T cell-based vaccine for la crosse virus

Abstract

Provided are compositions and methods of eliciting a T-cell mediated response to La Crosse virus (LACV). Also provided are T-cell vaccines, T-cell adjuvants, and methods for performing T-cell diagnostics.

Description

[0001] Docket No. 070439.01934

[0002] T CELL-BASED VACCINE FOR LA CROSSE VIRUS CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application is entitled to priority pursuant to 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 761,248, filed on February 21, 2025. The content of the application is incorporated herein by reference in its entirety.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (070439.01934SeqList.xml; Size: 45,868 bytes; and Date of Creation: February 11, 2026) is herein incorporated by reference in its entirety.

[0006] FIELD OF THE INVENTION

[0007] This disclosure relates generally to compositions and methods for eliciting a T-cell response to La Crosse virus (LACV).

[0008] BACKGROUND

[0009] La Crosse virus (LACV) is a single-stranded, negative-sense RNA virus belonging to the Bunyavirales order. Its genome is segmented into three strands referred to as small (S), medium (M), and large (L), distinguished by their relative sizes. The S segment encodes the nucleocapsid protein (N) and nonstructural protein s (NSs), the latter of which has been shown to modulate the host cell antiviral response through innate immune pathways. The M segment encodes a glycosylated polyprotein precursor (GPC) that is cleaved by host cell proteases to generate the envelope glycoproteins Gn and Gc. Cleavage of GPC also produces the nonstructural protein m (NSm), which has been implicated in viral assembly. The L segment encodes an RNA-dependent RNA polymerase (RdRp), responsible for both transcription and replication of the viral genome.

[0010] Human infection with LACV occurs predominantly through the bite of infected Eastern tree hole mosquitoes (Aedes triseriatus), which are endemic throughout North America. Nevertheless, additional mosquito species can harbor LACV, potentially facilitating viral spread into new regions and hosts. Following entry into the human host, LACV replicates in 1

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[0012] skin muscle cells, enabling subsequent viral dissemination to multiple tissues and organs. Although most L AC V-infected individuals experience only mild, febrile symptoms, the virus can invade the central nervous system (CNS) causing a subset to experience severe neurological manifestations, including altered behavior, seizures, coma, or even death. Pediatric populations, particularly children under the age of 16, exhibit the highest susceptibility to LACV encephalitis, implicating age-associated factors in disease severity.

[0013] Currently, there are no approved antiviral therapies or vaccines against LACV, necessitating reliance on supportive care alone to mitigate disease symptoms. The absence of licensed vaccines partly reflects a limited understanding of how the host immune response mediates protection, particularly against neurological sequelae. This gap in knowledge has been difficult to address in humans, where clinical samples, especially those collected during the acute phase of infection, are challenging to obtain. However, murine models accurately recapitulate the age-dependent susceptibility7seen in humans, with weanling mice (3 weeks old) succumbing to LACV-induced neuropathology, whereas adult mice (>8 weeks old) resist peripheral LACV infection.

[0014] Previous studies in adult mice indicate a critical role of the peripheral immune system in preventing LACV neuroinvasion, given that peripheral inoculation of C57BL / 6 wild-type mice via intraperitoneal (IP) or intradermal (ID) routes does not induce overt neurological disease, whereas intracranial inoculation does. Additional research reveals that type I interferon responses are indispensable for protecting mice from lethal peripheral LACV challenge at all ages, highlighting the importance of innate immunity7in early antiviral control. However, innate immune responses play7a dual role in LACV infection, offering both protective and pathogenic effects depending on the tissue context. In peripheral tissues, activation of antiviral recognition pathways induces type I interferon responses, such as IFN-0, which suppress viral replication, control LACV infection, and limit its spread to the CNS. Conversely, similar signaling in the CNS can lead to neuronal damage and cell death. Myeloid dendritic cells, which bridge innate and adaptive immunity, are critical for early protective responses in adult wild-type mice infected with LACV. Additionally7, treatment of young wild-type mice with t pe I interferon, particularly IFN- , enhances protection mediated by myeloid dendritic cells. Notably, both innate and adaptive immune responses appear to cooperate in controlling LACV infection in adult mice. The depletion of CD4 and CD8 T cells in adult mice reverses their resistance phenotype, emphasizing that T cell-mediated responses are critical for protection and 2

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[0016] prevention of neurological complications. Conversely, in resistant adult mice, the depletion of both T cells and B cells results in increased viral loads in the brain and heightened neurological disease. However, the precise immunological correlates of protective immunity are not understood, particularly with respect to identifying virus-specific T cell responses and their antigenic targets.

