Vaccines for kaposi's sarcoma-associated herpesvirus

Abstract

Provided herein are compositions, systems, kits, and methods for immunizing a subject against Kaposi's sarcoma-associated herpesvirus (KSHV) with a composition: i) comprising a plurality of nanoparticles self-assembled from a plurality of fusion proteins that comprise: a) at least a portion of a Ferritin protein, and b) an immunogenic protein comprising at least a portion of: KSHV K8.1 protein, or ii) polynucleotides encoding said fusion proteins (e.g., human codon optimized mRNA sequences present in lipid nanoparticles). In other embodiments, provided herein are compositions, systems, and kits, and methods for immunizing a subject against KSHV employing a human codon optimized nucleic acid sequence (e.g., mRNA) encoding a KSHV K8.1 protein.

Description

[0001] VACCINES FOR KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS

[0002] The present application claims priority to U.S. provisional application serial number 63 / 673,232, filed July 19, 2024, which is herein incorporated by reference in its entirety.

[0003] This invention was made with government support under CA200422, CA251275, DE023926 and DE028521 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] FIELD

[0005] Provided herein are compositions, systems, kits, and methods for immunizing a subject against Kaposi's sarcoma-associated herpesvirus (KSHV) with a composition: i) comprising a plurality of nanoparticles self-assembled from a plurality of fusion proteins that comprise: a) at least a portion of a Ferritin protein, and b) an immunogenic protein comprising at least a portion of: KSHV K8.1 protein, or ii) polynucleotides encoding said fusion proteins (e.g., human codon optimized mRNA sequences present in lipid nanoparticles). In other embodiments, provided herein are compositions, systems, and kits, and methods for immunizing a subject against KSHV employing a human codon optimized nucleic acid sequence (e.g., mRNA) encoding a KSHV K8.1 protein.

[0006] BACKGROUND

[0007] Kaposi's sarcoma-associated herpesvirus (KSHV), also known as Human Herpesvirus-8, is one of the human oncogenic viruses. KSHV is associated with human malignancies such as KS, PEL, and MCD (3). The virus was first identified in a KS lesion of an acquired immunodeficiency syndrome (AIDS) patient in 1994 (4). While the KSHV -infected population is globally distributed, the occurrence of KS and KSHV-associated diseases is disproportionately enhanced under certain environmental factors, such as co-inf ection with other pathogens and compromised host immunity (5, 6). The prognosis of KSHV-related diseases, such as PEL, remains poor, with an overall survival of around 12 months despite clinical management or therapy (7). Given the high seroprevalence of up to 80% in specific geographic regions, an effective vaccine is a crucial and promising option to promote public health by preventing the spread of KSHV. The life cycle of KSHV includes lytic and latent phases. Upon infection, KSHV typically maintains latency and expresses only a limited number of viral genes to sustain persistent infection and evade host immune detection (8). The current standard treatment for KSHV-rclatcd diseases is not entirely effective, partially due to viral evasion mechanisms (9, 10).

[0008] SUMMARY

[0009] Provided herein are compositions, systems, kits, and methods for immunizing a subject against Kaposi's sarcoma-associated herpesvirus (KSHV) with a composition: i) comprising a plurality of nanoparticles self-assembled from a plurality of fusion proteins that comprise: a) at least a portion of a Ferritin protein, and b) an immunogenic protein comprising at least a portion of: KSHV K8.1 protein, or ii) polynucleotides encoding said fusion proteins (e.g., human codon optimized mRNA sequences present in lipid nanoparticles). In other embodiments, provided herein are compositions, systems, and kits, and methods for immunizing a subject against KSHV employing a human codon optimized nucleic acid sequence (e.g., mRNA) encoding a KSHV K8.1 protein.

[0010] In some embodiments, provided herein are compositions comprising: a plurality of fusion proteins, and / or a polynucleotide encoding the fusion protein, wherein each of the plurality of fusion proteins comprises: a) at least a portion of a Ferritin protein, and b) an immunogenic protein comprising at least a portion of Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1 protein, wherein the plurality of fusion proteins are self-assembled into a plurality of nanoparticles, and wherein the plurality of nanoparticles each display the immunogenic protein on their surfaces.

[0011] In particular embodiments, the compositions further comprises an adjuvant. In additional embodiments, the at least a portion of the Ferritin protein comprises at least 25 consecutive amino acids from a Ferritin protein (e.g., at least 25 ... 30 ... 35 ... 45 ... or 50 amino acids). In additional embodiments, the at least a portion of the Ferritin protein comprise at least 25 consecutive amino acids, or at least 100 consecutive amino acids, or at least about 150 consecutive amino acids, or the entire amino acid sequence, from an amino acid sequence selected from: SEQ ID NOs: 1, 2, 3, 4, and 5. In other embodiments, the at least a portion of the Ferritin protein comprises an amino acid sequence at least about 90% identical, or at least 95% identical, or at least 99% identical, or 100% identical, to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, and 5. In additional embodiments, at least a portion of the Ferritin protein comprises an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5 or SEQ ID NOs: 1, 2, 3, 4, and 5 with one or two conservative amino acid changes. In other embodiments, the at least a portion of the Ferritin protein is a hybrid protein comprising at least a portion of a bullfrog ferritin protein joined to at least a portion of a ferritin protein selected from the group consisting of a Helicobacter pylori ferritin protein and an Escherichia coli ferritin protein.

[0012] In some embodiments, the at least a portion of the KSHV K8.1 protein comprises at least 25, or at least 50, or at least 75 consecutive amino acids, or the entire amino acid sequence; from an amino acid sequence selected from: SEQ ID NOs: 8, 10, 11, and 13, or SEQ ID NOs: 8, 10, 11, 13 with one or two conservative amino acid changes or end deletions. In other embodiments, the fusion proteins further comprises a linker sequence. In particular embodiments, the fusion protein comprises an amino acid sequence as shown in SEQ ID NOs: 15 or 16, or SEQ ID NOs: 15 or 16 with one or two conservative amino acid changes or end deletions.

[0013] In particular embodiments, the polynucleotide comprises at least 24 or 35 or 60 consecutive nucleotides from any of SEQ ID NOs:6, 7, 9, 12, and 14, and wherein T if present is optionally replaced with U, and / or any of the nucleotides, including U, are replaced with a modified base. In other embodiments, the polynucleotide comprises RNA or DNA. In further embodiments, the polynucleotide further comprises or encodes: a 5' untranslated region (UTR), a 5’ cap, a 3' UTR, an IRES, a 3’ tailing sequence, or any combination thereof. In certain embodiments, the 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.

[0014] In some embodiments, the polynucleotide comprises at least one chemically modified nucleotide (e.g., 1 ... 5 ... 10 ... 20 ... or more). In additional embodiments, the at least one chemically modified nucleotide comprises a modified uracil. In further embodiments, the at least 60% of the uracils in the polynucleotide are chemically modified. In certain embodiments, the at least one chemically modified nucleotide comprises 5-methylcytosine or Nl- methylpseudouridine (mlT). In additional embodiments, the polynucleotide comprises a nucleotide sequence: i) having at least 75%, or 85%, or 95% identity to SEQ ID NOs: 6, 7, 9, 12, and 14 , or a complement or reverse complement thereof, wherein T, if present, may be replaced by U or a modified U, and / or any of the nucleotides, including U, are replaced with a modified base.

[0015] In certain embodiments, provided herein are methods of immunizing a subject comprising at least one of the following: a) administering at least a portion of the plurality of nanoparticles described above or herein to a subject such that an immune response to the immunogenic protein is produced in the subject, and / or b) administering the polynucleotides described above or herein (e.g., as mRNA), optionally present in an expression vector or delivery vehicle, such that the nanoparticles are expressed in the subject and such that an immune response to the immunogenic protein is product in the subject.

[0016] In some embodiments, the subject is a human. In other embodiments, the delivery vehicle comprises a lipid nanoparticle encapsulating the composition. In further embodiments, the lipid nanoparticle comprises a cationic lipid, a neutral and / or non-cationic lipid, a sterol, or any combination thereof.

