycan occupancy. Of the two trimers, BG505 B46, reported here, had a superior binding profileto BG18 type I Fabs. In the third approach, BG505 B48 was designed to have improved N137 glycan occupancy using an optimize sequence described previously (S. S. Kulkami et al., 2009) that modifies residues adjacent to the glycosylation site but in this case removes the N133 glycosylation site due to its proximity to N137. Therefore, BG505 B48 does not contain an N133 glycosylation sequon. All three approaches produced trimers with near complete glycan occupancy in the VI loop when expressed in 293F cells (Fig. 39). Amino acid sequences of BG505 B23, BG505 B46, and BG505 B48 are provided in Fig. 46.

[0402] Site-specific glycan analysis

[0403] Method 1 : DeGlyPHER (M. Caskey et al., 2017) was used to ascertain site-specific glycan occupancy and processivity on the examined glycoproteins.

[0404] Proteinase K treatment and deglycosylation: HIV Env glycoprotein was exchanged to water using Microcon Ultracel PL-10 centrifugal filter. Glycoprotein was reduced with 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HC1) and alkylated with 10 mM 2- Chloroacetamide in 100 mM ammonium acetate for 20 min at room temperature (RT, 24°C). Initial protein-level deglycosylation was performed using 250 U of Endo H for 5 pg trimer, for 1 h at 37°C. Glycorotein was digested with 1 :25 Proteinase K (PK) for 30 min at 37°C. PK was denatured by incubating at 90°C for 15 min, then cooled to RT. Peptides were deglycosylated again with 250 U Endo H for 1 h at 37°C, then frozen at -80°C and lyophilized. 100 U PNGase F was lyophilized, resuspended in 20 pl 100 mM ammonium bicarbonate prepared in H218O, and added to the lyophilized peptides. Reactions were then incubated for 1 h at 37°C, subsequently analyzed by LC-MS / MS.

[0405] LC-MS / MS: Samples were analyzed on an Q Exactive HF-X mass spectrometer. Samples were injected directly onto a 25 cm, 100 pm ID column packed with BEH 1.7 pm C18 resin. Samples were separated at a flow rate of 300 nL / min on an EASY-nLC 1200 UHPLC. Buffers A and B were 0.1% formic acid in 5% and 80% acetonitrile, respectively. The following gradient was used: 1-25% B over 160 min, an increase to 40% B over 40 min, an increase to 90% B over another 10 min and 30 min at 90% B for a total run time of 240 min. Column was reequilibrated with solution A prior to the injection of sample. Peptides were eluted from the tip of the column and nanosprayed directly into the mass spectrometer by application of 2.8 kV at the back of the column. The mass spectrometer was operated in a data dependent mode. Full MSI scans were collected in the Orbitrap at 120,000 resolution. The ten most abundant ions per scanwere selected for HCD MS / MS at 25 NCE. Dynamic exclusion was enabled with exclusion duration of 10 s and singly charged ions were excluded.

[0406] Data Processing: Protein and peptide identification were done with Integrated Proteomics Pipeline (IP2). Tandem mass spectra were extracted from raw files using RawConverter (L. He et al., 2015) and searched with ProLuCID (T. Xu et al. et al., 2015) against a database comprising UniProt reviewed (Swiss-Prot) proteome for Homo sapiens (UP000005640), UniProt amino acid sequences for Endo H (P04067), PNGase F (Q9XBM8), and Proteinase K (P06873), amino acid sequences for the examined proteins, and a list of general protein contaminants. The search space included no cleavage-specificity. Carbamidomethylation (+57.02146 C) was considered a static modification. Deamidation in presence of H218O (+2.988261 N), GlcNAc (+203.079373 N), oxidation (+15.994915 M) and N-terminal pyroglutamate formation (-17.026549 Q) were considered differential modifications. Data was searched with 50 ppm precursor ion tolerance and 50 ppm fragment ion tolerance. Identified proteins were filtered using DTASelect2 (D. L. Tabb et al., 2002) and utilizing a target-decoy database search strategy to limit the false discovery rate to 1%, at the spectrum level (J. Peng et al., 2003). A minimum of 1 peptide per protein and no tryptic end per peptide were required and precursor delta mass cut-off was fixed at 15 ppm. Statistical models for peptide mass modification (modstat) were applied. Census2 (S. K. Park et al., 2008) label-free analysis w...

Claims

WHAT IS CLAIMED IS:

1. A non-naturally occurring protein comprising the sequence of:(a) 001428-T278M.2_gpl51VENLWVTVYYGVPVWKEARTTLFCASDAKAYETEVHNVWATHACVPTDPNPQEMVL GNVTENFNMWKNDMVDQMHEDVISLWAQSLKPCVKLTPLCVTLECTQVNATQGNTT QVNVTQVNGDEMKNCSFNTTTEIRDKKQKAYALFYRLDLVPLERENRGDSNSASKYILI NCNTSAITQACPKVNFDPIPIHYCTPAGYAILKCNNKTFNGTGSCNNVSTVQCTHGIKPV VSTQLLLNGSLAEEEIIIRSENLMDNVKTIIVHLDQSVEIVCTRPNNNTVKSIRIGPGQTFY YTGDIIGNIREAHCNISEKKWHEMLRRVSEKLAEHFPNKTIKFTSSSGGDLEITTHSFNCR GEFFYCNTSGLFNSTYMPNGTYMPNGTNNSNSTIILPCRIKQIINMWQEVGRAMYAPPIA GNITCNSNITGLLLVRDGGKNNNTEIFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTR CKRRVVGSHSGSGGSGSGGHAAVGLGAVSLGFLGAAGSTMGAASITLTVQARQLLSGI VQQQSNLLQAPEPQQHLLQDTHWGIKQLQTRVLAIEHYLKDQQLLGIWGCSGKLICCTA VPWNSSWSNKSLTDIWDNMTWMQWDREVSNYTGIIYRLLEDSQNQQERNEQDLLALD SWKNLWSWFDISNWLWYIKIFIMIVGGLIGLRIIFAVLSIVNRVR (SEQ ID NO: 2);(b) 001428.2_gpl51VENLWVTVYYGVPVWKEARTTLFCASDAKAYETEVHNVWATHACVPTDPNPQEMVL GNVTENFNMWKNDMVDQMHEDVISLWAQSLKPCVKLTPLCVTLECTQVNATQGNTT Q VNVTQ VNGDEMKNC SFNTTTEIRDKKQKAYALF YRLDLVPLERENRGD SNS ASKYILI NCNTSA1TQACPKVNFDPIPIHYCTPAGYAILKCNNKTFNGTGSCNNVSTVQCTHGIKPV VSTQLLLNGSLAEEEIIIRSENLTDNVKTIIVHLDQSVEIVCTRPNNNTVKSIRIGPGQTFYY TGDIIGNIREAHCNISEKKWHEMLRRVSEKLAEHFPNKTIKFTSSSGGDLEITTHSFNCRG EFFYCNTSGLFNSTYMPNGTYMPNGTNNSNSTIILPCRIKQIINMWQEVGRAMYAPPIAG NITCNSNITGLLLVRDGGKNNNTEIFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTRC KRRVVGSHSGSGGSGSGGHAAVGLGAVSLGFLGAAGSTMGAASITLTVQARQLLSGIV QQQSNLLQAPEPQQHLLQDTHWGIKQLQTRVLAIEHYLKDQQLLGIWGCSGKLICCTAV PWNSSWSNKSLTDIWDNMTWMQWDREVSNYTGIIYRLLEDSQNQQERNEQDLLALDS WKNLWSWFDISNWLWYIKIFIMIVGGLIGLRIIFAVLSIVNRVR (SEQ ID NO: 3);(c) lHD2-N276Q.2_gp!51TEQLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPSPQEVRLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDFTGNVINANSSA SSAQEKMEEGEVKNCSFKVTTNIRDKTQEEYALFYKLDLVAIDQTKNNSYRLINCNTSVI TQACPKVSFEPIPIHYCAPAGFAILKCNDEKFNGKGKCNNVSTVQCTHGIRPVVSTQLLL NGSLAEKEIVIRSDQFTNNAKIIIVQLNKSVEINCTRVNNNTVKSIPIGPGRAFYATGDITG DIRRAHCTISRTQWNNTLKQVVEKLRKQFKNKTIVFNRSSGGDLEIVTHSFNCGGEFFYC NTTQLFNSKWNSSGTWTGGSNNTEGNDTLILPCRIKQIINMWQEVGKAMYAPPIKGLIR C S SNITGLLLTRDGGS STDPIEIFRP AGGNMRDNWRSEL YKYKVVKIEPLGVAPTRCKRR VVQSHSGSGGSGSGGHAAVGIGALSLGFLGAAGSTMGAASVTLTVQARLLLSGIVQQQ NNLLRAPEPQQHMLQDTDWGIKQLQARVLAVEHYLKDQQLLGIWGCSGKLICCTAVP WNATWSNKSLEKIWDNMTWREWEKEIDNYTSLIYSLLEDSQNQQEKNEQELLELDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 4);(d) lHD2.2_gpl51:TEQLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPSPQEVRLV NVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDFTGNVINANSSA SSAQEKMEEGEVKNCSFKVTTNIRDKTQEEYALFYKLDLVAIDQTKNNSYRLINCNTSVI TQACPKVSFEPIPIHYCAPAGFAILKCNDEKFNGKGKCNNVSTVQCTHGIRPVVSTQLLL NGSLAEKEIVIRSDNFTNNAKinVQLNKSVEINCTRVNNNTVKSIPIGPGRAFYATGDITG DIRRAHCTISRTQWNNTLKQVVEKLRKQFKNKTIVFNRSSGGDLEIVTHSFNCGGEFFYC NTTQLFNSKWNSSGTWTGGSNNTEGNDTLILPCRIKQIINMWQEVGKAMYAPPIKGLIR C S SNITGLLLTRDGGS STDPIEIFRP AGGNMRDNWRSEL YKYKVVKIEPLGVAPTRCKRRVVQSHSGSGGSGSGGHAAVGIGALSLGFLGAAGSTMGAASVTLTVQARLLLSGIVQQQ NNLLRAPEPQQHMLQDTDWGIKQLQARVLAVEHYLKDQQLLGIWGCSGKLICCTAVP WNATWSNKSLEKIWDNMTWREWEKEIDNYTSLIYSLLEDSQNQQEKNEQELLELDKW ASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 5);(e) BS208-T278M.2_gpl51TDNLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEMDL ENVIEEFNMWKNNMVEQMHEDVISLWDQSLKPCVKLTPLCVTLDCSNHKSIMGEEVKN CSFNMTTELRDKKQKVYSLFYKLDVVPLDEANGNNNSSYRLVNCNTSAITQACPKVTF EPIPIHYCAPAGFAILKCKDKEFSGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEEHVIIRSENIMNNAKNILVQLNESVEIYCIRPSNNTVKSVRIGPGQTFYATGEIVGDIRQAHCNVS GSKWNKTLQQVADQLRKHFNTTTIIFANPLGGDLEITTHSFNCGGEFFYCNTSGLFNSTWPS STQES SDPITLQCRIKQIINMWQRVGQ AMY APPIQGEIRCNS SITGIILTRDGGYNNTNE TFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKCRRRVVESHSGSGGSGSGGHAAVT MGAVSLGFLGAAGSTMGAASLTLTVQARLLLSGIVQQQSNLLRAPEPQQQLLKDTDWG IKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSTWSNKSYSEIWDNMTWLQ WDKEVSNYTDIIYKLLEDSQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMI VGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 6);(f) BS208.2_gpl51TDNLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEMDL ENVIEEFNMWKNNMVEQMHEDVISLWDQSLKPCVKLTPLCVTLDCSNHKSIMGEEVKN CSFNMTTELRDKKQKVYSLFYKLDVVPLDEANGNNNSSYRLVNCNTSAITQACPKVTF EPIPIHYCAPAGFAILKCKDKEFSGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEEHVII RSENITNNAKNILVQLNESVEIYCIRPSNNTVKSVRIGPGQTFYATGEIVGDIRQAHCNVS GSKWNKTLQQVADQLRKHFNTTTIIFANPLGGDLEITTHSFNCGGEFFYCNTSGLFNSTW PS STQES SDPITLQCRIKQIINMWQRVGQAMYAPPIQGEIRCNS SITGIILTRDGGYNNTNETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKCRRRVVESHSGSGGSGSGGHAAVT MGAVSLGFLGAAGSTMGAASLTLTVQARLLLSGIVQQQSNLLRAPEPQQQLLKDTDWG IKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSTWSNKSYSEIWDNMTWLQ WDKEVSNYTDIIYKLLEDSQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMI VGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 7);(g) CNE40-T278M.2_gpl51VGNLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPADPNPQEMLL KNVTENFNMWKNEMVNQMHEDVISLWDQSLKPCVKLTPLCVTLNCTNVKINSTSNETC IDSGNNSTCNETYKEMRNCSFNATTVVRDKQQKMYALFYKLDIVPLNSGYKNSSDETY