Development and optimization of an mrna-vectored single-chain iga1 isotype monoclonal antibody to treat or prevent dengue virus infection

Abstract

Dengue virus (DENV) is a rapidly expanding infectious disease threat that causes an estimated 100 million symptomatic infections every year. A barrier to preventing DENV infections with traditional vaccines or prophylactic monoclonal antibody (mAb) therapies is the phenomenon of Antibody-Dependent Enhancement (ADE). IgG is not the only antibody isotype capable of binding and neutralizing DENV, as DENV-specific IgA1 isotype mAbs can bind and neutralize DENV while without exhibiting any ADE activity. Here we describe the development and optimization of a DENV-specific single-chain IgA construct that retains the desirable biological properties of the parental IgA mAb yet is compatible with efficient in vivo delivery with a novel / liver-tropic lipid nanoparticle. We propose that this platform is uniquely and exceptionally well suited for preventing and / or treating DENV infections and may have broad applicability in the greater infectious disease space in situations where the use of IgG isotype mAbs may be counter indicated.

Description

DEVELOPMENT AND OPTIMIZATION OF AN mRNA-VECTORED SINGLE-CHAIN lgA1 ISOTYPE MONOCLONAL ANTIBODY TO TREAT OR PREVENT DENGUE VIRUS INFECTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present disclosure claims priority or the benefit under 35 U.S.C. § 119 of U.S. provisional application No. 63 / 671 ,399, filed July 15, 2024, herein entirely incorporated by reference.BACKGROUND OF THE INVENTION

[0002] Dengue virus (DENV) is a vector-borne human pathogen transmitted via the bite of infected Aedes family mosquitos. An estimated 400 million people are infected with DENV every year, with 3.9 billion individuals living in regions of the world with active DENV transmission. DENV infection can result in a variety of clinical outcomes ranging from mild flu-like symptoms to life-threatening conditions such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). While the case fatality rate for DHF and DSS is low when appropriately managed with judicious fluid support and intensive monitoring, there is currently no accurate diagnostic or test available to predict who will progress to develop these severe manifestations of infection. Therefore, the standard of care in many DENV-endemic regions is to monitor dengue patients in an in-patient setting so that supportive care can be provided in the event of hemorrhage or decompensation. This places an extremely high burden on healthcare systems during DENV outbreaks, repeatedly and unpredictably stressing vulnerable healthcare networks.

[0003] Despite the significant and growing global healthcare challenge posed by dengue there are few licensed countermeasures available to treat or prevent acute DENV infections. There is no vaccine that offers durable and balanced protection from DENV infection in individuals without preexisting DENV immunity and no licensed antiviral compound. Therefore, there is an urgent and growing need for new DENV countermeasures that can be deployed in either a therapeutic or prophylactic fashion, thereby reducing the global clinical burden of the disease.

[0004] Monoclonal antibodies (mAbs) have emerged as a front-line infectious disease therapy, prophylactic, and biodefense tools, exhibiting clinical efficacy against multiple distinct viral pathogens including SARS-CoV-2, RSV, and Ebola virus. mAbtherapies have the advantage of providing pathogen-specific immunity immediately upon infusion and a generally robust safety profile. All currently licensed mAb therapies are based on a human IgG 1 backbone, as this antibody type is responsible for providing durable protection from most viral infections and exhibit a long in vivo half-life.

[0005] However, a barrier to treating / preventing DENV infections with therapeutic or prophylactic mAbs based on an IgG backbone is the phenomenon of Antibody-Dependent Enhancement (ADE), wherein sub-neutralizing levels of DENV- specific IgG antibodies can enhance infection and pathogenesis rather than providing protection from disease. Poorly neutralizing DENV-reactive IgG antibodies can promote viral uptake and replication via IgG receptors (FcyR)-expressing cells such as monocytes, macrophages, and dendritic cells, leading to increased viral load and disease severity. Therefore, the potential utility of mAb therapies targeting DENV must be balanced against the potential risk of immune-mediated disease enhancement and transmission.

[0006] Fortunately, IgG is not the only antibody isotype capable of binding and neutralizing DENV. Our group and others have demonstrated that DENV-specific lgA1 isotype mAbs can bind and neutralize DENV without exhibiting any ADE activity, even in cells that express the myeloid-restricted IgA Fc receptor (CD89).Furthermore, DENV-specific lgA1 isotype mAbs can antagonize IgG-mediated ADE by competing with infection-enhancing antibodies for binding sites on the infectious virion. Furthermore, multiple groups have demonstrated that IgA isotype antibodies are inherently anti-inflammatory, which is a highly desirable feature for any targeted DENV-therapy in light of the immunopathogenesis associated with severe dengue. These results suggested that DENV-specific lgA1 isotype mAb may be uniquely and exceptionally well suited for preventing and / or treating DENV infections, and are a tractable alternative to IgG-based mAb therapies.

[0007] Despite the many desirable properties of lgA1 isotype mAbs there are several considerations that limit the practicality of any lgA1 -based therapy. Most mAb therapies currently require intravenous infusion and are extremely costly to produce in a cGMP fashion and at a scale compatible with large-scale deployment during outbreak scenarios. The production hurdles associated with large-scale production of lgA1 isotype mAbs are exaggerated by the lack of efficient purification technologies, as lgA1 isotype mAbs cannot be purified using traditional protein A / G resinscommonly used in the production of IgG isotype mAb therapies. Nucleic-acid vectored delivery technologies circumvent many of these barriers, especially when coupled with next-generation lipid-nanoparticle (LNP) delivery platforms. While mRNA / LNP delivery platforms are most well-known for their ability to express vaccine-antigens, several groups have utilized this strategy to express viral pathogenspecific antibodies, or antibody derivatives with therapeutic activity. However, these efforts have overwhelmingly focused on expressing IgG isotype mAbs, with no prior study attempting to utilize an lgA1 isotype mAb targeting a viral pathogen.BRIEF SUMMARY OF THE INVENTION

[0008] Embodiments disclosed herein are directed to an isolated single-chain lgA1 (sclgA1) antibody construct comprising: a heavy chain variable region (VH), a light chain variable region (VL), a linker peptide connecting the VH and VL regions, an igA1 constant region, wherein the construct is capable of binding to the dengue virus (DENV) envelope (E) protein and neutralizing DENV infectivity without enhancing infectivity via antibody-dependent enhancement (ADE). According to other embodiments the linker peptide comprises a sequence selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, and SEQ. ID.4. Other embodiments of the sclgA1 antibody construct include that the lgA1 constant region is derived from human Ig A1 .