[0017] In particular embodiments, provided herein are compositions comprising: an engineered polynucleotide encoding at least a portion of Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1 protein, wherein the polynucleotide is at least partially human codon optimized. In certain embodiments, the polynucleotide comprises DNA or RNA, and optionally wherein the DNA or RNA is fully or nearly fully human codon optimized. In some embodiments, the polynucleotide further comprises or encodes: a 5' untranslated region (UTR), a 5’ cap, a 3' UTR, a 3’ tailing sequence, or any combination thereof. In additional embodiments, the 5’ UTR, 3’ UTR, or both are heterologous to the polynucleotide encoding at least a portion of KSHV K8.1 protein. In particular embodiments, the 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.

[0018] In certain embodiments, the polynucleotide comprises at least one chemically modified nucleotide. In other embodiments, the at least one chemically modified nucleotide comprises a modified uracil. In additional embodiments, at least 60% (e.g., 60 ... 65% ... 75% or at least 95%) of the uracil in the polynucleotide encoding at least a portion of KSHV K8.1 protein are chemically modified. In other embodiments, the at least one chemically modified nucleotide comprises 5 -methylcytosine or N1 -methylpseudouridine (m I ). In further embodiments, the polynucleotide comprises a nucleotide sequence having: i) at least 75% sequence identity to SEQ ID NOs: 6, 7, 9, and 12, or a complement or reverse complement thereof, wherein T, if present, may be replaced by U, and / or ii) at least 50 consecutive nucleotides from SEQ ID NOs: 6, 7, 9, and 12, or a complement or reverse complement thereof, wherein T may be replaced by U. In certain embodiments, the at least a portion of KSHV K8.1 protein comprises: i) an amino acid sequence having at least 90% identity to SEQ ID NO:8, 10, 11, and 13 and / or ii) at least 12, at least 15, or at least 20, or at least 30 consecutive amino acids from SEQ ID NO: 8, 10, 11, and 13. In further embodiments, the at least a portion of KSHV K8.1 protein comprises: i) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 8, 10, 11, and 13, and / or ii) at least 12 or 15 or at least 30 consecutive amino acids from any one of SEQ ID NOs: 8, 10, 11, and 13.

[0019] In some embodiments, provided herein is a messenger ribonucleic acid (mRNA) comprising: i) a 5' untranslated region (UTR); ii) an open reading frame encoding at least a portion of Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1 protein; and iii) a 3' UTR, wherein the mRNA is at least partially human codon optimized. In further embodiments, the 5’ UTR, 3’ UTR, or both are heterologous to the open reading frame encoding at least a portion of KSHV K8.1 protein, and / or wherein the RNA is fully or nearly fully human codon optimized. In particular embodiments, the mRNA further comprises or encodes: a 5’ cap, a 3’ tailing sequence, or both. In further embodiments, the 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.

[0020] In other embodiments, the mRNA comprises at least one chemically modified nucleotide. In additional embodiments, the at least one chemically modified nucleotide comprises a modified uracil. In additional embodiments, the at least 60% of the uracil in the open reading frame encoding the at least a portion of KSHV K8.1 protein are chemically modified. In some embodiments, the at least one chemically modified nucleotide comprises 5-methylcytosine or N1 -methylpseudouridine (m IT).

[0021] In other embodiments, the mRNA comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:7 or 12, and / or comprises at least 15, or at least 25, consecutive nucleotides from SEQ ID NO: 7 or 12. In additional embodiments, the at least a portion of KSHV K8.1 protein comprises: i) an amino acid sequence having at least 90% identity to SEQ ID NOs:8, 10, 11, or 13, and / or ii) at least 12 or 15 consecutive amino acids from SEQ ID NOs:8, 10, 11, or 13.

[0022] In particular embodiments, provided herein are vaccines comprising: any of the polynucleotides above or herein, and at least one adjuvant, a delivery vehicle, a physiological buffer, or any combination thereof. In further embodiments, the delivery vehicle comprises a lipid nanoparticle encapsulating the composition or the mRNA. In other embodiments, the lipid nanoparticle comprises a cationic lipid, a neutral and / or non-cationic lipid, a sterol, or any combination thereof. In some embodiments, the non-cationic lipid comprises a phospholipid. In additional embodiments, the sterol comprises cholesterol or a modification or ester thereof. In other embodiments, the lipid nanoparticlc comprises a polyethylene glycol (PEG)-lipid conjugate. DESCRIPTION OF THE FIGURES

[0023] Figure 1. K8.1 vaccine generation. Figure 1 (A) HEK293T (left panel) or NIH3T3 (right panel) cells were transfected with plasmid for encoding K8.1 in original codon (K8.1) or human codon (K8.1 Hm). Cells transfected with empty vector (EV) was included as negative control. Transfected cells were lysed and analyzed by immunoblotting with anti-K8.1 and anti- Actin antibodies. (B) HEK293T cells transfected with plasmid as described in (A) were analyzed by flow cytometry with anti-K8.1 antibody (#4A4) and FITC anti-mouse antibody. (C) HEK293T cells were transfected with lipid nanoparticle carrying K8.1 mRNA. 24 hours later, the cells were lysed and analyzed as described in (A). (D) Immuno-dominant region of K8.1 fused with ferritin (K8.126'87-FT) was purified from cultured medium of transfected HEK293T cell. Purified protein in sample buffer were analyzed for their molecular weights by SDS-PAGE without ("-" lane) or with (“+” lane) boiling at 95°C for five minutes. 0.5 ug and 1 ug of BSA were included as standards for protein quantification. (E) Purified K8.126‘87-FT at 250 ug / ml concentration was loaded to 300 mesh carbon grid and negatively stained by 1% uranyl acetate. Stained grid was observed by transmission electron microscopy. (F) Purified K8.126‘87-FT was analyzed by immunoblotting with anti-K8.1 antibody.

[0024] Figure 2. K8.1 vaccine immunization and immune response in vivo. (A) Six Balb / c female mice were immunized with PBS or K8.1 mRNA vaccine at week 0, 3 and 6. Blood collection for serum isolation was performed at week 0, 2, 5 and 8. Anti-K8.1 antibody titers in mouse serum were measured by ELISA. Titers in PBS are shown as empty dots and those in K8.1 group is shown as colored solid dots. (B) Six balb / c female mice were immunized with FT or K8.126-87-FT and collected for blood samples at the same time points as described in (A). Anti- K8.1 antibody titers were determined and shown as described in (A). (C and D) Neutralization activity of serum from mouse described in (A) and (B) were determined by rKSHV.219 infectivity in HEK293T cells (C) or MCI 16 cells (D). Infectivity was determined by flow cytometry and normalized using control group (PBS immunization) as 100% infection. Data points from four mice were included in each group. (E) Splenocyte from immunized mouse as described in (A) and (B) were isolated at week 8. Splenocyte were ex vivo stimulated with K8.1 overlapping peptide pool in presence of Brefeldin A for 12 hours. Surface markers of CD3, CD4 and CD8 were stained using respective FACS antibodies before permeabilization and intracellular cytokines staining for IFN-y and TNF-a. Stained splenocyte were analyzed by flow cytometry. Figure 3. Recombinant MHV68-K-K8.1 generation and characterization. (A and B) MHV68 wild type (WT) BACmid was engineered by replacing M7 with KSHV K8.1 by Red- mediated recombination in E. coli GS1783. MHV68 wild type (WT) BACmid or -K8.1 BACmid were extracted and digested with Bglll. Digested BACmid were separated by 0.7% agarose gel electrophoresis. Fragment with M7 in MHV68-WT BACmid and fragment with K8.1 in MHV68-K-K8.1 BACmid are indicated in yellow dots on the gel (A). The fragments sizes are 5,300 bp and 4,535 bp, respectively, as shown in (B). (C) BHK-21 cells were transfected with BACmid described in (A). Cell lysates from transfected BHK-21 were analyzed by immunoblotting with anti-K8.1 (#4A4) and anti-Actin antibodies. (D) NIH3T3 cells were infected with MHV68-WT or -K-K8.1 and harvested at 3, 6, 12, 24 and 48 hours post infection (hpi) for genomic DNA extraction. DNA copies were quantified with real-time qPCR with primer pairs targeting MHV68 ORF50 and ORF73 (sequences provided in figures). Quantification result of mouse L8 gene was included as internal control for relative expression level determination. DNA copies were presented as relative copies to MHV68 WT 3 hpi and showed on top of bar charts. (E) Four Balb / c mice were challenged with 500 PFU / mice of MHV68-WT or -K-K8.1 for 7 days and 28 days and determined virus titer in lung and splenocyte by real-time qPCR with MHV68 ORF50 targeting primer. Quantification result of serial diluted pUC57-MHV68 ORF50 plasmid served as standard.