RLINCNTSAITQACPKVSFDPIPIHYCTPAGYALLKCNNKTFNGTGPCNNVSTVQCTHGIK PVVSTQLLLNGSLAEEEIIIRSENLMNNAKTIIVHLKEPVNITCERPNNNTVKSIRIGPGQTF YYTGDIIGNIREAHCNISRSQWNKTLQGVGEKLAELFPNKTIVFKNSSGGDLEITTHSFNC RGEFF YCNTTDLFNSTYWSNGTYITQSNS S SINITLPCRIKQIINMWQEVGRAIYAPPIAGQITCISNITGLLLLRDGGKEANGTEIFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTKCK RRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASITLTVQARQLLSGIVQQ QSNLLKAPEPQQHLLQDTHWGIKQLQTRVLAIEHYLKDQQLLGIWGCSGKLICCTAVPWNSSWSNKSQTEIWNNMTWMQWDREINNYTDIIYRLLEESQNQQENNEEDLLALDSWKN LWNWFNITNWLWYIKIFIIIVGGLIGLRIIFTVLSVVNRVR (SEQ ID NO: 8);(h) V703-0739-T278M.2_gpl51MENLWVTVYYGVPVWREAKTTLFCASDAKAYSKEVHNIWATHACVPTDPNPQEILLE NVTENFNMWKNDMVDQMHEDIISLWDQSLRPCVKLTPLCVTLNCTTANVNVTKGAYD NVTEQGLKEEIKNCSFNMTTEIRDKKKTVYALFYELDVVKLDEVSLDNSTSKYRKYRLI NCNTSAISQICPKITFDPIPIHYCAPAGYAILKCNDKAFNGTGPCKNVSTVQCTHGIKPVV STQLLLNGSLAEEGIIIRSENLMDNVKTIIVHLNESVEIICVRPNNNTVKSIRIGPGQTFYVT GDIIGDIRQAHCNITREKWNKTVIRVKEKLKEHFNNKTINFAEHSGGDLEITTHTFNCRGE FFYCDTTELFNKTDEGNDTNITLPCRIKQIINMWQEVGRAIYAPPIAGNITCTSYITGLLLT RDGGHINNSETETFRPGGGDMRNNWRSELYKYKVVEIKPLGVAPTKCKRRVVESHSGS GGSGSGGHAAVGIGAVLLGFLGAAGSTMGAASNTLFVQARQLLSGFVQQQSNLPRAPE AQQHLLQLGVWGIKQLQTRVLAIERYLEVQQLLGLWGCSGKLICCTSVPWNNTWSSKS FNDIWDNMTWMQWDREIGNYSDTIYRLLFQSQFQQEINEVDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 9);(i) V703-0537-T278M.2_gpl51AEDLWVTVYYGVPVWRDAITTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLE NVTENFNMWKNNMVDQMHEDIISLWDQSLKPCVQLTPLCVTLNCTDVVGNNTATRRN NTATRRNNTATINDTDTGIKNCSFNITTEVRDKKKKVHALFYRLDIVPIDGKNSNSTEYIL INCNTSVITQICPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTIQCTHGIKPVV STQLLLNGSLAEEDIIISSENMMDNTKTIIVHFNESVGIECTRPNNNTVESIRIGPGQTFYVT GEIIGDIRQ AHCNISKGNW SKTLQRVKEKLKKYYHNKTIKFEP S SGGDLEITTHSFNCRGE FFYCYTSGLFNESIVNGTNGTITLPCRIKQIVNMWQKVGRAMYAPPIAGNITCSSNITGLL LTRDGGRGTNGTETFRPAGGNMKDNWRSELYKYKVVEIKPLGIAPTKCRRRVVGSHSG SGGSGSGGHAAVLGTAILGFLGAAGSTMGAASNALFVQTRQLLSGFVQQQSNLPKAPK AQQQLLQLGVWGIKQLQTRVLAIERYLEVQQFLGLWGCSGKAICCTAVPWNTTWSNKS EQEIWNNMTWMQWDREVGNYTDTIYKLLFVAQFQQEINEVDLLALNSWKNLWSWFDI SNWLWYIKIFIMIVGGLIGLRIIFAVLSIVNRVR (SEQ ID NO: 10);(j) CAP260-T278M.2_gpl51VGNLWVTVYYGVPVWTEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEILLG NVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDVERNVTYKNDMKNCSFNTTTELRDKRQRAYALFYKPDVVPLNKNNASDYILINCNTSTITQICPKVSFDPIPI HYCTPAGYAILKCNDKNFTGMGSCFNVSTVQCTHGIKPVVSTQLLLNGSLAEGGIIIRSE NLMDNTKTIIAHLNESINIECVRPGNNTVTSIRIGPGQTFYVNSIIGDIRQAHCNINLNKWT KIVEGVKEKLREYYLNRTIEFRPPSGGDLEITTHSFNCGGEFFYCNTTQLFNTTLFNTTHH ENDTITLQCRIKQIINMWQGVGRAMYAPPIAGNITCNSSITGLLLTRDGGQTNDTDTTEIFRPGGGNMKDNWRSELYKYKVVEIKPLGLAPTGCKRRVVESHSGSGGSGSGGHAAVGIGAVLLGFLGAAGSTMGAASNTLFAQARQLLSGFVQQQSNLPRAPEAQQHMLQLGVWGF KQLQARVLAIERYLEVQQLLGLWGCSGKLICCTNVPWNSTWSNKSEKEIWDNMTWMQ WEKEIGNYTGTIYRLLFDSQNQQEINEVDLLALDSWKNLWSWFDISNWLWYIKIFIMIV GGLIGLRIIFAVLSIVNRVR (SEQ ID NO: 11);(k) BG505-MD39.3-N276Q_gpl51AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGE LKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQA CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSL AEEEVIIRSEQITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNT SGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNIT GLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLL RAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSW SNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWN WFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 12);(l) CAP260.2_gpl51VGNLWVTVYYGVPVWTEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEILLG NVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDVERNVTYKNDM KNCSFNTTTELRDKRQRAYALFYKPDVVPLNKNNASDYILINCNTSTITQICPKVSFDPIPI HYCTPAGYAILKCNDKNFTGMGSCFNVSTVQCTHGIKPVVSTQLLLNGSLAEGGIIIRSENLTDNTKTIIAHLNESINIECVRPGNNTVTSIRIGPGQTFYVNSIIGDIRQAHCNINLNKWT KIVEGVKEKLREYYLNRTIEFRPPSGGDLEITTHSFNCGGEFFYCNTTQLFNTTLFNTTHH ENDTITLQCRIKQIINMWQGVGRAMYAPPIAGNITCNSSITGLLLTRDGGQTNDTDTTEIFRPGGGNMKDNWRSELYKYKVVEIKPLGLAPTGCKRRVVESHSGSGGSGSGGHAAVGIG AVLLGFLGAAGSTMGAASNTLFAQARQLLSGFVQQQSNLPRAPEAQQHMLQLGVWGF KQLQARVLAIERYLEVQQLLGLWGCSGKLICCTNVPWNSTWSNKSEKEIWDNMTWMQ WEKEIGNYTGTIYRLLFDSQNQQEINEVDLLALDSWKNLWSWFDISNWLWYIKIFIMIV GGLIGLRIIFAVLSIVNRVR (SEQ ID NO: 13);(m) CNE40.2_gpl51VGNLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPADPNPQEMLL KNVTENFNMWKNEMVNQMHEDVISLWDQSLKPCVKLTPLCVTLNCTNVKINSTSNETC IDSGNNSTCNETYKEMRNCSFNATTVVRDKQQKMYALFYKLDIVPLNSGYKNSSDETY RLINCNTSAITQACPKVSFDPIPIHYCTPAGYALLKCNNKTFNGTGPCNNVSTVQCTHGIK PVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLKEPVNITCERPNNNTVKSIRIGPGQTF YYTGDIIGNIREAHCNISRSQWNKTLQGVGEKLAELFPNKTIVFKNSSGGDLEITTHSFNC RGEFFYCNTTDLFNSTYWSNGTYITQSNSSSINITLPCRIKQIINMWQEVGRAIYAPPIAGQ ITCISNITGLLLLRDGGKEANGTEIFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTKCK RRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASITLTVQARQLLSGIVQQ QSNLLKAPEPQQHLLQDTHWGIKQLQTRVLAIEHYLKDQQLLGIWGCSGKLICCTAVPW NSSWSNKSQTEIWNNMTWMQWDREINNYTDIIYRLLEESQNQQENNEEDLLALDSWKN LWNWFNITNWLWYIKIFIIIVGGLIGLRIIFTVLSVVNRVR (SEQ ID NO: 14);(n) V703-0739.2_gpl51MENLWVTVYYGVPVWREAKTTLFCASDAKAYSKEVHNIWATHACVPTDPNPQEILLE