[0009] Embodiments disclosed herein are directed to a pharmaceutical compositions comprising the sclgA1 antibody construct and a pharmaceutically acceptable carrier. Other embodiments include the pharmaceutical composition comprising the sclgA1 antibody construct, wherein the construct is delivered via livertargeting lipid nanoparticles (LNPs), as well as the sclgA1 antibody construct being encoded by mRNA. Other embodiments are directed to the sclgA1 antibody construct, which is modified to reduce in vivo reactogenicity by the incorporation of 5- methyl-CTP and Pseudo-UTP.

[0010] Further embodiments are directed to methods of treating or preventing dengue virus infection in a subject, comprising administering to the subject an effective amount of the pharmaceutical compositions disclosed herein. Other embodiments are directed to the sclgA1 antibody construct, wherein the linker peptide comprises a Gly-Gly-Gly-Gly-Ser (GGGGS) repeat motif, the construct includes a signal peptide sequence for secretion from mammalian cells, and theantibody is dimerized through the formation of intermolecular disulfide bonds within the IgA Fc region.

[0011] Other embodiments are directed to the LNP of the pharmaceutical composition, which can contain an ionizable lipid such as, 113-01 OS, MC3, or SM- 102. Additionally, the mRNA further comprising a 5' cap structure, a 5' untranslated region (UTR), a poly(A) tail, and a 3' UTR optimized for translation in human cells.

[0012] According to embodiments disclosed herein the methods are directed to subjects that are human that are at risk of dengue virus infection and the administration can be either prophylactic or therapeutic after detection of dengue virus infection.

[0013] Further embodiments are directed to the disclosed sclgA1 antibody construct, wherein the lgA1 constant region includes the Ca2 and Co3 domains, the hinge region is derived from human lgG1, and the construct retains FcaRI (CD89) binding activity comparable to that of a full-length parental lgA1 antibody.

[0014] Still other embodiments are directed to an mRNA molecule encoding a single-chain lgA1 antibody construct, comprising: a 5' cap, a 5' UTR, a coding sequence encoding a signal peptide, a heavy chain variable region (VH), a peptide linker, a light chain variable region (VL), and lgA1 constant domains Ca2 and Ca3, a 3' UTR, and a polyadenylation tail, wherein the mRNA is modified with 5- methylcytidine triphosphate (5mCTP) and pseudouridine triphosphate C+’UTP) to reduce innate immune stimulation.

[0015] Additional embodiments are directed to a method of producing a single-chain lgA1 antibody in a mammalian cell comprising, transfecting the mammalian cell with a plasmid or mRNA encoding the construct disclosed herein, and culturing the cell under conditions that permit expression and secretion of the antibody. Other methods disclosed herein are directed to methods of preventing antibody-dependent enhancement (ADE) in dengue virus treatment comprising, administering to a subject an effective amount of the disclosed single-chain lgA1 antibody, wherein the antibody lacks ADE-promoting Fey receptor binding domains. Still other embodiments include a system for in vivo delivery of a therapeutic antibody, comprising: an in vitro transcribed mRNA encoding a single-chain lgA1 antibody construct, a liver-tropic lipid nanoparticle, wherein administration of the system results in expression of a DENV-neutralizing antibody in hepatic tissue. And still more embodiments include a kit for treating or preventing dengue virus infection. The kitcontains a vial containing mRNA encoding a single-chain lgA1 antibody, a lipid nanoparticle delivery reagent, and instructions for administration to a subject including, the use of intravenous, intradermal, intramuscular, and oral administration, as needed.

[0016] Thus, there are disclosed herein novel compositions and methods for the prevention and treatment of dengue virus (DENV) infection using a single-chain lgA1 (sclgA1) antibody construct that neutralizes the virus without promoting antibody-dependent enhancement (ADE). In particular, the embodiments relate to engineered antibody constructs, encoding nucleic acids (e.g., mRNA), and delivery systems, including lipid nanoparticles (LNPs), capable of selectively targeting various tissue types and immune cell populations.

[0017] While certain embodiments are directed toward liver-targeted delivery via intravenous administration, the invention extends beyond hepatic applications. The LNP formulation may be modified to direct mRNA expression in other tissues or cell types, including, but not limited to spleen, fibroblasts, myocytes (muscle cells), dendritic cells, macrophages, and other antigen-presenting or immune effector cells. Targeting strategies may include the use of specific ligands, peptides (e.g., RGD), sugar moieties (e.g., mannose), folate, or antibody fragments to facilitate selective uptake by desired cell types.

[0018] The present embodiments provide an advancement over the prior art in the field of dengue virus (DENV) therapeutics through the novel integration of an isolated single-chain lgA1 (sclgA1) antibody construct, mRNA-based expression platforms, and tissue-targeted lipid nanoparticle (LNP) delivery systems. Unlike the conventional use of IgG-based antibodies for viral neutralization that typically require Fc engineering to avoid antibody-dependent enhancement (ADE), the present embodiments leverage the unique structural and immunological properties of the lgA1 isotype. The sclgA1 format combines the variable regions of heavy and light chains via a peptide linker into a single polypeptide fused to lgA1 constant domains (Ca2 and Ca3), allowing for potent viral neutralization while inherently avoiding ADE due to IgATs inability to bind activating Fey receptors (e.g., FcyRlla and FcyRHIa) responsible for ADE in IgG therapies. This mechanism offers a fundamentally different, and non-obvious, approach to ADE avoidance that does not rely on Fc mutations or glycoengineering.