[0025] Figure 4. Protection effect of K8.1 immunization from MHV68-K-K8.1 challenge in vivo. Immunized mice as described in Fig. 3 were challenged with MHV68 WT or MHV68-K- K8.1 at week 9 with 104 PFU / mice. (A) One week post infection, lung tissue was harvested and MHV68 WT and MHV68-K-K8.1 titers were determined by real-time qPCR by primers targeting MHV68 ORF50. Titer was quantified based on Cq from serially diluted pUC57- MHV68 ORF50 plasmid as standard. (B) Splenocyte were harvested and serially diluted for coculturing with Vero E6 cells at four weeks post infection. Reactivated virus titer was quantified by plaque forming unit (PFU).

[0026] Figure 5A shows the amino acid sequence of Helicobacter pylori Ferritin protein (SEQ ID NO:1). Figure 5B shows the amino acid sequence of Escherichia coli Ferritin protein (SEQ ID NO:2).

[0027] Figures 6A shows the amino acid sequence of Rana catesbeiana (Bull Frog) Ferritin protein (SEQ ID NO:3). Figure 6B shows the amino acid sequence of H. pylori ferritin-bullfrog ferritin fusion (SEQ ID NO:4). Figure 7 shows the amino acid sequence of E. coli ferritin-bullfrog ferritin fusion (SEQ ID NO:5).

[0028] Figure 8 shows: A. KSHV K8.1 sequence full length (DNA template) (SEQ ID NO:6); B. KSHV K8.1 mRNA sequence full length (mRNA vaccine) (SEQ ID NO:7); C. KSHV K8.1 amino acid sequence (full length) (SEQ ID NO:8); D.KSHV K8.1 mRNA sequence (codes for 26-87 amino acids) (SEQ ID NO:9); E. KSHV K8.1 amino acid sequence (26-87 amino acids) (SEQ ID NO:10); F. KSHV K8.1 amino acid sequence (25-56 amino acids) (SEQ ID NO:11); G. KSHV K8.1 mRNA sequence (mRNA, codes for amino acids 1-56) (SEQ ID NO: 12); and H. KSHV K8.1 amino acid sequence (amino acids 1-56) (SEQ ID NO: 13).

[0029] Figure 9 shows: A. KSHV K8.1-Ferritin nucleic acid sequence (K8.1 (26-87 amino acids); Linker; Ferritin) (SEQ ID NO: 14); B. KSHV K8.1-Femtin amino acid sequence (K8.1 26-87 amino acids; Linker; Ferritin) (SEQ ID NO:15); and C. KSHV K8.1-Ferritin amino acid sequence (K8.1 25-56 amino acids; Linker; Ferritin) (SEQ ID NO: 16)

[0030] DETAILED DESCRIPTION

[0031] Provided herein are compositions, systems, kits, and methods for immunizing a subject against Kaposi's sarcoma-associated herpesvirus (KSHV) with a composition: i) comprising a plurality of nanoparticles self-assembled from a plurality of fusion proteins that comprise: a) at least a portion of a Ferritin protein, and b) an immunogenic protein comprising at least a portion of: KSHV K8.1 protein, or ii) polynucleotides encoding said fusion proteins (e.g., human codon optimized mRNA sequences present in lipid nanoparticles). In other embodiments, provided herein are compositions, systems, and kits, and methods for immunizing a subject against KSHV employing a human codon optimized nucleic acid sequence (e.g., mRNA) encoding a KSHV K8.1 protein.

[0032] In certain embodiments, the KSHV K8.1-Ferritin nanoparticles are provided in compositions that further comprises an adjuvant. Any suitable adjuvant may be employed. Examples of adjuvants include, but are not limited to, AddaVax, aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate.

[0033] The nanoparticle vaccine compositions herein may be introduced into a subject by any suitable route, such as intramuscular, intradermal, subcutaneous, subcutaneous, transdermal, or intravenous. In certain embodiments, the nanoparticle vaccine compositions are injected into the subject via intramuscular injection. The present disclosure is not limited by the type of Ferritin protein employed. In certain embodiments, the KSHV K8.1 protein (or immunogenic portion thereof) is joined (c.g., directly or via linker or other moiety) to at least 25 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, or at least 150 contiguous amino acids from an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, wherein the fusion protein is capable of self-assembling into nanoparticles. In certain embodiments, the KSHV K8.1 protein (or immunogenic portion thereof) is joined (e.g., directly or via linker or other moiety) to a protein that is at least 85%, at least 90%, at least 95%, at least 97% at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5. Amino acid sequences from representative ferritin proteins of the present disclosure are disclosed herein as SEQ ID NO:1 (H. pylori ferritin), SEQ ID NO:2 (E. coli ferritin), SEQ ID NO:3 (bullfrog ferritin), SEQ ID NO:4 (H. pylori ferritin-bullfrog ferritin fusion) and SEQ ID NO:5 (E. coli ferritin-bullfrog ferritin fusion).

[0034] In certain embodiments, sequences within the KSHV K8.1-Ferritin fusion protein are directly joined. In some embodiments, one may employ linkers, spacers or other types of sequences.

[0035] In certain embodiments, proteins of the present disclosure are encoded by nucleic acid molecules and are expressed by nucleic acid constructs. As used herein a nucleic acid construct is a recombinant expression vector (i.e., a vector linked to a nucleic acid molecule encoding a protein) such that the nucleic acid molecule can effect expression of the protein when the nucleic acid construct is administered to, for example, a subject or an organ, tissue or cell. The vector also enables transport of the nucleic acid molecule to a cell within an environment, such as, but not limited to, an organism, tissue, or cell culture. The nucleic acid construct can be DNA, RNA or variants thereof. The vector can be, for example, a DNA plasmid, a viral vector, or other vector. Examples of such vectors include, for example, cytomegalovirus (CMV), retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poliovirus, or any other DNA or RNA virus vector. The nucleic acid molecules herein may be functionally linked to a promoter. In certain embodiments, provided herein is a cell that includes the foregoing nucleic acid molecule.

[0036] In certain embodiments, the KSHV K8.1 protein employed in the fusion protein may be full-length (sec, c.g., Figure 8) or portions thereof that arc capable of eliciting an immune response in a subject. Examples of particular KSHV K8.1 proteins (and nucleic acid sequences encoding such proteins) arc shown in Figure 8.

[0037] In some embodiments, variants of such amino acid and nucleic acid sequences may be employed. A variant refers to a protein, or nucleic acid molecule, the sequence of which is similar, but not identical to, a reference sequence, wherein the activity of the variant protein (or the protein encoded by the variant nucleic acid molecule) is not significantly altered. These variations in sequence can be naturally occurring variations or they can be engineered through the use of genetic engineering technique known to those skilled in the ail. Examples of such techniques are found in Sambrook J, Fritsch E F, Maniatis T et al., in Molecular Cloning — A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, pp. 9.31-9.57), or in Current Protocols in Molecular- Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, both of which are incorporated herein by reference in their entirety. With regard to variants, any type of alteration in the amino acid, or nucleic acid, sequence is permissible so long as the resulting variant protein retains the ability to, for example, elicit neutralizing antibodies against an KSHV K8.1 virus. Examples of such variations include, but are not limited to, deletions, insertions, substitutions and combinations thereof. For example, with regard to proteins, it is well understood by those skilled in the art that one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10), amino acids can often be removed from the amino and / or carboxy terminal ends of a protein without significantly affecting the activity of that protein. Similarly, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids can often be inserted into a protein without significantly affecting the activity of the protein.

[0038] In some embodiments, the polynucleotides or mRNAs herein encoding the proteins herein, comprise at least one chemical modification or chemically modified base, nucleoside, or nucleotide. The chemical modifications may comprise any modification which is not naturally present in the nucleic acid sequences or any naturally-occurring modification of adenosine (A), guanosine (G), uridine (U), or cytidine (C) ribonucleosides. For example, a single polynucleotide or mRNA may include both naturally-occurring and non-naturally-occurring modifications. Chemical modifications may be located in any portion of the polynucleotide or mRNA molecule and the polynucleotide or mRNA molecule may contain any percentage of modified nucleosides (1-100%, such as at least 20% ... at least 40% ... or at least 60%). In some embodiments, every particular base or nucleoside may be modified (e.g., every uridine is a modified uridine). In some embodiments, at least 20%, or 50%, or 80% of any single nucleotide (e.g., uracil) in the of the polynucleotide or mRNA is chemically modified. In some embodiments, a particular modification is used for every particular type of nucleoside or base (e.g., every uridine is modified to a 1-mcthyl-pscudouridinc). Exemplary RNA modifications can be found in the RNA modification database (See, mods(dot)ma(dot)albany(dot)edu / home).