[0017] SUMMARY OF THE INVENTION

[0018] In one aspect, provided is an immunogenic composition comprising an antigen fused to a cytotoxin, wherein the cytotoxin is a bacterial cytotoxin with a catalytic infectious domain removed, and the antigen induces a T-cell response to LACV. In one embodiment, the bacterial cytotoxin is Bacillus anthracis lethal factor (LFn). In one embodiment, the cytotoxin comprises the amino acid sequence of any one of SEQ ID NOs: 4-6. In one embodiment, the antigen fused is fused to the cytotoxin via the amino acid sequence of SEQ ID NO: 7.

[0019] In one embodiment, the antigen is derived from a structural protein of LACV. In some embodiments, the antigen is a Gc protein or a nucleocapsid (N) protein. In one embodiment, the antigen is derived from anonstructural protein of LACV. In some embodiments, the antigen is NSm, NSs, or RNA-dependent RNA polymerase (RdRp).

[0020] In one embodiment, provided is a pharmaceutical composition comprising the composition described herein and a pharmaceutically acceptable carrier.

[0021] In one embodiment, provided is a kit comprising the composition described herein, wherein the kit comprises:

[0022] a) an immunoassay to quantify cytokine levels from a sample of blood for detecting a T-cell-mediated immune response; and

[0023] b) instructions for use.

[0024] In one aspect, provided is a method of monitoring T-cells, comprising the following steps:

[0025] a) mixing blood from a subject with the composition described herein; b) incubating the mixture of a) for at least 24 hours at about 37 °C; c) centrifuging the mixture of b) following the incubation period;

[0026] d) collecting the supernatant from c); and

[0027] e) quantifying cytokine levels with an immunoassay.

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[0030] In some embodiments, the cytokine levels are quantified with an ELISpot assay, an ELISA assay, or flow cytometry.

[0031] In one aspect, provided is a T-cell vaccine comprising the composition described herein, wherein the T-cell vaccine comprises an aqueous or liposomal formulation. In one embodiment, the T-cell vaccine further comprises an adjuvant.

[0032] In one aspect, provided is an isolated polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. In one embodiment, said sequence is fused to an antigen. In one embodiment, said sequence is fused to the antigen via a protein linker that comprises the amino acid sequence of SEQ ID NO: 7. In one embodiment, the antigen is derived from a structural protein of LACV. In some embodiments, the antigen is a Gc protein or a nucleocapsid (N) protein.

[0033] In one aspect, provided is a method of eliciting a T-cell response in a subj ect comprising administering to the subject a composition comprising an antigen fused to a cytotoxin, wherein the antigen induces a T-cell response to La Crosse virus (LACV). In one embodiment, the cytotoxin is a bacterial cytotoxin. In one embodiment, the bacterial cytotoxin is Bacillus anthracis lethal factor (LFn). In one embodiment, the cytotoxin comprises the amino acid sequence of SEQ ID NO: 5. In one embodiment, the antigen is derived from a structural protein of LACV. In some embodiments, the antigen is a Gc protein or a nucleocapsid (N) protein.

[0034] In one embodiment, the subject is a human.

[0035] In some embodiments, the method comprises administering at least two doses of the composition to the subj ect.

[0036] In some embodiments, the T-cell response:

[0037] a) enhances a second anti-viral therapeutic agent; and

[0038] b) serves as a T-cell vaccine; and / or enables T-cell diagnostics.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A and IB illustrate a LACV-specific ex vivo cellular response in wild-type mice. FIG. 1A Top: A graph of quantified spot-forming cells (SFCs) detected by ELISPOT assay using splenic leukocytes from adult and weanling mice infected with LACV at 6 days 4

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[0041] post-infection (dpi). FIG. 1A Bottom: splenic leukocytes were treated with LFn-LACV proteins and IFN-y responses were detected by ELISPOT assay. (-) negative control: cells stimulated with LFn alone, (+) positive control: cells stimulated with PMA (phorbol 12-myristate 13-acetate). Statistical significance was determined using ANOVA test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are presented as individual points with minimum and maximum values (n = 5 mice in each group, data are representative of two independent experiments). FIG. IB is a graph that shows the percent of total responses generated against each LFn-LACV protein in adult mice at 14 dpi. LFn comprises the sequence of SEQ ID NO: 5.