[0019] In a further unexpected advancement, the expression of the sclgA1 construct in vivo using in vitro transcribed (IVT) messenger RNA (mRNA), incorporating chemically modified nucleotides such as 5-methylcytidine triphosphate (5mCTP) and pseudouridine triphosphate (M^UTP) to reduce innate immune activation and enhance stability and translation efficiency. mRNA-based antibody delivery has traditionally focused on IgG formats due to their well-characterized folding and secretion pathways; however, the successful design and expression of an mRNA- encoded Ig A1 construct including signal peptides for secretion, proper disulfide bond formation, and retention of FcaRI (CD89) binding is a significant technical achievement. Given the complex glycosylation and multimerization requirements of lgA1 , this represents a non-trivial and unexpected extension of mRNA therapeutic technology.

[0020] Moreover, the embodiments provide for the expanded utility of mRNA- encoded sclgA1 therapeutics by employing lipid nanoparticle (LNP) formulations that are not limited to liver targeting. While liver-tropic LNPs are well-documented in the context of siRNA and mRNA therapeutics, the embodiments disclosed herein contemplate and enable LNP systems designed for preferential delivery to a variety of tissues and immune cells including spleen, dendritic cells, macrophages, myocytes, fibroblasts, and others. This includes the use of targeting ligands such as mannose, RGD peptides, folate, or antibody fragments to direct LNPs to non-hepatic cell populations that play critical roles in dengue virus infection, immune modulation, and systemic antibody expression. The expansion of mRNA-LNP delivery to diverse, nonliver tissues for the purpose of therapeutic antibody production is an unexpected and non-obvious feature that addresses the limitations of hepatic expression platforms, and provides more localized or systemic immunological control depending on the targeted tissue.

[0021] Taken together, the combination of an sclgA1 construct with anti-DENV neutralization and ADE avoidance, its delivery via chemically modified mRNA, and its encapsulation in tissue-targeted LNPs that extend beyond liver tropism, constitutes a novel and inventive approach. The prior art does not suggest or motivate this particular integration, nor does it provide reasonable expectation of success in expressing IgA-based antibodies via mRNA in vivo with effective tissue-targeted delivery and therapeutic benefit in dengue virus infection.SEQUENCE LISTING

[0022] The specification further incorporates by reference the Sequence Listing submitted herewith via Patent Center. The Sequence Listing .xml file, identified as 110-2337US01 , is 6,720 bytes in size and was created on June 25, 2025. The Sequence Listing, electronically filed herewith, does not extend beyond the scope of the specification, and does not contain new matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 presents the design and characterization of candidate DENV- specific scIgA constructs. FIG 1A presents a schematic representation of the scIgA constructs designed and characterized as part of this study. FIG. 1 B presents an assessment of in vitro scIgA production following plasmid transfection of 293T cells. Abundance of human IgA present in the culture supernatant 24, 48, and 72 hours post transfection was quantified by IgA capture ELISA. Each datapoint represents an independent biological experiment (n=3). FIG. 1C presents an assessment of scIgA production following in vitro transfection of IVT mRNA (0.1 ug) or purified DNA plasmid (0.25ug) encoding the lead scIgA construct in to 293T cells. FIG. 1 D presents a predicted 3D structure of the lead scIgA construct. FIG. 1 E presents a Western blot of purified lead scIgA construct in non-reducing and denature condition. Staining performed with anti-human IgA.

[0024] FIG. 2 presents a functional characterization of lead DENV-specific scIgA construct. FIG. 2A presents a quantification of recombinant DENV-1 E and NS- 1 binding activity of purified scIgA as determined by multiplexed antigen array. FIG. 2B presents an assessment of DENV-1 neutralization activity of parental IgA mAb and purified scIgA utilizing DENV-1 RVP and U937 DC-SIGN cells. All antibodies used at a final concentration of 100ng / ml. FIG. 2C presents an assessment of DENV-1 ADE activity of parental IgG mAb and purified scIgA utilizing DENV-1 RVP and K562 cells. All antibodies used at a final concentration of 100ng / ml. FIG. 2D presents an assessment of FcaR binding activity of parental Ig A1 mAb and purified scIgA. ****p<0.0001 , one-way ANOVA with correction for multiple comparisons.

[0025] FIG. 3 presents an evaluation of in vivo mRNA delivery by livertargeting LNP. FIG. 3A presents a visualization of mfLuc expression in animals infused with mfLuc alone or mfLuc / 113-01 OS liver-targeting LNP. B) Quantification ofliver-specific mfLuc expression in animals infused with mfLuc alone or mfLuc / 113- 01 OS liver-targeting LNP the expression. C) quantification of DENV-1 E protein specific human IgA antibody titers in mice infused with scIgA-encoding mRNA complexed with LNP or LNP alone. n=3 animals per group. D) quantification of DENV- 1 NS-1 protein specific human IgA antibody titers in mice infused with scIgA-encoding mRNA complexed with LNP or LNP alone. n=3 animals per group.

[0026] FIG. 4 presents annotated sequences of the scFv-Fc-lgA1 constructs disclosed herein, represented as SEQ. ID. Nos.: 1 , 2, 3, and 4, wherein the sequences have been separated to identify the signal peptide (secretion sequence), the heavy chain variable sequence, the light chain variable sequence, the linker, the IGHA1 CH1 and IGHA1 CH2, the native IGHA1 sequence and the IGHG1 sequence (hinge), within each sequence represented in the accompanying Sequence Listing.

[0027] FIG. 5 presents a graphic representation of the purified scIgA encoding mRNA. A graphic assessment by bioanalyzer of mRNA generated from DNA plasmid encoding sc-lgA1 .

[0028] FIG. 6 presents flow-cytometry based DENV-1 neutralization assay. FIG. 6A presents representative flow cytometry plots and FIG. 6B presents nonnormalized neutralization data.

[0029] FIG. 7 presents flow-cytometry based DENV-1 ADE assay. FIG. 7A presents representative flow cytometry plots and FIG. 7B presents non-normalized ADE data.DETAILED DESCRIPTION OF THE INVENTION

[0030] As used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms “comprising,” “including,” “containing,” “having,” and “involving” are used in an open- ended sense and should be interpreted to mean “including but not limited to.” The term “or” is generally used to mean “and / or” unless otherwise indicated.