[0039] In some embodiments, the at least one chemical modification comprises a modified uridine residue. Exemplary modified uridine residues include, but are not limited to, pseudouridine, 1 -methylpseudouridine, 1 -ethylpseudo uridine, 2-thiouridine, 4'- thiouridine, 5- methyluridine, 2-thio-l -methyl- 1-deaza-pseudouridine, 2- thio-l-methyl-pseudouridine, 2-thio- 5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio- pseudo uridine, 4- methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-l-methyl-pseudouridine, 4- thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-0-methyl uridine.

[0040] In some embodiments, the at least one chemical modification comprises a modified cytosine residue. Exemplary nucleosides having a modified cytosine include 5-aza-cytidine, 6- aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4- methyl-cytidine, 5-methyl-cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 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- 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, 4-methoxy- 1-methyl-pseudoisocytidine, lysidine, a-thio- cytidine, 2'-O-methyl-cytidine, 5,2'-O-dimethyl-cytidine, N4-acetyl-2'-O-methyl-cytidine, N4,2'- O-dimethyl-cytidine, 5-formyl-2'-O-methyl-cytidine, N4,N4,2'-O-trimethyl-cytidine, 1-thio- cytidine, 2'-F-aracytidine, 2'-F-cytidine, and 2'-OH-aracytidine.

[0041] In some embodiments, the at least one chemical modification comprises a modified adenine residue. Exemplary nucleosides having a modified adenine include 2-amino-purine, 2,6- diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido- adenosine, 7-deaza- adenine, 7-deaza-8-aza- adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2- amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl- adenosine, 2-methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6- isopentenyl-adenosine, 2-methylthio-N6-isopentenyl-adenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6- glycinylcarbamoyl-adcnosinc, N6-thrconylcarbamoyl-adcnosinc, N6-mcthyl-N6- threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6-dimethyl- adenosine, N6-hydroxynoryalylcarbamoyl-adenosine, 2-methylthio-N6- hydroxynoryalylcarbamoyl-adcnosinc, N6-acctyl-adcnosinc, 7-mcthyl-adcninc, 2-mcthylthio- adenine, 2-methoxy-adenine, a-thio-adenosine, 2'-O-methyl-adenosine, N6,2'-O-dimethyl- adenosine, N6,N6,2'-O-trimethyl-adenosine, l,2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine (phosphate), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara- adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)- adenosine.

[0042] In some embodiments, the at least one chemical modification comprises a modified guanine residue. Exemplary nucleosides having a modified guanine include inosine, 1 -methylinosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wybutosine, peroxy wybutosine, hydroxy wybutosine, undermodified hydroxy wybutosine, 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano-7-deaza- guanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 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-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2- methyl-guanosine, N2,N2-dimethyl-guanosine, N2,7-dimethyl-guanosine, N2,N2,7-dimethyl- guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl- 6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-O-methyl-guanosine, N2-methyl-2'-O-methyl-guanosine, N2,N2-dimethyl-2'-O-methyl-guanosine, l-methyl-2'-O- methyl-guanosine, N2,7-dimethyl-2'-O-methyl-guanosine, 2'-O-methyl-inosine, l,2'-O-dimethyl- inosine, and 2'-O-ribosylguanosine (phosphate).

[0043] In certain embodiments, the nucleic acid sequences encoding the proteins described herein are present in lipid nanoparticles (e.g., for intravenous delivery to a human). Lipid nanoparticle compositions may include one or more cationic and / or ionizable lipids, phospholipids, neutral or non-cationic lipids, polyethylene glycol (PEG)-lipid conjugates, and / or sterols. In some embodiments, the lipid nanoparticle comprises a cationic lipid and / or ionizable lipid, a neutral or non-cationic lipid, and cholesterol. Cationic and / or ionizable lipids include, for example, amine-containing lipids that can be readily protonated and may have a positive or partial positive charge at physiological pH due to a pKa value between pH 5 and 8. The polar headgroup of the cationic lipids preferably comprises amine derivatives such as primary, secondary, and / or tertiary amines, quaternary ammonium, various combinations of amines, amidinium salts, or guanidine and / or imidazole groups as well as pyridinium, piperazine and amino acid headgroups such as lysine, arginine, ornithine and / or tryptophan. Cationic lipids include, but are not limited to, l ,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC),

[0044] 1.2-di-O-octadcccnyl-3-trimcthylammonium propane (DOTMA) and / or l,2-diolcoyl-3- trimethylammonium propane (DOTAP), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium bromide (DMRIE), didodecyl(dimethyl)ammonium bromide (DDAB), l,2-dioleyloxypropyl-3-dimethyl- hydroxyethyl ammonium bromide (DORIE), 3P-[N — (N\N'-dimethylamino- ethane)carbamoyl]cholesterol (DC-Chol) or dioleyl ether phosphatidylcholine (DOEPC). Ionizable lipids include, but are not limited to, l,2-dioleyloxy-3-dimethylamino-propane (DODMA).

[0045] In some embodiments, the lipid nanoparticle comprises a polyethylene glycol (PEG)-lipid conjugate. A PEG-lipid conjugate may include, but is not limited to, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG- modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-DMG (l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol), PEG-c-DOMG (R-3-[(co-methoxy poly(ethylene glycol)2000)carbamoyl)]-l,2-dimyristyloxlpropyl-3-amine), PEG-DMA (PEG-dimethacrylate), PEG-DLPE (l,2-didodecanoyl-sn-glycero-3-phosphoethanolamine-PEG), PEG-DMPE (PEG-

[0046] 1.2-dimyristoyl-sn-glycero-3-phosphoethanolamine), PEG-DPPC (PEG-dipalmitoyl phosphatidylcholine), PEG-N,N-di(tetradecyl)acetamide, or a PEG-DSPE (1, 2-distearoyl-sn- glycero-3-phosphoethanolamine-poly(ethylene glycol)) lipid. In some embodiments, the lipid nanoparticle comprises PEG-DMG and / or PEG-N,N-di(tetradecyl)acetamide.

[0047] The sterol may comprise cholesterol, fecosterol, ergosterol, campesterol, sitosterol, stigmasterol, brassicasterol or a sterol ester, such as cholesteryl hemisuccinate, cholesteryl sulfate, or any other derivatives of cholesterol. A neutral or non-cationic lipid may include one or more phospholipids. Phospholipids include a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety may include, but is not limited to, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2- lysophosphatidyl choline, and sphingomyelin. A fatty acid moiety may include, but is not limited to, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0048] Phospholipids suitable for use in the compositions may include, but arc not limited to, phosphatidylglycerol (PG) including dimyristoyl phosphatidylglycerol (DMPG) and 1,2- dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG); phosphatidylcholine (PC), including egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC), 1 ,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diarachidonoyl-sn-glycero-3-phosphocholine; phosphatidylethanolamine (PE) including 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine; phosphatidic acid (PA); phosphatidylinositol (PI); phosphatidylserine (PS); and sphingomyelin (SM).

[0049] The positively charged lipid structures described herein may also include other components typically used in the formation of vesicles (e.g., for stabilization). Examples of such other components includes, without being limited thereto, fatty alcohols, fatty acids, and / or any other pharmaceutically acceptable excipients which may affect the surface charge, the membrane fluidity and assist in the incorporation of the lipid into the lipid assembly.