[0042] FIGS. 2A and 2B depict LACV-induced CD4+and CD8+T cell expansion in LACV-infected mice. FIG. 2A is a graph of the total percentage of CD4+T cells among total splenocytes of adult and weanling mice at 6 and 14 dpi with LACV analyzed by flow cytometry. FIG. 2B is a graph of the total percentage of CD8+T cells among total splenocytes of adult and weanling mice at 6 and 14 dpi with LACV analyzed by flow cytometry. Statistical significance was determined using ANOVA test, *p < 0.05, **p < 0.01, ***p < 0.001 , ****p < 0.0001. Data are presented as individual points with mean ± standard deviation (n = 6 mice in each group, data are combined from two independent experiments).

[0043] FIGS. 3A-3D illustrate LACV-specific ex vivo splenic CD4+and CD8+T cell functional response in LACV-infected mice. FIG. 3A is a graph of the functional LACV-specific CD4+T cell responses shown as the percent of CD4 cells positive for IFNy in CD3+gate in FC / ICS in adult mice at 6 and 14 dpi compared to age-matched uninfected controls.

[0044] FIG. 3B is a graph of the functional LACV-specific CD8+T cell responses shown as the percent of CD8 cells positive for IFNy in CD3+gate in FC / ICS in adult mice at 6 and 14 dpi compared to age-matched uninfected controls. FIG. 3C is a graph of the functional LACV-specific CD4+T cell responses shown as the percent of CD4 cells positive for IFNy in CD3+gate in FC / ICS in weanling mice at 6 and 14 dpi compared to age-matched uninfected controls.

[0045] FIG. 3D is a graph of the functional LACV-specific CD8+T cell responses shown as the percent of CD8 cells positive for IFNy in CD3+gate in FC / ICS in weanling mice at 6 and 14 dpi compared to age-matched uninfected controls. (-) negative control: cells stimulated with LFn alone; (+) positive control: cells stimulated with PMA (phorbol 12-myristate 13-acetate). Statistical significance was determined using ANOVA test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are presented as individual points with minimum and maximum 5

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[0047] values, (n = 9 adult mice, data are combined from three independent experiments, 5 weanling mice, data are combined from two independent experiments). FIGS. 4A-4D show polyfunctional profiles of LACV-specific CD8+and CD4+T cells at 6 dpi. FIG. 4A shows pie charts depicting the distribution of LACV-specific CD4+T cell populations with single or dual functional profiles, defined by cytokine production (n = 9 mice, data are combined from three independent experiments). FIG. 4B is a bar graph showing the total percentage of LACV-specific CD4+T cells producing one or two cytokines in response to infection. Data are represented as mean with minimum and maximum values (n = 9 mice, data are combined from three independent experiments). FIG.4C show s pie charts depicting the distribution of LACV-specific CD8+T cell populations with single or dual functional profiles, defined by cytokine production (n = 9 mice, data are combined from three independent experiments). FIG. 4D is a bar graph show ing the total percentage of LACV-specific CD8+T cells producing one or two cytokines in response to infection (n = 9 mice, data are combined from three independent experiments).

[0048] FIGS. 5A-5E illustrate in vivo cytotoxicity and protection mediated by LACV-specific T cells. FIG.5A is a schematic representation of the in vivo cytotoxicity assay. Equal numbers of naive splenocytes pulsed with LFn-LACV-Gc or LFn-LACV-N (CFSEHlghtarget cells) and splenocytes pulsed with an irrelevant protein, LFn-ZIKV-NS3 (CFSELowmarker cells), were transferred into either naive or LACV-immune recipient mice. Percent killing was quantified by flow cytometry in recipient spleens 4 h post-transfer. FIG. 5B is a schematic of the LFn-LACV immunization strategy. One-week-old mice were immunized with 50 qg of either LFn-LACV-Gc or LFn-LACV-N. A booster dose was administered 7 days later, followed by LACV challenge at weanling age. FIG.5C is a graph of survival curves of weanling mice immunized with LFn-LACV-Gc or LFn-LACV-N compared with LFn-ZIKV-NS3 and subsequently challenged with LACV (n = 5 mice per group, data are representative of a single experiment). Statistical significance was determined using log rank test. FIG.5D is a bar chart that quantifies in vivo cytotoxicity in previously LACV-infected adult and weanling mice, as well as in LFn-LACV-Gc- or LFn-LACV-N-immunized weanling mice. Percent killing was determined by flow cytometry. Statistical significance was determined using ANOVA test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are presented as individual points with mean ± standard deviation (n = 5 mice per group, data are representative of a single experiment).