[0031] Where ranges are stated (e.g., concentrations, sequence lengths, molecular weights, pH, temperature), all intervening values and subranges are intended to be encompassed. Where specific sequences (e.g., nucleic acid or amino acid sequences) are disclosed, variants having at least 70%, 80%, 90%, or 95% sequence identity, or functional equivalents thereof, are intended to be included unless otherwise limited.

[0032] The invention is not limited to the specific nucleic acid sequences, protein sequences, organisms, vectors, host cells, methods of administration, dosages, or formulations explicitly disclosed herein. Rather, it is intended to cover all modifications, substitutions, analogs, derivatives, fragments, fusion constructs, homologs, orthologs, or functional equivalents that perform substantially the same function in substantially the same way to achieve substantially the same result.

[0033] Any features described with respect to one embodiment or aspect may be applied to any other embodiment or aspect, unless expressly stated otherwise or technically incompatible. The claims are intended to cover all such variations, equivalents, and alternatives that fall within the true spirit and scope of the invention.

[0034] In embodiments disclosed herein, we report the generation, optimization, and in vivo delivery via tissue-targeting LNP of a novel single-chain Ig A1 (scIgA) isotype antibody. These optimized constructs exhibited efficient expression in both a DNA and mRNA vectored format and retained the ability to bind / neutralize DENV without enhancing infectivity via ADE. The sclgA1 constructs can be expressed in vivo following optimized LNP delivery, highlighting the potential therapeutic utility of such a construct.

[0035] Dengue virus (DENV) is a rapidly expanding infectious disease threat that causes an estimated 100 million symptomatic infections every year. A barrier to preventing DENV infections with traditional vaccines or prophylactic monoclonal antibody (mAb) therapies is the phenomenon of Antibody-Dependent Enhancement (ADE), wherein sub-neutralizing levels of DENV-specific IgG antibodies can enhance infection and pathogenesis rather than providing protection from disease.

[0036] IgG is not the only antibody isotype capable of binding and neutralizing DENV, as DENV-specific lgA1 isotype mAbs can bind and neutralize DENV while without exhibiting any ADE activity. However, the development of lgA1-based mAb therapies is currently hindered by inefficient in vitro expression systems and the lack of saleable purification platforms. Accordingly, alternative delivery modalities are required to realize the therapeutic potential of IgA-based infectious-disease therapies. Herein, embodiments describe the development and optimization of a DENV-specific single-chain IgA construct that retains the desirable biological properties of the parental IgA mAb, yet is compatible with efficient in vivo delivery with a novel / liver-tropic lipid nanoparticle. This platform is uniquely and exceptionally well suited for preventing and / or treating DENV infections and may have broadapplicability in the greater infectious disease space in situations where the use of IgG isotype mAbs may be contraindicated.

[0037] To enable efficient nucleic-acid vectored delivery we first sought to convert a DENV-specific lgA1 mAb into a single-chain format compatible with both DNA and RNA expression platforms. To this end, we generated multiple single-chain IgA (scIgA) constructs with differing variable heavy domain and variable heavy domain, i.e. , VH / VL orientations separated by a flexible peptide sequence composed of repeating glycine-serine units, i.e., GS3 linker, terminating with the Ca2 and Co3 domains from the constant region of the human lgA1 heavy chain (FIG. 1A). The Co2 and Ca3 domains were fully retained in all constructs to enable the engagement of FcaR. We additionally utilized either the native lgA1 hinge region to separate the VH / VL sequences from the Ca2 and Ca3 domains, or the shorter human lgG1 hinge region sequence. All constructs were codon-optimized for expression in human cells and transfected into 293T cells to assess expression efficiency in a DNA-launched format. Cell culture supernatant harvested at 24h, 48h, and 72h after transfection and the abundance of human lgA1 quantified by ELISA (FIG. 1B). A two-plasmid expression system encoding the corresponding native lgA1 antibody construct was utilized as a positive control. Human lgA1 was readily detected in the cell cultures transfected with the constructs with a VH / VL domain configuration, while minimal protein production was observed in the cultures transfected with constructs with a VL / VH orientation. In addition, higher lgA1 signals were observed in the VH / VL construct with containing the human IgG 1 hinge region than for the constructs containing the native lgA1 hinge region (FIG. 1B). This lead construct (VH / VL + lgG1 hinge) was additionally determined to be compatible with mRNA-vectored expression using transient transfection of in vitro transcribed mRNA (FIG. 1C). Structural prediction analysis suggested that this construct forms a stable dimer with both the Fab and truncated lgA1 Fc domain accessible (FIG. 1D). This assumption was supported by western blot analysis of the purified protein product (FIG. 1 E), which under non-reducing conditions demonstrated a dominant product of ~120kD (the predicted weight of the assembled scIgA dimer), and was subsequently condensed to a single band with a molecular weight of ~60kD under reducing conditions.

[0038] sclgA1 maintains the same biological function of the parental Ig A1 mAb. Having identified a single-chain construct sequence that afforded maximal lgA1 production efficiency in vitro, we next sought to confirm that this scIgA moleculeretained the biological activity as the parental Ig A1 antibody. We observed that purified scIgA exhibited specific DENV E protein binding activity (FIG. 2A), with an affinity near what is observed using the parental mAb. Furthermore, the scIgA construct exhibited similar DENV neutralization activity as the parental IgA mAb (FIG. 2B), while also exhibiting no DENV infection enhancing activity (FIG. 2C). Finally, the scIgA construct was observed to retain its ability to bind its cognate Fc receptor (FcaR / CD89) at a magnitude similar to what was observed for the parental Ig A1 mAb. Altogether, these results indicate that this scIgA construct retains all the desirable biologic features of the parental IgA antibody and may have therapeutic or prophylactic value for treating or preventing DENV infection.

[0039] scIgA can be delivered in vivo by liver-targeting lipid nanoparticles. Our primary motivation for developing a scIgA construct was to facilitate and streamline the in vivo expression of the recombinant protein for therapeutic implementation. mRNA and DNA vectored delivery platforms have been previously utilized for in vivo expression of candidate mAb therapies with varying degrees of success, and the functionality of these platforms are strongly impacted by the composition of the in vivo delivery and expression system. Accordingly, we leveraged the unique properties of our organ-targeting LNP platform to mediate and enhance the delivery and expression of scIgA mRNA in the liver of mouse model. This approach represents a new modality of mRNA-based mAb therapy for DENV treatment.