[0050] EXAMPLE

[0051] EXAMPLE 1

[0052] KSHV vaccine platforms

[0053] In this example, we report the development of new vaccine platforms against KSHV infection and a chimeric MHV68 carrying the replacement of KSHV glycoprotein as a surrogate challenge virus for testing the efficacy of KSHV vaccines in an in vivo mouse model. Among KSHV virion glycoproteins, K8.1 is the most abundant envelope glycoprotein with the highest immunogenicity to mediate infection of human B cells and subsequent establishment of persistent infections. We developed two types of KSHV K8.1 vaccines: K8.1 mRNA-lipid nanoparticle (LNP) and K8.126-87-Ferri tin (FT) self-assembling nanoparticle vaccines. They both successfully induced strong humoral immune responses in immunized mice, whereas K8.1 mRNA LNP also strongly induced T cell responses.

[0054] The MHV68 M7 (gpl50) gene was replaced by the KSHV K8.1 gene via Red-mediated homologous recombination to generate chimeric MHV68-K-K8.1 virus. Recombinant MHV68- K-K8.1 established acute and latent infection in the lungs and spleens of infected mice, respectively. Mice immunized with K8.1 mRNA LNP or K8.126-87-FT showed a reduction of MHV68-K-K8.1 titer but not MHV68 wildtype (WT) titer in the lung. In addition, MHV68-K- K8.1 reactivation was also significantly reduced in K8.1 mRNA LNP-immunized mice. This example demonstrates two vaccines providing immunity against KSHV and a new surrogate MHV68 system to characterize the vaccine efficacy in vivo.

[0055] This example utilized a chimeric murine gamma herpesvirus 68 (MHV68) bearing K8.1 (MHV68-K-K8.1) as a challenge virus in a mouse model to evaluate the protective efficacy of K8.1 vaccines. By replacing a homologous gene on the MHV68 genome with KSHV K8.1, MHV68-K-K8.1 was able to establish acute infection and replication in mouse lung tissues and latent infection in mouse splenocytes. Immunization of mice with K8.1 mRNA-LNP and K8.1- FT vaccines induced robust immune responses, leading to reduced virus titers in lung tissue and impaired reactivation of viral titer in splenocytes upon challenge with MHV68-K-K8.1 virus.

[0056] RESULTS

[0057] Development of K8.1 vaccines

[0058] We developed mRNA vaccine and self-assembling nanoparticle vaccine carrying KSHV K8.1. We synthesized a human codon-optimized K8.1 sequence for cloning into the mRNA expression construct. We compared the expression level of K8.1 in the original viral codon and human codon in transfected HEK293T or NIH3T3 cells. Human codon-optimized K8.1 showed a higher expression level than the original viral codon (Fig. 1A). We also checked K8.1 surface expression by flow cytometry (Fig. IB) and fluorescence microscopy to replicate surface presentation, enabling recognition by immune cells after vaccine delivery. Additionally, we generated K8.1 mRNA with human codon via in vitro transcription (IVT and transfected HEK293T cells with mRNA to test for K8.1 expression). To deliver the mRNA into cells, it was encapsulated in lipid nanoparticle (LNP), and the delivery efficiency was analyzed via immunoblotting of HEK293T cell lysates transfected with mRNA-LNP, demonstrating efficient expression of K8.1 (Fig. 1C).

[0059] In addition to the mRNA-LNP vaccine, we generated an FT-based K8.1 nanoparticle vaccine. We generated the immunodominant region (amino acid residues from 26 to 87) of K8.1 (16, 35) fused with ferritin (FT) (K8.126-87-FT) in a mammalian expression vector for protein vaccine purification from transiently transfected HEK293T cells. K8.I26-87-FT nanoparticle was purified via ion exchange chromatography from the transfected cell culture medium and evaluated for its purity by SDS-PAGE with or without boiling at 95°C. With boiling, the purified sample showed the expected molecular weight of K8.I26-87-FT monomer, including glycosylation on K8.1 (Fig. ID). Without boiling, the purified protein maintained the higher order of the expected 24mer, as demonstrated by minimal migration in SDS-PAGE (Fig. ID). To visualize the nanoparticle structure of purified K8.I26-87-FT, we performed negative staining and transmission electron microscopy, showing spherical nanoparticles (Fig. IE), as seen in our previous FT-based nanoparticles (24). Lastly, the presence and antigenicity of K8.1 from the purified K8.1 6-87 -FT nanoparticle were analyzed by immunoblotting with anti-K8.1 antibody (Fig. IF).

[0060] K8.1 vaccine immunization and host immune responses

[0061] To investigate whether K8.1 mRNA and K8.I26-87-FT vaccines induce immune responses, we immunized Balb / c mice with K8.1 vaccine at weeks 0, 3, and 6. Anti-K8.1 antibody titers in mouse sera were quantified by EEISA at weeks 0, 2, 5, and 8. Anti-K8.1 antibody titers increased immediately at weeks 2, 5, and 8 in K8.I26-87-FT immunized mice (Fig. 2B), whereas they robustly increased only at weeks 5 and 8 in K8.1 mRNA-immunized mice (Fig. 2A). To further examine whether anti-K8.1 antibodies in mouse serum show neutralization activity to block virus infection, we performed a neutralization assay using rKSHV.219 for the infection of HEK293T epithelial cells, or MCI 16 B cells. Because rKSHV.219-infected cells express GFP (36), we analyzed the neutralization activity by flow cytometry. Diluted sera from K8.1 mRNA-immunized mice or K8.126-87-FT-immunized mice reduced KSHV infectivity in MCI 16 B cells by approximately 30% or 40%, respectively, although they showed weak neutralizing activity in HEK293T epithelial cells (Fig. 2C, D). Neutralization activity from both vaccines was stronger in MCI 16 cells than in HEK293T cells, which may be attributable to a difference in the virological significance of K8.1 in KSHV infection (17, 18). In addition, neutralization activity was slightly higher in K8.126-87-FT-immunized mice sera, implying that the FT-bascd vaccine induces a more potent antibody response than mRNA vaccine.

[0062] In addition to the vaccine-induced humoral immunity, we also investigated T cell responses from splenocytes harvested at week 8. After 12 hours of ex vivo splenocyte stimulation with the K8.1 overlapping peptide (OLP) pool, we stained splenocytes with antibodies of T cell surface markers, including CD3, CD4 and CD8 to distinguish subtypes of T cells surface markers. We also performed intracellular antibody staining to investigate the expression levels of IFN-y and TNF-a, which play a critical role in the anti-viral response (37- 39). Cell populations were analyzed by flow cytometry. Among the CD3+T cells, IFN-y+or IFN-y+ / TNF-a+populations were significantly increased in CD8+T cells but showed slight enhancement in TNF-a+or IFN-y+ / TNF-a+in CD4+T cells, without significant difference in K8.1 mRNA-immunized group (Fig. 2E, left panel). Surprisingly, cytokine production did not significantly increase in the K8.126-87-FT-immunized group (Fig. 2E, right panel). Secreted IFN-y and TNF-a also showed similar result, whereas secreted IFN-y in the culture medium significantly increased in the K8.126-87-FT-immunized group. This suggests that although IFN-y production by T cells didn’t increase in the K8.126-87-FT-immunized group, there might be other cell types in the spleen secreting IFN-y after K8.1 overlapping peptide pool stimulation (38). Finally, the supernatants of OLP-stimulated splenocytes from immunized mice were subjected to a multiplex cytokine array. These results also showed that K8.1 mRNA LNP immunization, but not K8.126-87-FT-immunization, strongly induced the productions of many cytokines and chemokines upon K8.1 OLP stimulation, showing robust T cell responses. Comparing the K8.126-87-FT-immunization group to the K8.1 mRNA-immunization group, we found weaker T cell responses in the K8.126-87-FT-immunized group. This may be attributable to the differences between the antigens covered by K8.I26-87-FT and the full-length K8.1 peptide pool used for the ex vivo stimulation. This finding also corresponds to a previous study demonstrating that protein vaccines tend to induce a low T cell response (40).