[0049] FIG.5E is a graph of the fold increase in LACV RNA in the brain of vaccinated mice at day 4

[0050] 6

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[0052] post-challenge. Weanling mice were immunized with either LFn-LACV-Gc or LFn-LACV-N and then boosted and challenged as described in FIG.5B. Brains were harvested at day 4 postinfection and viral RNA was quantified by RT-qPCR. Data are shown as individual values with mean ± standard deviation (n = 5 mice per group). Statistical significance was determined using ANOVA test, ****p < 0.0001. Data shown are representative of two independent experiments.

[0053] DETAILED DESCRIPTION OF THE INVENTION

[0054] This disclosure relates to an immunogenic composition comprising an antigen fused to a cytotoxin, wherein the antigen induces a T-cell response to LACV. Also provided are methods of performing T-cell diagnostics using the immunogenic composition described herein. There currently are no treatments for LACV. Thus, there remains a long-felt need to develop vaccines and therapies for LACV.

[0055]

[0056] In one aspect, provided is an immunogenic composition comprising an antigen fused to a cytotoxin, wherein the antigen induces a T-cell response to LACV.

[0057] As used herein, the term '‘immunogenic composition’7refers to a composition that comprises an antigen that induces an immune response.

[0058] As used herein, the term “T-cell response’’ refers to an antigen-specific response by T- cells that is a key component of an adaptive immune response to a pathogen. A T-cell response can be measured by methods as known in the art, including, but not limited to a cytotoxicity assay, a proliferation assay, enzyme-linked immunospot analysis, or a delayed-type hypersensitivity test.

[0059] As used herein, the term “pathogen” refers to an organism, including a microorganism, which causes disease in another organism (e.g., animals and plants) by directly infecting the other organism, or by producing agents that cause disease in another organism (e.g., bacteria that produce pathogenic toxins and the like). As used herein, pathogens include, but are not limited to bacteria, protozoa, fungi, nematodes, viroids and viruses, or any combination thereof, wherein each pathogen is capable, either by itself or in concert with another pathogen, of eliciting disease in vertebrates including but not limited to mammals, and including but not limited to humans. As used herein, the term “pathogen” also encompasses microorganisms which may not ordinarily be pathogenic in a non-immunocompromised host.

[0060] As used herein, the term “cytotoxin” refers to a substance that has a toxic effect on a cellular function. In some embodiments, the cytotoxin is a bacterial cytotoxin with the catalytic infectious domains removed. In some embodiments, the bacterial cytotoxin is Bacillus 7

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[0062] anthracis, Bacillus thuringiensis (Bt), or Bacillus shihchuchen. Bacillus thuringiensis is a soil-borne bacteria that produces a variety of insecticidal proteins. Bacillus shihchuchen is related to Bacillus thuringiensis (Cheng et al. Int. J. Mol. Sci. 2023. 24( 11): 9636). Bacillus anthracis. Bacillus thuringiensis, and Bacillus shihchuchen are Gram-positive bacteria.

[0063] In some embodiments, the cytotoxin comprises an amino acid sequence as shown in Table 1. In some embodiments, the cytotoxin comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NOs: 2 or 4 such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or any percent therebetween or in a range of any high value and low value selected from these values. Truncated SEQ ID NOs: 2 and 4 were generated based on domains believed to be involved in protein trafficking into the cell.

[0064] In one embodiment, the cytotoxin comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the cytotoxin comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 5 such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or any intermediate values therebetween. In some embodiments, the cytotoxin comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 6 such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or any percent therebetween or in a range of any high value and low value selected from these values. SEQ ID NO: 6 was generated based on domains known to traffic antigens into the cytosol.

[0065] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the nonlimiting examples below.

[0066] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two 8

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[0068] amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.

[0069] 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0070] Table 1. Representative amino acid sequences of the cytotoxin fused to an antigen.