NVTENFNMWKNDMVDQMHEDIISLWDQSLRPCVKLTPLCVTLNCTTANVNVTKGAYD NVTEQGLKEEIKNCSFNMTTEIRDKKKTVYALFYELDVVKLDEVSLDNSTSKYRKYRLI NCNTSAISQICPKITFDPIPIHYCAPAGYAILKCNDKAFNGTGPCKNVSTVQCTHGIKPVV STQLLLNGSLAEEGIIIRSENLTDNVKTIIVHLNESVEIICVRPNNNTVKSIRIGPGQTFYVT GDIIGDIRQAHCNITREKWNKTVIRVKEKLKEHFNNKTINFAEHSGGDLEITTHTFNCRGE FFYCDTTELFNKTDEGNDTNITLPCRIKQIINMWQEVGRAIYAPPIAGNITCTSYITGLLLT RDGGHINNSETETFRPGGGDMRNNWRSELYKYKVVEIKPLGVAPTKCKRRVVESHSGS GGSGSGGHAAVGIGAVLLGFLGAAGSTMGAASNTLFVQARQLLSGFVQQQSNLPRAPE AQQHLLQLGVWGIKQLQTRVLAIERYLEVQQLLGLWGCSGKLICCTSVPWNNTWSSKS FNDIWDNMTWMQWDREIGNYSDTIYRLLFQSQFQQEINEVDLLALDKWASLWNWFDIS NWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 15);(o) V703-0537.2_gpl51AEDLWVTVYYGVPVWRDAITTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLE NVTENFNMWKNNMVDQMHEDIISLWDQSLKPCVQLTPLCVTLNCTDVVGNNTATRRN NTATRRNNTATINDTDTGIKNCSFNITTEVRDKKKKVHALFYRLDIVPIDGKNSNSTEYIL INCNTSVITQICPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTIQCTHGIKPVV STQLLLNGSLAEEDIIISSENMTDNTKTIIVHFNESVGIECTRPNNNTVESIRIGPGQTFYVT GEIIGDIRQ AHCNISKGNW SKTLQRVKEKLKKYYHNKTIKFEP S SGGDLEITTHSFNCRGE FFYCYTSGLFNESIVNGTNGTITLPCRIKQIVNMWQKVGRAMYAPPIAGNITCSSNITGLL LTRDGGRGTNGTETFRPAGGNMKDNWRSELYKYKVVEIKPLGIAPTKCRRRVVGSHSG SGGSGSGGHAAVLGTAILGFLGAAGSTMGAASNALFVQTRQLLSGFVQQQSNLPKAPK AQQQLLQLGVWGIKQLQTRVLAIERYLEVQQFLGLWGCSGKAICCTAVPWNTTWSNKS EQEIWNNMTWMQWDREVGNYTDTIYKLLFVAQFQQEINEVDLLALNSWKNLWSWFDI SNWLWYIKIFIMIVGGLIGLRIIFAVLSIVNRVR (SEQ ID NO: 16);(p) BG505 MD39 N332B1 l_mCAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYTPNLTSMMRG ELKNC SFNMTTELRDKKQK VYSLF YRLD VVQINENQGNRSNNSNKE YRLINCNT S AITQ ACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGS LAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIR MAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYC NTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVS NITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVV GRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQ QHLLKDTHWGIKQLQ ARVLAVEHYLRDQQLLGIWGC SGKLICC TNVPWNS S WSNRNL S EIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 17);(q) BG5O5_MD39.3_N332B1 l_gpl51_mAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYTPNLTSMMRG ELKNC SFNMTTELRDKKQK VYSLF YRLD VVQINENQGNRSNNSNKE YRLINCNT S AITQ ACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIR MAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYC NTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVS NITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVV GSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSN LLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNS SWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLW NWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 18);(r) BG505_MD39_N332B16_mCAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYTEKLRSMMKG ELKNC SFNMTTELRDKKQK VYSLF YRLD VVQINENQGNRSNNSNKEYRLINCNT S AITQ ACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGS LAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGHIRQ AHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNT SGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNIT GLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGR RRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQH LLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 19);(s) BG505 MD39 N332B 16_gpl5 l_L14_mAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYTEKLRSMMKG ELKNC SFNMTTELRDKKQK VYSLF YRLD VVQINENQGNRSNNSNKE YRLINCNT S AITQ ACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGS LAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGHIRQ AHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNT SGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNIT GLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGS HSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLL RAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWN WFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 20);(t) BG505_MD65_congly_N332B46_mAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNFTLNLTSMMRGE IKNC SFNMTTELRDKKQKVYSLF YRLD VVQINENQGNRSNNSNKE YRLINCNT S AITQ A CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSL AEEEVIIRSENITNNAKNILVQLNTSVQINCTRPSNNTVKSIRIGPGQAFYYFGDVLGHVR MAHCNISKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCN TSGLFNSTWISNTSVQGSNSTGSNDSLILPCWIKQIINMWQRIGQAMYAPPIQGVIRCVSN ITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRTVG RRRRRRAAGIGASSDGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQ HLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSE IWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 21);(u) BG505_MD65.2_N332B46_gpl51_mAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNFTLNLTSMMRGE IKNCSFNMTTELRDKKQKVYSLFYRLD VVQINENQGNRSNNSNKE YRLINCNT S AITQ A CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSL AEEEVIIRSENITNNAKNILVQLNTPVQINCTRPSNNTVKSIRIGPGQAFYYFGDVLGHVR MAHCNISKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCN TSGLFNSTWISNTSVQGSNSTGSNDSLILPCWIKQIINMWQRIGQAMYAPPIQGVIRCVSN ITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRTVG SHSGSGGSGSGGHAAAGIGASSDGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNL LRAPEPQQHLLKDTHWGIKQLQ ARVLAVEHYLRDQQLLGIWGC SGKLICCTNVPWNS S WSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLW NWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 22);(v) BG505_MD65_N332B48_mCAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYMSNGTSMMRG EIKNC SFNMTTELRDKKQKVYSLF YRLD VVQINENQGNRSNNSNKE YRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPSNNTVKSIRIGPGQAFYYFGDVLGHVRMAHCNISKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYC NTSGLFNSTWISNTSVQGSNSTGSNDSLILPCWIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRTV GRRRRRRAAGIGASSDGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQ QHLLKDTHWGIKQLQ ARVLAVEHYLRDQQLLGIWGC SGKLICCTNVPWNS S W SNRNL S EIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 23);(w) BG505_MD65.2_N332B48_gpl51_mAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYMSNGTSMMRG EIKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQ ACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPSNNTVKSIRIGPGQAFYYFGDVLGHVRMAHCNISKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYC NTSGLFNSTWISNTSVQGSNSTGSNDSLILPCWIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRTVGSHSGSGGSGSGGHAAAGIGASSDGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSN LLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLW NWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 24);(x) BG505_MD65_N332B38_mCAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITSMMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQA CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPSNNTVKSIRIGPGQAFYYFGDVLGHVRMAHCNISKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCN TSGLFNSTWISNTSVQGSNSTGSNDSLILPCWIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRTVG RRRRRRAAGIGASSDGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSE IWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 25);(y) BG505_MD65.2_N332B38_gpl51_mAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITSMMRGE LKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQA CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSL AEEEVIIRSENITNNAKNILVQLNTPVQINCTRPSNNTVKSIRIGPGQAFYYFGDVLGHVRMAHCNISKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCN TSGLFNSTWISNTSVQGSNSTGSNDSLILPCWIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRTVG SHSGSGGSGSGGHAAAGIGASSDGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNL LRAPEPQQHLLKDTHWGIKQLQ ARVLA VEHYLRDQQLLGIWGC SGKLICCTNVPWNS S WSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLW NWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 26);(z) BG505 MD65 N332B23 mCAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYTPNLTSMMRG EIKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQ ACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGS LAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPSNNTVKSIRIGPGQAFYYFGDVLGHVRMAHCNISKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYC NTSGLFNSTWISNTSVQGSNSTGSNDSLILPCWIKQIINMWQRIGQAMYAPPIQGVIRCVS NITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRTVGRRRRRRAAGIGASSDGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQ QHLLKDTHWGIKQLQ ARVLA VEHYLRDQQLLGIWGC SGKLICCTNVPWNS SW SNRNL S EIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 27);(aa) BG505_MD65.2_N332B23_gpl51_mAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYTPNLTSMMRG EIKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGS LAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPSNNTVKSIRIGPGQAFYYFGDVLGHV RMAHCNISKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYC NTSGLFNSTWISNTSVQGSNSTGSNDSLILPCWIKQIINMWQRIGQAMYAPPIQGVIRCVS NITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRTV GSHSGSGGSGSGGHAAAGIGASSDGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSN LLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNS SWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLW NWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 28);(ab) BG505_MD39_congly_N332B54_mAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNFTLNLTSMMRGE LKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQA CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSL AEEEVIIRSENITNNAKNILVQLNTSVQINCTRPSNNTVKSIRIGPGQAFYYTGDIIGHVRM AHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNT SGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNIT GLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGR RRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQH LLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEI WDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 29);(ac) BG505_MD39.3_N332B54_gpl51_m2AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNFTLNLTSMMRGE LKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQA CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSL AEEEVIIRSENITNNAKNILVQLNTSVQINCTRPSNNTVKSIRIGPGQAFYYTGDIIGHVRM AHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNT SGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNIT GLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSW SNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWN WFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 30);(ad) 001428 MD39 L14 N332B50 m2VENLWVTVYYGVPVWKEARTTLFCASDAKAYETEVHNVWATHACVPTDPNPQEMVL GNVTENFNMWKNDMVDQMHEDVISLWAQSLKPCVKLTPLCVTLECTNFTLNLTSMMR GELKNCSFNTTTEIRDKKQKAYALFYRLDLVPLERENRGDSNSASKYILINCNTSAITQA CPKVNFDPIPIHYCTPAGYAILKCNNKTFNGTGSCNNVSTVQCTHGIKPVVSTQLLLNGS LAEEEIIIRSENLTDNVKTIIVHLDQSVEIVCTRPSNNTVKSIRIGPGQTFYYTGDIIGHVRM AHCNISEKKWHEMLRRVSEKLAEHFPNKTIKFTSSSGGDLEITTHSFNCRGEFFYCNTSG LFNSTYMPNGTYMPNGTNNSNSTLILPCRIKQIINMWQEVGRAMYAPPIAGNITCNSNIT GLLLVRDGGKNNNTEIFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTRCKRRVVGSH SGSGGSGSGGHAAVGLGAVSLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLQ APEPQQHLLQDTHWGIKQLQTRVLAIEHYLKDQQLLGIWGCSGKLICCTAVPWNSSWS NKSLTDIWDNMTWMQWDREVSNYTGIIYRLLEDSQNQQERNEQDLLALD (SEQ ID NO: 31);(ae) 001428_MD39.6_N332B 50_gp 151 _m2 VENLWVTVYYGVPVWKEARTTLFCASDAKAYETEVHNVWATHACVPTDPNPQEMVL GNVTENFNMWKNDMVDQMHEDVISLWAQSLKPCVKLTPLCVTLECTNFTLNLTSMMR GELKNCSFNTTTEIRDKKQKAYALFYRLDLVPLERENRGDSNSASKYILINCNTSAITQA CPKVNFDPIPIHYCTPAGYAILKCNNKTFNGTGSCNNVSTVQCTHGIKPVVSTQLLLNGS LAEEEIIIRSENLTDNVKTIIVHLDQSVEIVCTRPSNNTVKSIRIGPGQTFYYTGDIIGHVRM AHCNISEKKWHEMLRRVSEKLAEHFPNKTIKFTSSSGGDLEITTHSFNCRGEFFYCNTSG LFNSTYMPNGTYMPNGTNNSNSTLILPCRIKQIINMWQEVGRAMYAPPIAGNITCNSNIT GLLLVRDGGKNNNTEIFRPGGTDMRDNWRSELYKYKVVEIKPLGVAPTRCKRRVVGSH SGSGGSGSGGHAAVGLGAVSLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLQ APEPQQHLLQDTHWGIKQLQTRVLAIEHYLKDQQLLGIWGCSGKLICCTAVPWNSSWS NKSLTDIWDNMTWMQWDREVSNYTGIIYRLLEDSQNQQERNEQDLLALDSWKNLWS WFDISNWLWYIKIFIMIVGGLIGLRIIFAVLSIVNRVR (SEQ ID NO: 32);(af) SF162P3_MD64_B20.1_mCVEKLWVTVYYGVPAWKEATTTLFCASDAKAYDTEVHNVWATHECVPTDPNPQEIVLE NVTENFNMWKNNMVEQMHEDIIELWDQSLEPCVKLTPLCVTLHCTNLENATNTTSSNW KSMMRGEIKNCSFNVTTSIGNKMQKEYALFYRLDVVPIDNDNTSYNLINCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNDKKFNGSGPCINVSTVQCTHGIRPVVSTQLLLNGSLA EEGVURSENFTDNVKTIIVQLKESVEFNCTRPNNNTVKSIPIGPGKAFYYTGDIIGDIRMAH CNISGEKWNNTLKQIVTKLQAQFENKTIVFKQSSGGDPEIVMHSFNCGGEFFYCNSTQLF NSTWNNTIGPNNTNGTITLPCRIKQIINRWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDG GREVGNTTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKCKRRVVQRRRRRRAV TLGAVSLGFLGAAGSTMGAASLTLTVQARQLLSGIVQQQNNLLRAPEPQQRLLQLTVW GIKQLQARVLAVEHYLKDQQLLGIWGCSGKLICCTAVPWNASWSNKSLDQIWNNMTW MEWEREIGNYTNLIYTLIEESQNQQEKNEQELLELD (SEQ ID NO: 33);(ag) SF162P3_MD64.3_gpl51_B20.1VEKLWVTVYYGVPAWKEATTTLFCASDAKAYDTEVHNVWATHECVPTDPNPQEIVLE NVTENFNMWKNNMVEQMHEDIIELWDQSLEPCVKLTPLCVTLHCTNLENATNTTSSNW KSMMRGEIKNCSFNVTTSIGNKMQKEYALFYRLDVVPIDNDNTSYNLINCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNDKKFNGSGPCINVSTVQCTHGIRPVVSTQLLLNGSLA EEGVIIRSENFTDNVKTIIVQLNESVEINCTRPNNNTVKSIPIGPGKAFYYTGDIIGDIRMAH CNISGEKWNNTLKQIVTKLQAQFENKTIVFKQSSGGDPEIVMHSFNCGGEFFYCNSTQLF NSTWNNTIGPNNTNGTITLPCRIKQIINRWQEVGKAMYAPPIRGQIRC S SNITGLLLTRDG GREVGNTTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKCKRRVVQSHSGSGGS GSGGHAAVTLGAVSLGFLGAAGSTMGAASLTLTVQARQLLSGIVQQQNNLLRAPEPQQ RLLQLTVWGIKQLQARVLAVEHYLKDQQLLGIWGCSGKLICCTAVPWNASWSNKSLD QIWNNMTWMEWEREIGNYTNLIYTLIEESQNQQEKNEQELLELDKWASLWNWFDISK WLWYIKIFIMIVGGLVGLRIVFTVLSIVNRVR (SEQ ID NO: 34);(ah) 10E8-GT1NAMQGIHFRRHYVRHLPKEVSQNDIIKALASPLINDGMVVSDFADHVITREQNFPTGLPV EPVGVAIPHTDSKYVRQNAISVGILAEPVNFEDAGGEPDPVPVRVVFMLALGNWADITN VLWWIKAVIQDEDFMQQLLVMNDDEIYQSIYTRISELE (SEQ ID NO: 35);(ai) 10E8-GT2NAMQGIHFRRHYVRHLPKEVSQNDIIKALASPLINDGMVVSDFADHVITREQNFPTGLPV EPVGVAIPHTDGAYVRQNAISVGILAEPVNFEDAGGEPDPVPVRVVFMLALGNWDDITN VLWWIKAVIQDEDFMQQLLVMNDDEIYQSIYTRISELE (SEQ ID NO: 36);(aj) 10E8-GT3.1NAMQGIHFRRHYVRHLPKEVSQNDIIKALASPLINDGMVVSDFADHVITREQNFPTGLPV EPVGVAIPHTDQAYVRQNAISVGILAEPVNFEDAGGEPDPVPVRVVFMLALGNWDDITN VLWWIKAVIQDEDFMQQLLVMNDDEIYQSIYTRISELE (SEQ ID NO: 37);(ak) 10E8-GT3.2NAMQGIHFRRHYVRHLPKEVSQNDIIKALASPLINDGMVVSDFADHVITREQNFPTGLPVEPVGVAIPHTDGAYVRQNAISVGILAEPVNFEDMGGEPDPVPVRVVFMLALGNWDDIT NVLWWIKAVIQDEDFMQQLLVMNDDEIYQSIYTRISELE (SEQ ID NO: 38);(al) 10E8-GT4NAMQGIHFRRHYVRHLPKEVSQNDIIKALASPLINDGMVVSDFADHVITREQNFPTGLPVEPVGVAIPHTDGEYVRQNAISVGILAEPVNFEDATGEPDPVPVRVVFMLALSNWDDITN VLYWIKAVIQDEDFMQQLLVMNDDEIYQSIYTRISELE (SEQ ID NO: 39);(am) 10E8-GT5NAMQGIHFRRHYVRHLPKEVSQNDIIKALASPLINDGMVVSDFADHVITREQNFPTGLPV EPVGVAIPHTLGEYVRQNAISVGILAEPVNFEDMSGEPDPVPVRVVFMLALSTWDDITN VLYWIKAVILDEDFMQQLLVMNDDEIYQSIYTRISELE (SEQ ID NO: 40);(an) 10E8-GT7NAMQGIHFRRHYVRHLPKEVSQNDIIKALASPLINDGMVVSDFADHVITREQNFPTGLPV EPVGVAIPHTLGEYVRQNAISVGILAEPVNFEDMSGEPDPVPVRVVFMLAIRTWDDITNV LYWIKAVILDEDFMQQLLVMNDDEIYQSIYTRISELE (SEQ ID NO: 41);(ao) T298EVSQNDIIKALASPLINDGMVVSDFADHVITREQNAPTGLPVEPVGVAIPHTDSKYVRQN AISVGILAEPVNFEDAGGEPDPVPVRVVFMLALGNWFDITNVLWWIMDVIQDADFMQQ LLVMNDDEIYQSIYTRISEAAGMAGIHFRRHYVRHLPLE (SEQ ID NO: 42); or(ap) MPER-B1NVSQEDIIRALAEPLIDDGMVEKEFADHVIEREKQTPTGLQAEPVGVAIPHTMGEYVREN AISVGILTKPVNFTGWYQSEEPVPVRVVFMLAIRNWFDITNVLNWIKRVIQDRDFMRRL LTMNDEEIYEEIYKKIKQAPNLTGIHFTKKYVRHLNGTK (SEQ ID NO: 53).