[0063] Generation of MHV68-K-K8.1

[0064] To test for vaccine-induced immunity in vivo, we engineered a chimeric murine gammaherpesvirus, MHV68, by replacing M7 with KSHV K8.1 via Red-mediated homologous recombination, where MHV68 M7 is homologous to KSHV K8.1. We verified the resultant sequence by PCR with Sanger sequencing and confirmed the genome structure of the recombinant BACmid by restriction enzyme digestion. After Bglll digestion, the 5.3 kb band in MHV68-WT BACmid migrated to 4.5 kb in MHV68-K8.1 BACmid, as expected (Fig. 3A and 3B). To examine whether the engineered virus expressed KSHV K8.1, we utilized BHK-21 cells for BACmid transfection to produce the chimeric virus. Cell lysates showed K8.1 expression only upon transfection with the recombinant BACmid (Fig. 3C). We further amplified the chimeric virus in Vero E6 cells and observed that MHV68 ORF59 and ORF61 expressions were not affected by the BACmid recombination. In addition, we purified virions by sucrose gradient ultracentrifugation and demonstrated the intact virion structure under TEM. Immunoblotting of purified virions also showed that KSHV K8.1 was incorporated into the recombinant virion particles. Compared to MHV68 WT, MHV68-K-K8.1 showed lower replication efficiency in an in vitro cell culture system (Fig. 3D) and in an in vivo mouse infection model (Fig. 3E). Although recombinant virus MHV68-K-K8.1 showed a lower titer at 7 dpi in mouse lung (Fig. 3E, left panel), it successfully established latent infection in splenocytes (Fig. 3E, right panel). This indicates that while MHV68-K-K8.1 has lower efficiency in viral replication, it still retains the capability to establish latent viral infection in a mouse model.

[0065] K8.1 vaccines provide immunity against MHV68-K-K8.1 infection in vivo

[0066] Based on the efficient induction of humoral and cellular immunity upon immunization with K8.1 vaccine candidates (Fig. 2), we investigated if they provide strong immunity against MHV68-K-K8.1 infection. MHV68 WT was included as a negative control, lacking KSHV K8.1. Two weeks after the last immunization, mice were intranasally challenged with MHV68 WT or MHV68-K-K8.1 in serum-free DMEM (IxlO4PFU / mouse). After 4-7 dpi, mouse lungs were harvested to determined viral titers using real-time qPCR of genomic DNAs. K8.1 mRNA or K8.126-87-FT immunization considerably lowered MHV68-K-K8.1 DNA copy number (Fig. 4A). In addition, ORF61 immunohistochemistry (IHC) of the lung tissues from K8.1 -immunized mice showed the considerably reduced expression in MHV68-K-K8.1-infected mice but not in MHV68 WT-infected mice. To further test whether K8.1 vaccinations affect viral latency, we examined the reactivation frequency of MHV68 WT or MHV68-K-K8.1 in mouse splenocytes one month after virus infection. This showed that the K8.1 mRNA-immunized group showed a lower reactivation frequency of MHV68-K-K8.1 but not MHV68 WT (Fig. 4B). Interestingly, K8.126-87-FT immunization showed no effect on the reactivation frequency of either MHV68 WT or -K-K8.1 (Fig. 4B), which may correlate with the weaker T cell response of K8.I26-87-FT immunization (Fig. 2E). KSHV is a human oncogenic virus associated with KS and B cell lymphoma. Current treatments for KSHV-associatcd diseases include surgery, chemotherapy, immunotherapy, and anti-viral drug (10, 41); however, these treatments have failed to clear KSHV. This failure is attributed to KSHV’s manipulation of host gene expression (42) and mutations in the viral genome during long-term treatments (43, 44) which result in drug resistance. Given the limitation of current therapies, an effective method to block initial infection with KSHV is one of the most promising strategies to improve public health against KSHV.

[0067] In this study, we designed a KSHV vaccine targeting the most immunogenic glycoprotein, K8.1, using two different vaccine platforms: K8.1 mRNA-encapsulated LNP and K8.126-87-FT self- assembling nanoparticles. K8.1 is a glycoprotein expressed on the viral surface during the lytic phase of KSHV infection. Current studies show that K8.1 play a significant role in viral entry by associating with heparan sulfate (HS) (45, 46) and facilitating other glycoproteins, such as gB and gH / gL, to efficiently recognize their receptors on the host cell surface for viral entry (11, 12). Although KSHV utilizes multiple glycoproteins for the entry process, recent studies report that K8.1 has a cell type-specific contribution to KSHV infection (17, 18). Since K8.1 is one of the most immunogenic antigens among viral glycoproteins (15, 16), both mRNA and FT nanoparticle vaccines induced a strong antibody response in vivo (Fig. 2A-D). On the other hand, the K8.1 mRNA vaccine successfully induced T cell responses in immunized mice, whereas the K8.I26-87-FT vaccine induced only minimal T cell responses (Fig 2E). This may be associated with different K8.1 regions included in the two vaccine platforms; the mRNA vaccine covers the full length (228 amino acids) of K8.1, while the FT nanoparticle vaccine covers only the immunodominant 62 amino acids of K8.1 (16). Since full-length K8.1- FT was unstable, the segment of K8.1 comprising 26 aa to 87 aa, which covers a major immunodominant region, was fused with FT. While the present disclosure is not limited to any particular mechanism, and an understanding of the mechanism is not necessary to practice the invention, this suggests that the minimal T cell responses observed with the K8.I26-87-FT vaccine were due to only parts of the K8.1 overlapping peptide pool successfully stimulating splenocytes ex vivo from K8.126-87-FT-immunized mice.

[0068] Several studies have reported KSHV vaccine immunogenicity in in vivo mouse model (14, 28-31), where the neutralization activity of those vaccines was primarily measured by in vitro cell culture systems. In addition, the study of vaccine-mediated protection efficacy has been exclusively performed in humanized mouse model (23, 47, 48). While humanized mice can provide significant advancements for immune response research, they still have certain limitations. Even the most advanced humanized mice do not perfectly replicate a normal immune system. Their immune system often remain immature or functionally compromised compared to a fully developed human immune system. The level of human-derived cell reconstitution is low, and the lifespan of human-derived T cells is short. For these reasons, we developed chimeric MHV68 carrying the replacement of KSHV glycoprotein as a surrogate challenge virus to evaluate the immunogenicity and efficacy of KSHV vaccine candidates in an in vivo WT mouse model.

[0069] Using the MHV68 BAC, we created chimeric MHV68-K-K8.1, which carries the replacement of its M7 with the K8.1. Although the MHV68-K-K8.1 replicates at a slower rate, it retains the capability for lytic replication and establishes viral latency in infected mice. K8.1 mRNA or K8.I26-87-FT immunization considerably lowered the DNA copy numbers and viral gene expressions of MHV68-K-K8.1, but not MHV68 WT, in lung tissues (Fig.4A). On the other hand, reactivation virus titers were significantly lower in K8.1 mRNA-immunized group, but not in K8.126-87-FT-immunized group (Fig. 4B). This differences in reactivation frequency may be due to the stronger cellular immunity induced in the K8.1 mRNA vaccine-immunized group. Nevertheless, we demonstrate that MHV68-K-K8.1 serves as a surrogate virus to evaluate the effectiveness of KSHV vaccine candidates in a mouse model.

[0070] MATERIAL AND METHODS

[0071] Mice experiment

[0072] Five-week-old female BALB / c mice were purchased from Jackson laboratory. All mouse experiment were performed in accordance with Institutional Animal Care and Use Committee (lACUC)-approved protocol (Protocol number 2427). Animals were quarantined for 1 week before starting experiment and housed at Cleveland Clinic Lerner Research Institute’s Biological Resource Unit equipped with 12 hours dark / light cycle throughout the experiment.

[0073] Cell line

[0074] HEK293T, iSLK.219, NIH3T3, BHK-21, Vero E6 were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco) containing 10% fetal bovine serum (Gibco, FBS), 1% penicillin / streptomycin (Gibco) at 37°C and 5% CO2. MCI 16 cells was cultured in Roswell Park Memorial Institute (RPMI) 1640 (Gibco) supplied with 10% FBS, 1% penicillin / streptomycin at 37°C. For KSHV selection, puromycin (Gibco) was added into culture medium of iSLK.219 to the concentration of 10 ug / ml. For rKSHV.219 production, iSLK.219 were treated with doxycycline at the concentration of 1 ug / ml for five days. KSHV was harvested following a previous publication (30). MHV68 was amplified via infection of Vero E6 cells, and the supernatant was centrifuge to remove cell debris and harvest the virus. Vims was stored at -80°C until further use.

[0075] BACmid engineering

[0076] BACmid engineering was performed as described in a previous manuscript (50).