[0071]

[0072] In some embodiments, the amino acid sequence of the cytotoxin is no greater than 78 amino acids in length such as 50 amino acids, 55 amino acids, 60 amino acids, 65 amino acids,

[0073] 9

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[0075] 70 amino acids, 75 amino acids, 78 amino acids, or any number of amino acids in-between. In some embodiments, the amino acid sequence of the cytotoxin is no greater than 90 amino acids in length such as 70 amino acids, 75 amino acids, 80 amino acids, 85 amino acids, 90 amino acids, or any number of amino acids in-between. In some embodiments, the amino acid sequence of the cytotoxin is no greater than 104 amino acids in length such as 80 amino acids, 85 amino acids, 90 amino acids, 95 amino acids, 100 amino acids, 101 amino acids, 102 amino acids, 103 amino acids, 104 amino acids, or any number of amino acids in-between. In some embodiments, the amino acid sequence of the cytotoxin is no greater than 250 amino acids in length, such as about 100 amino acids, 150 amino acids, 200 amino acids, 250 amino acids, or any number of amino acids in-between. In some embodiments, the amino acid sequence of the cytotoxin is no greater than 350 amino acids in length, such as about 200 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, or any number of amino acids in-between. In some embodiments, the amino acid sequence of the cytotoxin is no greater than 450 amino acids in length, such as about 300 amino acids, 350 amino acids, 400 amino acids, 450 amino acids, or any number of amino acids in-between.

[0076] Notably, the cytotoxin may or may not bind the target antigen, but it will elicit a T-cell mediated immune response. As used herein, the term "‘target antigen” refers to a molecular target for which it would be desirable to elicit a T-cell mediated immune response.

[0077] As used herein, “antigen” refers to a substance that induces an immune response. The antigen can be a protein, peptide, polysaccharide, lipid, or a nucleic acid. In one embodiment, the antigen is an exogenous antigen. In one embodiment, the antigen is derived from LACV. In preferred embodiments, the antigen is derived from a structural protein such as a Gc glycoprotein, a Gn glycoprotein, or a nucleocapsid (N) protein of LACV. In some embodiments, the antigen is derived from the nonstructural proteins NSm, NSs and the RNA-dependent RNA polymerase (RdRp) of LACV. Exemplary sequences of nonstructural and structural proteins of LACV are shown in Table 2.

[0078] In some embodiments, the antigen fused to the cytotoxin comprises an amino acid sequence as shown in Table 2. In some embodiments, the antigen fused to the cytotoxin comprises an amino acid sequence having at least 80% sequence identity to any one of the sequences shown in Table 2 such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or any intermediate values therebetween.

[0079] 10

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[0081] Table 2. Representative sequences of nonstructural and structural proteins of LACV.

[0082]

[0083] In some embodiments, the immunogenic composition is prepared according to known methods in the art.

[0084] In some embodiments, the cytotoxin is fused to the antigen of interest via a protein linker as known in the art. In one embodiment, the protein linker is an enzyme-cleavable linker. In one embodiment, the protein linker is a protease-sensitive linker. In one embodiment, the protein linker has moderate flexibility such as containing AP (Alanine-Proline) repeat linkers. In some embodiments, the protein linker is hydrophilic. In one embodiment, the protein linker is a rigid linker (such as a-helical). In one embodiment, the protein linker is a helix-forming linker for maintaining structural integrity. In one embodiment, the protein linker comprises the 11

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[0086] amino acid sequence of SEQ ID NO: 7 (GGSGGS). In some embodiments, the protein linker comprises a sequence as shown in Table 3.

[0087] Table 3. Representative protein linker sequences.

[0088] <<<

[0089]

[0090] “n” indicates any number of repeating sequence.

[0091] In some embodiments, the protein linker comprises a sequence as described in U.S. Patent No. 11,833,212, which is incorporated herein by reference in its entirety.

[0092] As used herein, the term “protein linker’" or “linker” refers to short peptide sequences that occur between protein domains. In one embodiment, a protein linker comprises flexible amino acid residues such as glycine and serine.

[0093] Pharmaceutical Composition

[0094] In some embodiments, provided is a pharmaceutical composition comprising the immunogenic composition described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition is particularly suitable for the treatment or prophylaxis of LAC V described herein. In some embodiments, the pharmaceutical composition is in the form of a tablet, capsule, or parenteral. In some embodiments, the pharmaceutical composition is a 12

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[0096] liposome. In some embodiments, the pharmaceutical composition is administered to a subject in need thereof via various delivery modes including, but not limited to intramuscular, intravenous, intraperitoneal, intradermal injection, oral, sublingual, parenteral, and subcutaneous.

[0097] As used herein, the term “subject” refers to a mammal. The term “mammal” as used herein is intended to include, but is not limited to, humans, laboratory- animals, domestic pets, and farm animals. Mammals include, but are not limited to, a human or non-human mammal, such as a canine, bovine, equine, ovine, orfeline, etc. Individuals and patients are also subjects herein.