[0040] The chalcogen-based, polyamine-containing, ionizable lipid (113- 01 OS) was synthesized following our previously reported procedures. LNPs were fabricated using 113-01 OS, cholesterol, phospholipid DOPC and PEG-lipid (DMG- PEG2k) through self-assembly, with cargo mRNA encapsulated via electrostatic interactions. To confirm the organ-specificity and potential therapeutic utility of the 113-01 OS LNP formulation, we first assessed the ability of the platform to deliver luciferase-encoding mRNA (mfLuc) in vivo. Mice were intravenously injected with mfLuc / LNP and the expression of luciferase assessed using the In Vivo Imaging System (I VIS). Robust luciferase activity was observed in the liver of all animals injected with mfLuc / LNP, while no signal above background was observed in any anatomical location for mice infused with luciferase-encoding mRNA alone (FIG. 3A and FIG. 3B).

[0041] In light of these positive initial observations, we next sought to deliver our solgA construct in vivo using the same platform. We generated in vitro transcribed (IVT) mRNA encoding our scIgA construct, modified to reduce in vivo reactogenicity by the incorporation of 5-methyl-CTP and Pseudo-UTP. This modified IVT mRNA construct was complexed with the 113-01 OS LNP formulation and delivered i.v. into C57BI / 6 mice. The abundance of DENV E and NS1 protein specific human IgA antibodies in mouse serum was quantified using a custom multiplexed electrochemiluminescence assay based on the MSD QuickPlex SQ 120 platform. Animals infused with modified mRNA encoding the scIgA construct exhibited robust serum expression of human IgA antibodies specific for DENV E protein for 3-4 days after infusion, with an in vivo half-life of the DENV-specific IgA signal of ~19 hours (FIG. 30). No signal was detected in animals infused with the 113-01 OS LNP formulation alone, and no off-target DENV NS1 binding was observed in the animals infused with the scIgA mRNA + 113-01 OS LNP (Figure 3D). Collectively, these results demonstrate the technical feasibility of utilizing an mRNA / LNP expression platform for the in vivo administration of a single-chain, DENV-specific IgA isotype mAb.

[0042] Here we report the generation, optimization, and in vivo delivery of a novel single-chain lgA1 (sclgA1 ) isotype antibody construct. Thus, the disclosed optimized constructs exhibited efficient expression in both a DNA and mRNA vectored format and retained the ability to bind / neutralize DENV without enhancing infectivity via ADE. This sclgA1 construct was additionally able to be delivered in vivo via a liver-tropic LNP, highlighting the potential therapeutic utility of such a construct.

[0043] Our study is the first to describe the in vivo delivery and expression of an mRNA-vectored single-chain IgA construct, multiple prior studies have demonstrated the technical feasibility of in vivo delivery of IgG isotype antibodies utilizing similar mRNA expression modalities. Indeed, these platforms have been used in multiple preclinical studies to treat or prevent infections with viral pathogens as diverse as HIV, ZIKV, and SAR-CoV-2, while early-state clinical studies have commenced utilizing mRNA vectored antibodies specific for pathogens such as CHIKV. Notably, a similar approach has been previously utilized to deliver mRNA constructs encoding for the IgG isotype anti-HIV-1 antibody VRC01 , with LNP- vectored delivery of mRNA encoded-antibodies showing prophylactic protection fromHIV-1 challenge. The VRC01 antibody achieved peak 24h after injection and was detectible in serum of mice for 11 days after injection, providing complete protection from HIV infection in humanized mouse models of HIV infection. Similarly, lungtargeting LNPs have been used to deliver the mRNA encoded, SARS-Cov-2 specific IgG mAbs. This treatment was demonstrated to result in high levels of mAb production in the lung of infused animals, preventing SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model

[0028] , Finally, an alphavirus replicon system has been previously utilized to deliver and express a potently-neutralizing ZlKV-specific mAb (ZIK-117), demonstrating robust protection against lethal Zika virus infection in both pre-exposure prophylaxis and post-exposure therapeutic preclinical assays.

[0044] While the development of IgA-based mAb therapies is a nascent field of research, there is prior work demonstrating the potential utility of mRNA-based expression systems for delivery of such therapeutic constructs. Notably, Deal et al. have demonstrated the feasibility of delivering mRNA-encoded Pseudomonas- and Salmonella- specific IgA mAbs in vivo utilizing an LNP delivery platform. In contrast to the platform described in our study, this study utilized a system designed to produce dimeric IgA, intended to target mucosal surfaces to prevent colonization and invasion by mucosal pathogens. LNP mediated delivery of this tripartite expression system (heavy chain, light chain, and J chain) resulted in robust mucosal localization of both Pseudomonas- and Salmonella- specific IgA mAbs, providing significant protection from infection by both pathogens. While highly encouraging, additional optimization of this platform appears to be required, as the half-life of the mRNA- encoded IgA utilized in these studies was limited and the mRNA dose required to achieve protective levels of dimeric lgA2 in mucosal surfaces exceeds what has been currently tested and approved for use in humans.

[0045] While the results described in this study demonstrate the general feasibility of in vivo delivery a DENV-specific single-chain IgA mAb via liver-tropic LNP, there a few limitations that must be considered. First, this half-life of our scIgA mAb remains well-below what is observed for IgG-based mAb therapies. This is presumably due both to the inherently shorter half-life of IgA compared to IgG, due to its inability to interact with FcRn, and due to the slightly smaller hydrostatic size of our scIgA construct relative to a native antibody. There are several orthogonal approaches that can be utilized to overcome this limitation, including the addition of albumin-binding domains to the scIgA construct or the utilization of self-replicating / degradation-resistant RNA constructs for in vivo delivery. Second, there is the possibility that an immune response may be mounted against the scIgA construct, limiting the ability to repeated administer the therapy. While we believe this is unlikely due to the anti-inflammatory nature of the LNP system utilized in our study, addition study is required.