2. The protein of claim 1 , comprising the sequence of any one of the sequences of (a)-(o).

3. The protein of claim 1, comprising the sequence of any one of the sequences of(p)-(ao).

4. The protein of claim 1, comprisng the sequence of NVSQEDIIRALAEPLIDDGMVEKEFADHVIEREKQTPTGLQAEPVGVAIPHTMGEYVREN AISVGILTKPVNFTGWYQSEEPVPVRVVFMLAIRNWFDITNVLNWIKRVIQDRDFMRRL LTMNDEEIYEEIYKKIKQAPNLTGIHFTKKYVRHLNGTK (SEQ ID NO: 53).

5. A protein having at least 90% sequence identity with the sequence of the protein of any one of claims 1-4.

6. A protein having at least 95% sequence identity with the sequence of the protein of any one of claims 1-4.

7. A multimeric protein comprising the protein of any one of claims 1-6.

8. The protein of any one of claims 1-6 further comprising an additional cysteine.

9. The protein of claim 7 fused to a multimerization motif.

10. The protein of any one of claims 1-9, wherein the protein is formulated in a nanoparticle.

11. The protein of claim 10, wherein the nanoparticle is a lipid nanoparticle.

12. The protein of claim 10, wherein the nanoparticle is a saponin / monophosphryl lipid A nanoparticle (SMNP).

13. The protein of any one of claims 10-12, wherein the nanoparticle has the sequence of:(aq) 10E8-GT9.2 12mer NGSVLILNGPNLNLLGRREPEVYGNTTLEELNASAEAWGAELGLGVVFNQTNYEGQLIE WVQNASQEGFLAIVLNPGALTHYSYALLDAIRAQPLPVVEVHLTNLHAREEFRRHSVTA PAARGIVSGFGPLSYKLALVYLAETLEVGGEGFGGSNGSGAKFVAAWTLKAAAGGSNVTQEDIIRALASPLIKDGMVDEDFAEHVIEREKRVPTGLPVKGVGVAIPHTLGEYVRDNAI SVGILDKPVNFSGWYQSPDPVPVRVVFMLAGRTWDDIVNVLKWIKDVILDEEFMKRLL NMSDEEIYRQIYTRISKAPNLSGINFSREYVRHLNGTK (SEQ ID NO: 70);(ar) 10E8-GT9KO 12merNGSVLILNGPNLNLLGRREPEVYGNTTLEELNASAEAWGAELGLGVVFNQTNYEGQLIE WVQNASQEGFLAIVLNPGALTHYSYALLDAIRAQPLPVVEVHLTNLHAREEFRRHSVTA PAARGIVSGFGPLSYKLALVYLAETLEVGGEGFGGSNGSGGGSNVTQEDIIRALAAPLIK DGMVDEDFAEHVIEREKREPTGLPVKGVGVAIPHTLGEYVRDNAISVGILDKPVLFEGWYQSPDPVPVRVVFMLAGRTARDIRNVLEWIDDVILDEEFMKRLLTMSDEEIYRQIYAIIA KAPGMRGIHFKLEYVRHLNGTK (SEQ ID NO: 71);(as) 10E8-GT10.2 12merNGSVLILNGPNLNLLGRREPEVYGNTTLEELNASAEAWGAELGLGVVFNQTNYEGQLIE WVQNASQEGFLAIVLNPGALTHYSYALLDAIRAQPLPVVEVHLTNLHAREEFRRHSVTA PAARGIVSGFGPLSYKLALVYLAETLEVGGEGFGGSNGSGAKFVAAWTLKAAAGGSNV TQEDIIRALASPLIKDGMVDEDFAEYVIARENRSPTGLQAKGVGVAIPHTLGDYVRDNAIS VGILDKP VNF SGWYQ SPDP VP VRVVFML AGRTWDDIVIVLKWIKD VILDEEFMKRLLN MSDEEIYRQIYTRISKAPNLSGINFSREYVRHLNGTK (SEQ ID NO: 72);(at) 10E8-GT10.3 12merNGSVLILNGPNLNLLGRREPEVYGNTTLEELNASAEAWGAELGLGVVFNQTNYEGQLIE WVQNASQEGFLAIVLNPGALTHYSYALLDAIRAQPLPVVEVHLTNLHAREEFRRHSVTA PAARGIVSGFGPLSYKLALVYLAETLEVGGEGFGGSNGSGAKFVAAWTLKAAAGGSNV TQEDIIRALASPLIKDGMVDEDFAEYVIAREDRSPTGLQAKGVGVAIPHTLGDYVRDNAIS VGILDKP VNF SGWYQ SPDP VP VRVVFML AGRTWDDIVIVLKWLKD VILDEEFMKRLL NMSDEEIYRQIYTRISKAPNLSGINFSREYVRHLNGTK (SEQ ID NO: 73);(au) 10E8-GT12 12merNGSVLILNGPNLNLLGRREPEVYGNTTLEELNASAEAWGAELGLGVVFNQTNYEGQLIE WVQNASQEGFLAIVLNPGALTHYSYALLDAIRAQPLPVVEVHLTNLHAREEFRRHSVTA PAARGIVSGFGPLSYKLALVYLAETLEVGGEGFGGSNGSGAKFVAAWTLKAAAGGSNV TQEDIIRALASPLIKDGMVDEDFAEKVIENESRYPSGLQAKGVGFAIPHVLGDYVRDNAIS VGILDKP VNF SEW YQ SPDP VP VRVVFML AIRT WDDITNVMNWIKD VVLDEEFMKRLL NMSDEEIYRQIYTRISKAPNLSGINFSREYVRHLNGTK (SEQ ID NO: 74);(av) 10E8-GT1224merNVTQEDIIRALASPLIKDGMVDEDFAEKVIENESRYPSGLQAKGVGFAIPHVLGDYVRDN AISVGILDKPVNFSEWYQSPDPVP VRVVFML AIRTWDDITNVMNWIKDVVLDEEFMKR LLNMSDEEIYRQIYTRISKAPNLSGINFSREYVRHLNGTKGGSATPHFDYIASEVSKGLADLGGSNGSVLILNGPNLNLLGRREPEVYGNTTLEELNASAEAWGAELGLGVVFNQTNYEGQLIEWVQNASQEGFLAIVLNPGALTHYSYALLDAIRAQPLPVVEVHLTNLHAREEFRRHSVTAPAARGIVSGFGPLSYKLALVYLAETLEVGGEGFGGSNGSGATPHFDYIASEVSKGLADLGGSFGVITADTLEQAIEGGSNVTQEDIIRALASPLIKDGMVDEDFAEKVIENESRYPSGLQAKGVGFAIPHVLGDYVRDNAISVGILDKPVNFSEWYQSPDPVPVRVVFMLAIRTWDDITNVMNWIKDVVLDEEFMKRLLNMSDEEIYRQIYTRISKAPNLSGINFSREYVRHLNGTK (SEQ ID NO: 75); or(aw) 10E8-GT10.2 60merNVTQEDIIRALASPLIKDGMVDEDFAEYVIARENRSPTGLQAKGVGVAIPHTLGDYVRDNAISVGILDKPVNFSGWYQSPDPVPVRVVFMLAGRTWDDIVIVLKWIKDVILDEEFMKRLLNMSDEEIYRQIYTRISKAPNLSGINFSREYVRHLNGTGGSGAKFVAAWTLKAAAGGSNGSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVRHGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLR (SEQ ID NO: 76).

14. A nucleic acid encoding the protein of any one of claims 1-13.

15. A nucleic acid having at least 90% homology or identity with the sequence of the nucleic acid of claim 14.

16. A nucleic acid having at least 95% homology or identity with the sequence of the nucleic acid of claim 14.

17. The nucleic acid of any one of claims 14-16, wherein the nucleic acid is formulated in a nanoparticle.

18. The nucleic acid of claim 17 wherein the nanoparticle is a lipid nanoparticle (LNP).

19. The nucleic acid of claim 17, wherein the nanoparticle is a saponin / monophosphryl lipid A nanoparticle (SMNP).

20. A non-naturally occurring mRNA encoding the protein of any one of claims 1-13.

21. A non-naturally occurring mRNA encoding a protein having at least 90% homology or identity with the sequence of the protein of any one of claims 1-13.

22. A non-naturally occurring mRNA encoding a protein having at least 95% homology or identity with the sequence of the protein of any one of claims 1-13.

23. The mRNA of any one of claims 20-24 wherein the mRNA is formulated in a nanoparticle.

24. The mRNA of claim 23 wherein the nanoparticle is a lipid nanoparticle (LNP).

25. A method for eliciting an immune response comprising systemically administering to an animal an effective amound of the protein of any one of claims 1-13, the nucleic acid of any one of claims 14-19, or the mRNA of any one of claims 20-24.

26. The method of claim 25, wherein the animal is a mammal.

27. The method of claim 26, wherein the mammal is a human.

28. The method of any one of claims 25-27, comprising administration of an additional booster dose.

29. The method of claim 28, wherein the booster dose is from a HIV pseudo virus (PSV).

30. The protein of any one of claims 1-13, the nucleic acid of any one of claims 15-19, or the mRNA of any one of claims 20-24 for use in a method of treating HIV in an animal comprising administering the protein, the nucleic acid, or the mRNA to the animal.

31. The protein of any one of claims 1-13, the nucleic acid of any one of claims 15-19, or the mRNA of any one of claims 20-24 for use according to claim 30, wherein the animal is a mammal.

32. The protein of any one of claims 1-13, the nucleic acid of any one of claims 15-19, or the mRNA of any one of claims 20-24 for use according to claim 31, wherein the animal is a mammal.

33. The protein of any one of claims 1-13, the nucleic acid of any one of claims 15-19, or the mRNA of any one of claims 20-24 for use according to any one of claims 30-32, further comprising administration of an additional booster dose.

34. The protein of any one of claims 1-13, the nucleic acid of any one of claims 15-19, or the mRNA of any one of claims 20-24 for use according to claim 33, wherein the booster dose is from a HIV pseudo virus (PSV).