[0077] Plasmid

[0078] K8.1 original codon was PCR-amplified from pcDNA3-myc-K8.1 and subcloned to pZMV (20) vector (pZMV-K8.1). Full length K8.1 in human codon (K8.1 Hm) was synthesized (GeneScript) and cloned into pZMV (pZMV-K8.1 Hm) in identical method. For protein subunit vaccine, K8.I26-87-FT expression construct was PCR-amplified from pZMV-K8.1 Hm and subcloned into modified pFUSE (Invivogen) vector as described in our previous publication (24, 51). K8.126-198-FC expression construct was also PCR-amplified from pZMV-K8.1 Hm and subcloned into a commercially available pFUSE vector.

[0079] Transmission electron microscopy (TEM)

[0080] 2 ug of purified proteins were loaded to glow-discharged formvar film (Electron Microscopy Science) and incubated for one minute. Sample-adsorbed grids were washed by filtered 5 mM Tris-HCl (pH 7.0) and distilled water. Residual water was removed by blotting against filter paper and the grid was stained with 1% uranyl formate (Electron Microscopy Science) for 1 minute. Images were collected with FEI Tecnai G2 Spirit BioTwin TEM.

[0081] Virion purification with sucrose gradient ultracentrifugation

[0082] In short, Vero E6 cells were infected with MHV68 wild-type (WT) or MHV68-K-K8 for two to four days and until 90% cytopathic effect (CPE) was observed. Cell debris were remove and harvested viruses were loaded onto sucrose gradient mixture for separation by centrifugation at 24,000 rpm. K8.1 mRNA vaccine generation

[0083] K8.1 mRNA generation followed our previous study (20). In brief, K8.1 mRNA was generated from linearized pZMV-K8.1 (or pZMV-K8.1 Hm) by using T7 RNA synthesis kit (New England Biolabs). DNA template was digested after transcription by DNase I and RNA was purified by Monarch RNA cleanup kit (New England Biolabs). Linear mRNA was enzymatically polyadenylated with E. coli Poly (A) polymerase kit (New England Biolab) and encapsulated in LNP.

[0084] K8.126-87-FT vaccine and K8.126-i98-Fc purification

[0085] Briefly, HEK293T cells were transfected with protein expression construct carrying signal peptide for secretion into the culture media. After harvesting medium, cell debris were removed by centrifugation before further purification by Akta pure chromatography system (Cytiva) or protein A / G Agarose beads (Thermo Fisher).

[0086] Mouse immunization

[0087] Six to eight-week-old mouse were intramuscularly immunized with 1 ug of mRNA vaccine diluted in PBS or 1 ug protein vaccine mixed with equal volume of AddaVax adjuvant (InvivoGen). Mouse blood was collected through retro-orbital route and incubated at room temperature for one to three hours to remove blood clotting. Isolated serum was heated at 55°C for 30 minutes and kept in -20°C until further characterization. Eight weeks after the last immunization, splenocyte were isolated and frozen in FBS with 10% v / v Dimethyl Sulfoxide (MP biomedical; DMSO).

[0088] Mouse viral challenging with MHV68-K-K8.1

[0089] Mice was anesthetized by ketamine / xylazine (100 mg / kg; 10 mg / kg) mixture in PBS (Gibco) and intranasally inoculated with MHV68 WT or MHV68-K-K8.1 in serum-free DMEM (I X 104PFU / mice). One week after infection, a group of mice were sacrificed to harvest lung tissues and the tissues were separated into two fractions for freezing at -80°C and fixation with 4% paraformaldehyde. Fixed tissues were embedded in paraffin and sectioned. Remaining mice were sacrificed three to four weeks after viral challenge and splenocyte were isolated to made serial dilution for overnight co-culturing with Vero E6 cells and reactivation assay. Flow cytometry

[0090] Cells were harvested and washed in FACS buffer (PBS supplied with 2% FBS, 1 mM EDTA and 1% sodium azide). Cells were treated with Fc blocker (BD Biosciences) for 15 minutes and stained with FACS antibodies for 30 minutes at 4°C. After staining, cells were washed and resuspended in FACS buffer before applying to BD FACSCelesta (BD Biosciences). Result was analyzed by FlowJo™ vlO Software (BD Biosciences).

[0091] Enzyme-Linked Immunosorbent Assay (ELISA)

[0092] 96-well ELISA plate (Greiner) coated with purified K8.1-Fc antigen was blocked with 3% BSA. Diluted mouse serum was added for two hours binding and washed with PBS-T. HRP- conjugated mouse IgG was then added for 90 minutes incubation and washed with PBS-T. TMB Substrate (3,3 ',5,5' - tetramethylbenzidine; BD Biosciences) was applied to each well and stopped by sulfuric acid. Absorbance was measured with optical density (OD) at 450 nm. To determine concentration of secreted IFN-y and TNF-a, harvested media were applied to human IFN-y or TNF-a quantikine ELISA Kit (R&D Systems) accordingly to manufacturer’s suggested protocol.

[0093] Neutralization assay

[0094] HEK293T or MCI 16 cells were seeded in 96-well-plate 24 hours or 1 hour before assay respectively. Diluted serum from mouse was mixed with KSHV.219 and incubated at 37°C for 1 hour. 50 ul of the mixture was added into cells in 50 ul of culture media. 50 ul fresh culture medium was added in the next day and incubated for another day. Cells were then washed and fixed with 1 % paraformaldehyde (Sigma) in FACS buffer. Infectivity was determined by flow cytometry.

[0095] T cell response

[0096] Splenocyte were seeded in 96-well-plate and treated with K8.1 overlapping peptide pool in the presence of BD GolgiPlug (BD Biosciences). Cells were harvested after 12 hours stimulation and followed with surface staining and intracellular staining to characterize induction of cellular immunity via flow cytometry.

[0097] DNA extraction

[0098] Cell / Splenocyte Cells were resuspended in lysis buffer (NP-40 diluted to 1 % v / v in PBS) and incubated at 4°C for 15 minutes. Insoluble debris were removed by centrifugation at 21,000 g for 15 minutes. DNA was extracted by phenol: chloroform: isoamyl alcohol (pH 8), 25 : 24 : 1 in v / v (Sigma). Nucleic acid in aqueous phase was precipitated with 100% ethanol and washed by 75% ethanol to remove residual salt. DNA pellet was dissolved in nuclease-free water.

[0099] Lung

[0100] Lung tissues were thawed for DNA extraction by DNA extraction kit (IBI Scientific) accordingly to manufacture’s guideline.

[0101] IHC staining

[0102] In short, slides were incubated at 72°C overnight and subjected to the process of dewaxing, rehydration, and antigen presentation. Endogenous peroxidase activity was quenched with 3% H2O2 (sigma) before blocking. Tissues were then stained with primary antibody, MHV68 ORF61, followed by secondary antibody. Images of stained slides were captured with slide scanner Aperio AT2 (Leica) and analyzed by QuPath (52).

[0103] Real-time quantitative PCR (RT-qPCR)

[0104] DNA mix with 0.6 mmole primer and SsoAdvanced universal SYBR Green Supermix (Bio-Rad) in 15 ul. PCR reaction performed by CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with suggested protocol.

[0105] Statistical Analysis

[0106] All asterisks in figures indicate statistical significance between control and the respectively immunized group. Statistical analysis was performed by GraphPad Prism with student’s t test. (* indicates p<0.05, ** indicates p<0.01, *** indicates p<0.005, **** indicates p<0.001)

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[0156] All publications and patents mentioned in the specification and / or listed below are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the ail without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope described herein.

Claims

CLAIMSWc Claim:

1. A composition comprising: a plurality of fusion proteins, and / or a polynucleotide encoding said fusion protein, wherein each of said plurality of fusion proteins comprises: a) at least a portion of a Ferritin protein, and b) an immunogenic protein comprising at least a portion of Kaposi's sarcoma- associated herpesvirus (KSHV) K8.1 protein, wherein said plurality of fusion proteins are self-assembled into a plurality of nanoparticles, and wherein said plurality of nanoparticles each display said immunogenic protein on their surfaces.

2. The composition of Claim 1, wherein said composition further comprises an adjuvant.

3. The composition of Claim 1, wherein said at least a portion of said Ferritin protein comprises at least 25 consecutive amino acids from a Ferritin protein.

4. The composition of Claim 1, wherein said at least a portion of said Ferritin protein comprise at least 25 consecutive amino acids, or at least 100 consecutive amino acids, or at least about 150 consecutive amino acids, or the entire amino acid sequence, from an amino acid sequence selected from: SEQ ID NOs: 1, 2, 3, 4, and 5.