[0046] Overall, the mRNA-launched single chain lgA1 isotype antibody format has shed new light on treating and preventing DENV infection. However, addressing the existing challenges described previously, such as delivery efficiency, short halflife, and immunogenicity, will be required for a realizing the full prophylactic and therapeutic potential of this new treatment modality.EXAMPLES

[0047] Cell lines. 293T cells were maintained in DMEM (Gibco, 11965118) containing 10% heat-inactivated FBS (Corning, 35016CV), 1 % L-glutamine, penicillin, and streptomycin (Gibco). U937 DC-SIGN cells were maintained in RPMI (Corning 10-040-CM) containing 10% FBS, 1 % L-glutamine, penicillin, and streptomycin. K562 cells were obtained from ATCC (CCL-243) and were maintained in IMDM (Gibco, 12440061 ) containing 10% FBS, 1% penicillin, and streptomycin.

[0048] In vitro expression of scIgA constructs. 6-well plates were seeded with 80,000 293T cells in 2ml of DMEM media and incubated at 37°C in 5% CO2 overnight. Next day, cell media was replaced with 250uL Opti-MEM media (Gibco) 30 minutes before the transfection. For one well of cell, 5ug of DNA mixed with 125uL Opti-MEM and 10uL of P3000 reagent (Invitrogen, L3000008) for 5 minutes. In a separate tube, lipofectamine Invitrogen, L3000008was diluted in Opti-MEM in a 3:100 ration. After incubation, 125uL of DNA / P3000 dilution and 125uL of lipofectamine dilution were mixed, vortexed and incubated for an additional 15 minutes. 250uL of transfection solution was added directly to the well and incubated overnight at 37°C in 5% CO2. Cell media was replaced the next day with 2mL of DMEM. Following overnight cell incubation, cell supernatant was harvested at 24, 48, and 72h.

[0049] IgA- Capture ELISA. 96-well NUNC MaxSorb flat-bottom plates were coated with 2 ug / ml Goat Anti-Human Ig UNLB (SouthernBiotech, 2010-01 ) diluted in borate saline buffer. Plates were incubated overnight at 40C. Plates were washed with 1X PBS and 0.1% Tween-20 and then blocked with 0.25% BSA and 1% NormalGoat Serum in PBS in 30 minutes. Serially diluted cell supernatant was incubated on the plate for 2h at room temperature. Plates were washed with 1X PBS and 0.1% Tween-20. Goat Anti-Human IgA HRP (BioLegend, 411002) was diluted T.2000 and added into the plate. Following 2h incubation at room temperature, the amount of secondary antibody was quantified by using TMB Substrate Kit (Thermo Scientific) and Synergy HT plate reader at 450 nm (BioTek, Winooski, VT). Standard curve was generated using purified monoclonal recombinant IgA antibody.

[0050] mRNA in vitro transcription. DNA plasmids encoding the scIgA construct was linearized by EcoRI digestion purified by using QIAquick PCR Purification kit (QIAGEN, Cat. #28104). After purification, IVT mRNA was generated using the HiScribe T7 ARCA mRNA kit with tailing (New England BioLabs). The modified mRNA version was generated by adding 5-methyl-CTP (APExBIO, Cat. #67967) and Pseudo-UTP (APExBIO, Cat. #B7972) at final concentration of 1.25 mM. After IVT reaction, the mRNA was purified by using RNeasy Mini Kit (QIAGEN, Cat. #74104) and eluted in 100 uL of nuclease-free water. The mRNA integrity and concentration were checked by using Agilent 2100 Bioanalyzer.

[0051] mRNA in vitro transfection. HEK293T cells were grown to 60% confluence in T75 tissue culture treated flask. We seeded 48-well plate with 15,000 cells in 250 uL of DMEM media and incubated at 37°C in 5% CO2 overnight. For one well of cell, 0.2 ug of mRNA was mixed with lipofectamine at ratio 1 :5. Optimem (ThermoFisher, Cat. #51985034) is added to make 20 uL total volume of transfection solution. The solution was incubated for 15 minutes at RT before transferring to the cell. The plate was incubated overnight at 37°C in 5% CO2. Cell supernatant was harvested on the next day. The amount of sc-lgA1 protein was measured by running IgA-capture ELISA.

[0052] scIgA purification: scIgA antibody protein was purified from 293T cell supernatant using CaptureSelect Biotin Anti-lgA Conjugate (ThermoFisher Scientific, Cat. #7102882100) and Pierce Streptavidin Magnetic Bead (ThermoFisher Scientific, Cat. #88817) After conjugation and extensive washing, the IgA capture beads were mixed with cell supernatant containing scIgA and incubated with gentle agitation for 30 minutes at room temperature. The captured scIgA protein was eluted from the capture beads by using 0.1 M Glycine, pH 2, and neutralized by 15% 1M Tris pH 9.0, followed by buffer exchange into PBS. The concentration of sc-lgA1 was quantified by IgA-capture ELISA.

[0053] Western blot. Purified scIgA protein was mixed with NuPAGE LDS Sample Buffer (ThermoFisher, Cat. #NP0007) and DI water + / - betamercaptoethanol (Millipore Sigma). Samples were incubated at 700C for 10 minutes, then loaded into a NuPAGE 4-12%Bis-Tris Gels (Thermo Fisher) and run with NuPAGE SDS Running Buffer (Thermo Fisher). Samples were transferred to nitrocellulose membrane and blocked in PBS-T with 5% BSAin 30 minutes.Samples were washed with PBS-T and immunoblotted with 1:10,000 diluted goat anti-human IgA HRP (BioLengend, Cat. #411002) with constant shaking overnight at 40C. Blots were developed using NovexTM ECL HRP.

[0054] Chemiluminescent Substrate Reagent kit (Thermo Fisher, Cat. #WP20005) and imaged using a Bio-Rad ChemiDoc MP Imaging System: DENV-1 RVP. DENV-1 dsRed Reporter virus (RVP) was prepared by co-transfecting 293T cells with DENV-1 (strain WP74) CprME structural DNA plasmid and dsRed WNVII- replicon plasmid. Cells were transfected using Lipofectamine 3000 (Invitrogen, cat.# L3000001). Cells incubated at 30oC to promote DENV virion assembly. Transfected cell supernatant was harvested, centrifuged at 5000g to pellet suspended cells and debris, filtered using 0.2uM syringe filter, aliquoted, and stored at -80C. Dengue viruses were titrated on U937 DC-SIGN cells, infectious units (IU) calculated based on the dilution of virus needed to infect 10-15% of cells.