35. A non-naturally occurring protein comprising the sequence of:(I) T298v2_RSFEVTQEDIIRALAAPLIKDGMVDEDFAEHVIEREKREPTGLPVKGVGVAIPHTDSKYVRDN AISVGILDKPVLFEDASGSPDPVPVRVVFMLALGNWFDITNVLWWIKAVIQDEEFMKRL LTMSDEEIYRQIYAIIAKAPGMRGIHFKLEYVRHLVLE (SEQ ID NO: 43);(II) 10E8-GT8.1EVTQEDIIRALAAPLIKDGMVDEDFAEHVIEREKREPTGLPVKGVGVAIPHTLGEYVRDN AISVGILDKPVLFEDMMGSPDPVPVRVVFMLAIRTWDDITNVLYWIKAVILDEEFMKRL LTMSDEEIYRQIYAIIAKAPGMRGIHFKLEYVRHLVGTK (SEQ ID NO: 44);(III) 10E8-GT8.2NVTQEDIIRALAAPLIKDGMVDEDFAEHVIERENRSPTGLPVKGVGVAIPHTLGEYVRDNAISVGILDKPVLFEDMNGSPDPVPVRVVFMLAIRTWDDITNVLYWIKAVILDEEFMKRLLNMSDEEIYRQIYAIIAKAPGMRGIHFKLEYVRHLVGTK (SEQ ID NO: 45);(IV) 10E8-GT9.1NVTQEDIIRALAAPLIKDGMVDEDFAEHVIERENRSPTGLPVKGVGVAIPHTLGEYVRDNAISVGILDKPVLFEGWYQSPDPVPVRVVFMLAGRTWDDIVIVLKWIKDVILDEEFMKRL LNMSDEEIYRQIYAIIAKAPGMRGIHFKLEYVRHLVGTK (SEQ ID NO: 46);(V) 10E8-GT9.2NVTQEDIIRALASPLIKDGMVDEDFAEHVIEREKRVPTGLPVKGVGVAIPHTLGEYVRDNAISVGILDKPVNFSGWYQSPDPVPVRVVFMLAGRTWDDIVNVLKWIKDVILDEEFMKRL LNMSDEEIYRQIYTRISKAPNLSGINFSREYVRHLNGTK (SEQ ID NO: 47);(VI) 10E8-GT10.1NVTQEDIIRALASPLIKDGMVDEDFAEYVIERENRSPTGLQVKGVGVAIPHTLGEYVRDNAISVGILDKPVNFEGWYQSPDPVPVRVVFMLAGRTWDDIVNVLKWIKDVILDEEFMKRL LNMSDEEIYRQIYTRISKAPGMRGIHFKREYVRHLNGTK (SEQ ID NO: 48);(VII) 10E8-GT10.2NVTQEDIIRALASPLIKDGMVDEDFAEYVIARENRSPTGLQAKGVGVAIPHTLGDYVRDNAISVGILDKPVNFSGWYQSPDPVPVRVVFMLAGRTWDDIVIVLKWIKDVILDEEFMKR LLNMSDEEIYRQIYTRISKAPNLSGINFSREYVRHLNGTK (SEQ ID NO: 49);(VIII) 10E8-GT10.3NVTQEDIIRALASPLIKDGMVDEDFAEYVIAREDRSPTGLQAKGVGVAIPHTLGDYVRDNAISVGILDKPVNFSGWYQSPDPVPVRVVFMLAGRTWDDIVIVLKWLKDVILDEEFMKR LLNMSDEEIYRQIYTRISKAPNLSGINFSREYVRHLNGTK (SEQ ID NO: 50);(IX) 10E8-GT11NVTQEDIIRALASPLIKDGMVDEDFAEKVIENESRYPTGLQAKGVGVAIPHTLGDYVRD NAISVGILDKPVNFSGWYQSPDPVPVRVVFMLAIRTWDDITNVLNWIKDVILDEEFMKR LLNMSDEEIYRQIYTRISKAPNLSGINFSREYVRHLNGTK (SEQ ID NO: 51); or(X) 10E8-GT12 NVTQEDIIRALASPLIKDGMVDEDFAEKVIENESRYPSGLQAKGVGFAIPHVLGDYVRDN AISVGILDKPVNFSEWYQSPDPVPVRVVFMLAIRTWDDITNVMNWIKDVVLDEEFMKR LLNMSDEEIYRQIYTRISKAPNLSGINFSREYVRHLNGTK (SEQ ID NO: 52).

36. A protein having at least 90% sequence identity with the sequence of the protein of claim 35.

37. A protein having 95% sequence identity with the sequence of the protein of claim 35.

38. The protein of any one of claims 35-37, wherein the protein is a multimeric protein.

39. The protein of any one of claims 35-38, wherein the protein further comprises an additional cysteine.

40. The protein of any one of claims 35-39, wherein the protein comprises a multimeric protein fused to a multimerization motif.

41. The protein of any one of claims 35-40, wherein the protein is formulated in a nanoparticle.

42. The protein of claim 41, wherein the nanoparticle is a lipid nanoparticle.

43. The protein of claim 41, wherein the nanoparticle is a saponin / monophosphryl lipid A nanoparticle (SMNP).

44. The protein of claim 41, wherein the nanoparticle has the sequence:(XI) 10E8-B1 24mer NVSQEDIIRALAEPLIDDGMVEKEFADHVIEREKQTPTGLQAEPVGVAIPHTMGEYVREN AISVGILTKPVNFTGWYQSEEPVPVRVVFMLAIRNWFDITNVLNWIKRVIQDRDFMRRL LTMNDEEIYEEIYKKIKQAPNLTGIHFTKKYVRHLNGTKGGSATPHFDYIASEVSKGLAD LGGSNGSVLILNGPNLNLLGRREPEVYGNTTLEELNASAEAWGAELGLGVVFNQTNYE GQLIEWVQNASQEGFLAIVLNPGALTHYSYALLDAIRAQPLPVVEVHLTNLHAREEFRR HSVTAPAARGIVSGFGPLSYKLALVYLAETLEVGGEGFGGSNGSGATPHFDYIASEVSK GLADLGGSFGVITADTLEQAIEGGSNVSQEDIIRALAEPLIDDGMVEKEFADHVIEREKQT PTGLQAEPVGVAIPHTMGEYVRENAISVGILTKPVNFTGWYQSEEPVPVRVVFMLAIRN WFDITNVLNWIKRVIQDRDFMRRLLTMNDEEIYEEIYKKIKQAPNLTGIHFTKKYVRHLN GTK (SEQ ID NO: 77).

45. A nucleic acid encoding the protein of any one of claims 35-44.

46. A nucleic acid, wherein the protein encoded by the nucleic acid has at least 90% sequence identity with the protein of any one of claims 35-44.

47. A nucleic acid, wherein the protein encoded by the nucleic acid has at least 95% sequence identity with the protein of any one of claims 35-44.

48. The nucleic acid of any one of claims 45-47, wherein the nucleic acid is formulated in a nanoparticle.

49. The nucleic acid of claim 48, wherein the nanoparticle is a lipid nanoparticle (LNP).

50. A non-naturally occurring mRNA encoding the protein of any one of claims 35-44.

51. A non-naturally occurring mRNA, wherein the protein encoded by the mRNA has at least 90% sequence identity with the protein of any one of claims 35-44.

52. A non-naturally occurring mRNA, wherein the protein encoded by the mRNA has at least 95% sequence identity with the sequence of the protein of any one of claims 35-44.

53. The mRNA of any one of claims 50-52, wherein the mRNA is formulated in a nanoparticle.

54. The mRNA of claim 53, wherein the nanoparticle is a lipid nanoparticle (LNP).

55. A method for eliciting an immune response comprising systemically administering to an animal an effective amound of the protein of any one of claims 35-44, the nucleic acid of any one of claims 45-49, or the mRNA of any one of claims 50-54.

56. The method of claim 55, wherein the animal is a mammal.

57. The method of claim 56, wherein the mammal is a human.

58. The method of any one of claims 55-57, comprising administration of an additional booster dose.

59. The method of claim 58, wherein the booster dose is from a HIV pseudo virus (PSV).

60. The protein of any one of claims 35-44, the nucleic acid of any one of claims 45- 49, or the mRNA of any one of claims 50-54 for use in a method of treating HIV in an animal comprising administering the protein, the nucleic acid, or the mRNA to the animal.

61. The protein of any one of claims 35-44, the nucleic acid of any one of claims 45- 49, or the mRNA of any one of claims 50-54 for use according to claim 60, wherein the animal is a mammal.

62. The protein of any one of claims 35-44, the nucleic acid of any one of claims 45- 49, or the mRNA of any one of claims 50-54 for use according to claim 61 , wherein the animal is a mammal.

63. The protein of any one of claims 35-44, the nucleic acid of any one of claims 45- 49, or the mRNA of any one of claims 50-54for use according to any one of claims 60-62, further comprising administration of an additional booster dose.

64. The protein of any one of claims 35-44, the nucleic acid of any one of claims 45- 49, or the mRNA of any one of claims 50-54 for use according to claim 63, wherein the booster dose is from a HIV pseudo virus (PSV).

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