5. The composition of Claim 1, wherein said at least a portion of said Ferritin protein comprises an amino acid sequence at least about 90% identical, or at least 95% identical, or at least 99% identical, or 100% identical, to an amino acid sequence selected from SEQ ID NOs: 1,2, 3, 4, and 5.

6. The composition of Claim 1, wherein said at least a portion of said Ferritin protein comprises an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5 or SEQ ID NOs: 1, 2,3, 4, and 5 with one or two conservative amino acid changes.

7. The composition of Claim 1 , wherein said at least a portion of said Ferritin protein is a hybrid protein comprising at least a portion of a bullfrog ferritin protein joined to at least a portion of a ferritin protein selected from the group consisting of a Helicobacter pylori ferritin protein and an Escherichia coli ferritin protein.

8. The composition of Claim 1, wherein said at least a portion of said KSHV K8.1 protein comprises at least 25, or at least 50, consecutive amino acids, or the entire amino acid sequence; from an amino acid sequence selected from: SEQ ID NOs: 8, 10, 11, and 13, or SEQ ID NOs: 8, 10, 11, and 13 with one or two conservative amino acid changes or end deletions.

9. The composition of claim 1, wherein each of said fusion proteins further comprises a linker sequence.

10. The composition of claim 1, wherein said polynucleotide comprises at least 24 or 35 consecutive nucleotides from any of SEQ ID NOs:6, 7, 9, 12, and 14, and wherein T is optionally replaced with U, and / or any of the nucleotides, including U, are replaced with a modified base.

11. The composition of claim 1, wherein said polynucleotide comprises RNA.

12. The composition of claim 1, wherein the polynucleotide further comprises or encodes: a 5' untranslated region (UTR), a 5’ cap, a 3' UTR, an IRES, a 3’ tailing sequence, or any combination thereof.

13. The composition of claim 12, wherein the 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.

14. The composition of claim 1, wherein said polynucleotide comprises at least one chemically modified nucleotide.

15. The composition of claim 14, wherein the at least one chemically modified nucleotide comprises a modified uracil.

16. The composition of claim 15, wherein at least 60% of the uracils in the polynucleotide arc chemically modified.

17. The composition of claim 14, wherein the at least one chemically modified nucleotide comprises 5-methylcytosine or N1 -methylpseudouridine (mlT).

18. The composition of claim 1, wherein said polynucleotide comprises a nucleotide sequence: i) having at least 75%, or 85%, or 95% identity to SEQ ID NOs: 6, 7, 9, 12, or 14, or a complement or reverse complement thereof, wherein T may be replaced by U or a modified U, and / or any of the nucleotides, including U, are replaced with a modified base.

19. A method of immunizing a subject comprising at least one of the following: a) administering at least a portion of said plurality of nanoparticles of any of Claims 1-9 to a subject such that an immune response to said immunogenic protein is produced in said subject, and / or b) administering said polynucleotide of any of Claims 1-19, optionally present in an expression vector or delivery vehicle, such that said nanoparticles are expressed in said subject and such that an immune response to said immunogenic protein is product in said subject.

20. The method of claim 19, wherein said subject is a human.

21. The method of claim 19, wherein the delivery vehicle comprises a lipid nanoparticle encapsulating the composition.

22. The method of claim 21, wherein the lipid nanoparticle comprises a cationic lipid, a neutral and / or non-cationic lipid, a sterol, or any combination thereof.

23. A composition comprising: an engineered polynucleotide encoding at least a portion of Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1 protein, wherein the polynucleotide is at least partially human codon optimized.

24. The composition of claim 23, wherein the polynucleotide comprises RNA, and optionally wherein said RNA is fully or nearly fully human codon optimized.

25. The composition of claims 23 or 24, wherein the polynucleotide further comprises or encodes: a 5' untranslated region (UTR), a 5’ cap, a 3' UTR, a 3’ tailing sequence, or any combination thereof.

26. The composition of claim 25, wherein the 5’ UTR, 3’ UTR, or both are heterologous to the polynucleotide encoding at least a portion of KSHV K8.1 protein.

27. The composition of claims 25 or 26, wherein the 3’ tailing sequence comprises a poly A tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.

28. The composition of any of claims 23-28, wherein the polynucleotide comprises at least one chemically modified nucleotide.

29. The composition of claim 28, wherein the at least one chemically modified nucleotide comprises a modified uracil.

30. The composition of claim 29, wherein at least 60% of the uracil in the polynucleotide encoding at least a portion of KSHV K8.1 protein are chemically modified.

31. The composition of any of claims 28-30, wherein the at least one chemically modified nucleotide comprises 5-methylcytosine or N1 -methylpseudouridine (m I ).

32. The composition of any of claims 23-31, wherein the polynucleotide comprises a nucleotide sequence having: i) at least 75% sequence identity to SEQ ID NOs: 6, 7, 9, 12, or 14,or a complement or reverse complement thereof, wherein T may be replaced by U, and / or ii) at least 50 consecutive nucleotides from SEQ ID NOs: 6, 7, 9, 12, or 14, or a complement or reverse complement thereof, wherein T may be replaced by U.

33. The composition of any of claims 23-32, wherein said at least a portion of KSHV K8.1 protein comprises: i) an amino acid sequence having at least 90% identity to SEQ ID NO: 8, 10, 11, or 13 and / or ii) at least 12, at least 15, or at least 20, consecutive amino acids from SEQ ID NO: 8, 10, 11, or 13.

34. The composition of any of claims 23-31, wherein said at least a portion of KSHV K8.1 protein comprises: i) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 8, 10, 11, or 13, and / or ii) at least 12 or 15 consecutive amino acids from any one of SEQ ID NOs: 8, 10, 11, or 13.

35. A messenger ribonucleic acid (mRNA) comprising: i) a 5' untranslated region (UTR); ii) an open reading frame encoding at least a portion of Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1 protein; and iii) a 3' UTR, wherein the mRNA is at least partially human codon optimized.

36. The mRNA of claim 35, wherein the 5’ UTR, 3’ UTR, or both are heterologous to the open reading frame encoding at least a portion of KSHV K8.1 protein, and / or wherein the RNA is fully or nearly fully human codon optimized.

37. The mRNA of claim 35 or 36, wherein the mRNA further comprises or encodes: a 5’ cap, a 3’ tailing sequence, or both.

38. The mRNA of claim 37, wherein the 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.

39. The mRNA of any of claims 35-38, wherein the mRNA comprises at least one chemically modified nucleotide.

40. The mRNA of claim 39, wherein the at least one chemically modified nucleotide comprises a modified uracil.

41. The mRNA of claim 40, wherein at least 60% of the uracil in the open reading frame encoding said at least a portion of KSHV K8.1 protein are chemically modified.

42. The mRNA of any of claims 39-41, wherein the at least one chemically modified nucleotide comprises 5-methylcytosine or N1 -methylpseudouridine (m I T).

43. The mRNA of any of claims 35-42, wherein the mRNA comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:7, 9, or 12, and / or comprises at least 15, or at least 25, consecutive nucleotides from SEQ ID NO:7, 9, or 12.

44. The mRNA of any of claims 35-42, wherein said at least a portion of KSHV K8.1 protein comprises: i) an amino acid sequence having at least 90% identity to SEQ ID NOs:8, 10, 11, or 13, and / or ii) at least 12 or 15 consecutive amino acids from SEQ ID NOs:8, 10, 11, or 13.

45. A vaccine comprising: the composition of any of claims 23-34 or the mRNA of any of claims 35-44; and at least one adjuvant, a delivery vehicle, a physiological buffer, or any combination thereof.

46. The vaccine of claim 45, wherein the delivery vehicle comprises a lipid nanoparticle encapsulating the composition or the mRNA.

47. The vaccine of claim 46, wherein the lipid nanoparticle comprises a cationic lipid, a neutral and / or non-cationic lipid, a sterol, or any combination thereof.

48. The vaccine of claim 47, wherein the non-cationic lipid comprises a phospholipid.

49. The vaccine of claim 47 or 48, wherein the sterol comprises cholesterol or a modification or ester thereof.

50. The vaccine of any of claims 46-49, wherein the lipid nanoparticle comprises a polyethylene glycol (PEG)-lipid conjugate.

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