[0055] DENV-1 Neutralization Assay. Purified scIgA or the parental IgA isotype mAb were diluted in PBS to a final concentration of 100 ng / ml and mixed with a concentration of DENV-1 RVP determined to achieve infection rate of -10% on U937 DC-SIGN cells. The antibody and dengue RVP were mixed (1 :1 ratio) in triplicate and incubated in 96-well plate at 37oC for 1h. This solution was then added to an equal volume of media containing 5 x 104 U937 DC-SIGN cells followed by a 24hr incubation. After incubation, the cells were washed twice with PBS + 2% FBS and analyzed by flow cytometry for the expression of dsRed.

[0056] DENV-1 ADE Assay. VDB11-lgG that we generated previously and purified sc-lgA1 was diluted in PBS to a single concentration point of 0.1 ug / ml. The antibody was incubated with an equal volume of dengue RVP that infects 20% of K562 cells in absence of antibody. The mixture antibody-dengue RVP was incubated in 1h at 37°C, and then added to 96-well plate containing 5 x 104 K562 cells in triplicate. Following 24h incubation at 37°C in 5% CO2, the cells were washed and resuspended in 200 uL of FACs buffer. The percentage of infected cellswere detected by dsRed fluorescence on Attune flow cytometer using yellow 2 filter. Data was analyzed in FlowJo.

[0057] IgA / FcOR binding assay: The 96-well NUNC MaxSorb flat-bottom plates were coated overnight at 4°C with 50ul of a 10 ug / ml solution of human FcaR (CD89) or bovine serum albumin (BSA) in PBS. After coating, plates were washed with 1X PBS and 0.1% Tween-20, and then blocked with 0.25% BSA and 1% Normal Goat Serum diluted in PBS in 30 minutes. Purified scIgAand native lgA1 mAb were incubated on the plate for 2h at room temperature. After extensive washing, the plates were stained with goat anti-human IgA HRP (BioLegend, 411002, 1 :2,000 dilution), followed by incubation with TMB Substrate (Thermo Scientific). Plates were read using a Synergy HT plate reader at 450 nm (BioTek, Winooski, VT).

[0058] Liver-targeting LNP formulation and production: To prepare the LNP formulation, ionizable lipids (70.4%, weight ratio) were mixed with cholesterol (16.9%), DOPC (8.5%), and DMG-PEG2k (4.2%) in pure ethanol. Lipid nanoparticles were prepared by adding the lipid mix solution to sodium acetate buffer (pH 5.0) under vortex. The sLNP was then purified by dialysis against DI water (Slide-A- LyzerTM dialysis cassette; ThermoFisher). To prepare mRNA-loaded LNP, precalculated mRNA and LNP were mixed in nuclease-free water (LNP / mRNA = 14 / 1 , weight ratio). After a brief pipette mixing, the solution was stored at room temperature for 20 min before use.

[0059] mRNA in vivo expression - mouse studies: Six C57BL / 6J female mouses (age of six week) were purchased from Jackson Laboratory. They were divided into two groups of three. One group received mixture of LNP and modified mRNA(10:1 ratio, respectively) through retro-orbital injection. Another group received the same amount of LNP dose only. The blood was obtained one day before the injection (day 0), and then 1 , 2, 3, 5, and 7 days after injection. To collect serum, the blood was placed at RT in 30 minutes to clot, and then centrifuged at 10,000 rpm in 10 minutes. The amount of antibody in the serum was assessed through the DENV1-E protein MSD binding assay.

[0060] DENV1-E / NS1 protein binding assay: The binding activity of purified sc-lgA1 and serum antibody detection was assessed by Meso Scale Discovery (MSD) U-Plex Development Packs. DENV1-NS1 protein (Native Antigen, Western Pacific strain) and DENV1-E protein produced in insect cells (Native Antigen, 31679- 100?) were biotinylated and coated on U-Plex plate using U-Plex Linkers. Either 1ug / ml of purified sc-lgA1or 1 :10 diluted mouse sera were added to each well of the plate and incubated for 1h at RT with 700 rpm on the shaker, followed by detection with SULFO-TAG anti-human IgA antibody. The plate was loaded into MSD QuickPlex SQ120 instrument to detect electrochemiluminescence.

[0061] All publications, patent applications, patents, database entries and other references cited herein are hereby incorporated by reference in their entirety for all purposes as if each were individually and explicitly incorporated by reference. However, incorporation by reference is made solely for the purpose of providing additional disclosure and background, and no incorporated material is to be construed as essential to the invention unless expressly stated to be so. In the event of any inconsistency or conflict between the incorporated material and the present disclosure, the present disclosure shall govern and take precedence.REFERENCES

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Claims

WHAT WE CLAIM IS:

1. An isolated single-chain lgA1 (sclgA1) antibody construct comprising: a heavy chain variable region (VH), a light chain variable region (VL), a linker peptide connecting the VH and VL regions, an lgA1 constant region, wherein the construct is capable of binding to the dengue virus (DENV) envelope (E) protein and neutralizing DENV infectivity without enhancing infectivity via antibody-dependent enhancement (ADE).

2. The sclgA1 antibody construct of claim 1 , wherein the linker peptide comprises a sequence selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, and SEQ. ID.4.

3. The sclgA1 antibody construct of claim 1 , wherein the Ig A1 constant region is derived from human lgA1.

4. A pharmaceutical composition comprising the sclgA1 antibody construct of claim 1 and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition of claim 4, wherein the sclgA1 antibody construct is delivered via liver-targeting lipid nanoparticles (LNPs).

6. The pharmaceutical composition of claim 4, wherein the sclgA1 antibody construct is encoded by mRNA.

7. The pharmaceutical composition of claim 6, wherein the mRNA encoding the sclgA1 antibody construct is modified to reduce in vivo reactogenicity by the incorporation of 5-methyl-CTP and Pseudo-UTP.

8. A method of treating or preventing dengue virus infection in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 4.

9. The sclgA1 antibody construct of claim 1 , wherein the linker peptide comprises a Gly-Gly-Gly-Gly-Ser (GGGGS) repeat motif.

10. The sclgA1 antibody construct of claim 1 , wherein the construct includes a signal peptide sequence for secretion from mammalian cells.11 . The sclgA1 antibody construct of claim 1 , wherein the antibody is dimerized through the formation of intermolecular disulfide bonds within the IgA Fc region.

12. The pharmaceutical composition of claim 5, wherein the LNP comprises an ionizable lipid selected from the group consisting of 113-01 OS, MC3, and SM- 102.

13. The mRNA of claim 7, further comprising a 5' cap structure, a 5' untranslated region (UTR), a poly(A) tail, and a 3' UTR optimized for translation in human cells.

14. The method of claim 8, wherein the subject is a human at risk of dengue virus infection and the administration is prophylactic.

15. The method of claim 8, wherein the administration is therapeutic and occurs after detection of dengue virus infection.

16. The method of claim 8, wherein the administration results in detectable levels of human lgA1 in the serum for at least 3 days post-injection.

17. The sclgA1 antibody construct of claim 1 , wherein the lgA1 constant region includes the Ca2 and Ca3 domains, and the hinge region is derived from human lgG1.

18. The sclgA1 antibody construct of claim 1 , wherein the construct retains FcaRI (CD89) binding activity comparable to that of a full-length parental Ig A1 antibody.

19. An mRNA molecule encoding a single-chain lgA1 antibody construct, comprising: a 5' cap, a 5' UTR, a coding sequence encoding a signal peptide, a heavy chain variable region (VH), a peptide linker, a light chain variable region (VL), and lgA1 constant domains Ca2 and Ca3, a 3' UTR, and apolyadenyiation tail, wherein the mRNA is modified with 5-methylcytidine triphosphate (5mCTP) and pseudouridine triphosphate (H^UTP) to reduce innate immune stimulation.

20. A lipid nanoparticle (LNP) formulation comprising the mRNA of claim 19 and a cationic ionizable lipid, wherein the LNP targets the liver upon intravenous administration.21 . A method of producing a single-chain IgA 1 antibody in a mammalian cell, comprising: transfecting the mammalian cell with a plasmid or mRNA encoding the construct of claim 1 , and culturing the cell under conditions that permit expression and secretion of the antibody.

22. A method of preventing antibody-dependent enhancement (ADE) in dengue virus treatment, comprising administering to a subject an effective amount of a single-chain lgA1 antibody according to claim 1 , wherein the antibody lacks ADE-promoting Fey receptor binding domains.

23. A system for in vivo delivery of a therapeutic antibody, comprising: an in vitro transcribed mRNA encoding a single-chain lgA1 antibody construct, a livertropic lipid nanoparticle, wherein administration of the system results in expression of a DENV-neutralizing antibody in hepatic tissue.

24. A kit for treating or preventing dengue virus infection, comprising: a vial containing mRNA encoding a single-chain lgA1 antibody, a lipid nanoparticle delivery reagent, and instructions for administration to a subject including, use of one or more of intravenous, intradermal, intramuscular, and oral administration.

25. A lipid nanoparticle (LNP) composition comprising the mRNA of claim 19, wherein the LNP comprises: (a) an ionizable lipid; (b) cholesterol; (c) a phospholipid; and (d) a polyethylene glycol (PEG)-lipid; wherein the LNP is formulated to target liver tissue upon intravenous administration.

26. The composition of claim 25, wherein the ionizable lipid comprises a chalcogen-based polyamine-containing lipid selected from the group consisting of 113-01 OS.

27. A method of treating or preventing dengue virus (DENV) infection in a subject in need thereof, comprising: administering to the subject an effective amount of the lipid nanoparticle composition of claim 25, wherein the expressed antibody neutralizes DENV and does not induce antibody-dependent enhancement (ADE) of infection.

28. The method of claim 27, wherein the subject is administered the composition intravenously, intradermally, intramuscularly, or orally.

29. The method of claim 27, wherein the subject is at risk of DENV infection.

30. The method of claim 27, wherein the subject has been diagnosed with acute DENV infection.31 . The pharmaceutical composition of claim 4, wherein the lipid nanoparticle (LNP) is formulated to target the spleen.

32. The pharmaceutical composition of claim 4, wherein the LNP is formulated to deliver the mRNAto myocytes.

33. The pharmaceutical composition of claim 4, wherein the LNP is formulated to deliver the mRNA to fibroblasts.

34. The pharmaceutical composition of claim 4, wherein the LNP is formulated to deliver the mRNAto dendritic cells.

35. The pharmaceutical composition of claim 4, wherein the LNP is formulated to deliver the mRNA to macrophages.

36. The pharmaceutical composition of claim 4, wherein the LNP is engineered for selective uptake by non-hepatic immune cells.

37. The pharmaceutical composition of claim 6, wherein the mRNA is encapsulated in a lipid nanoparticle targeted to one or more tissue selected from the group consisting of: liver, spleen, lymph nodes, muscle, and lung.

38. The composition of claim 19, wherein the lipid nanoparticle is modified with a targeting ligand that binds to receptors expressed on dendritic cells.

39. The composition of claim 19, wherein the lipid nanoparticle is modified with a targeting moiety selected from the group consisting of: mannose, folate, RGD peptide, or an antibody fragment that binds to a tissue-specific receptor.

40. The system of claim 23, wherein the lipid nanoparticle is modified to enable targeting of spleen-resident antigen-presenting cells.

41. The pharmaceutical composition of claim 4, wherein the LNP comprises a targeting ligand for CD206 for selective delivery to macrophages.

42. The pharmaceutical composition of claim 4, wherein the mRNA encoding the sclgA1 antibody construct is co-formulated with an adjuvant that promotes uptake and translation in myocytes.

43. The pharmaceutical composition of claim 4, wherein the LNP is formulated for intramuscular or subcutaneous administration to facilitate delivery to lymphoid and peripheral tissues.

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