[0098] In some embodiments, the pharmaceutical composition is formulated with a mixture of active and inactive ingredients. The active ingredients of the pharmaceutical composition can be in an amount from about 0.5% to about 15% w / w. For example, the active ingredients can be in an amount of about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or any percent therebetyveen or in a range of any high value and loyv value selected from these values.

[0099] In some embodiments, the pharmaceutical composition further comprises an adjuvant. In some embodiments, the adjuvant is selected from the group comprising alum, calcium phosphate, poly-IC, Freund’s adjuvant, MF59, and AS03.

[0100] In some embodiments, the pharmaceutical composition can comprise lubricants, binders, diluents, and glidants as known in the art.

[0101] In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils, or injectable organic esters. The choice of the pharmaceutically acceptable carrier depends on the route of administration of the composition. Furthermore, the pharmaceutically acceptable carrier can contain ingredients that stabilize, increase solubility or increase the uptake of the immunogenic composition as disclosed herein.

[0102] Kit

[0103] In some embodiments, provided is a kit that comprises the immunogenic composition described herein. In one embodiment, the kit is used for T-cell diagnostics, wherein the kit detects cytokine levels (indicative of a T-cell response) via an assay such as an immunoassay.

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[0106] In some embodiments, the kit comprises an immunogenic composition described herein, blood draw tubes, and antibodies for flow cytometry or intracellular cytokine staining.

[0107] In some embodiments, the kit comprises an immunogenic composition described herein, blood draw tubes, and an immunoassay (such as for detecting cytokine levels). In some embodiments, the kit comprises an immunogenic composition described herein, blood draw tubes, and an ELISpot (enzyme-linked immunosorbent spot) assay or an ELISA assay. For example, the kit can comprise Vacutainer™ venous blood collection tubes, wherein the inner walls of the tubes are coated with a single or multiple (multiplex test) fusion immunogen(s). Blood from a subject is collected in the tubes, mixed, and incubated at 37 °C for about 24 to about 48 hours. Following the incubation period, blood is spun down and the resulting supernatant is analyzed via an anti-cytokine ELISA assay.

[0108] In some embodiments, the kit comprises an immunogenic composition described herein and an immunoassay to detect cytokine levels such as an ELISpot assay. In some embodiments, the kit comprises an immunogenic composition described herein, cells of interest, and an immunoassay to detect cytokine levels. For example, PBMCs (human peripheral blood mononuclear cells) can be treated with the immunogenic composition in a 96-well plate coated with the anti-cytokine antibodies. The cytokine levels can then be measured via an ELISpot assay.

[0109] In some embodiments, the kit comprises an LFn (N-terminal domain of lethal factor) protein fused to a cytotoxin described herein that is used to treat cells. For example, PBMCs can be treated with an LFn fusion protein in a 96-well plate coated with the anti-cytokine antibodies. Resulting cytokine production can then be measured by ELISpot or ELISA assay.

[0110] The kit can further include instructions for use.

[0111] Methods of Use

[0112] Also provided are methods of using the immunogenic composition described herein. The immunogenic composition can be used as a T-cell vaccine, a T-cell adjuvant, and for T-cell diagnostics.

[0113] I. T-cell Vaccine

[0114] In some embodiments, the immunogenic composition described herein is used in a T-cell vaccine to elicit a T-cell response in a subject. In one embodiment, the subject has LACV.

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[0117] In some embodiments, the T-cell vaccine comprises an aqueous formulation. In some embodiments, the T-cell vaccine is a liposomal vaccine. In some embodiments, the T-cell vaccine further comprises an adjuvant. In some embodiments, the adjuvant is selected from the group comprising alum, calcium phosphate, poly-IC, Freund’s adjuvant, MF59, and AS03. In some embodiments, the adjuvant is alum.

[0118] In some embodiments, the T-cell vaccine is administered to a subject in need thereof intramuscularly, intravenously, intraperitoneally, intradermally, orally, sublingually, parenterally, or subcutaneously.

[0119] In some embodiments, the T-cell vaccine is administered at a dose of about 0.1 mg / kg of body weight to about 100 mg / kg of body weight of the subject in need thereof. For example, the T-cell vaccine can be administered to a subj ect in need thereof at a dose of about 0.1 mg / kg, about 1.0 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg of body weight, or any dose in-between. In other embodiments, the T-cell vaccine is administered in an amount of about 50 mg to about 1000 mg to the subject in need thereof. For example, the T-cell vaccine can be administered to a subject in need thereof in an amount of about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg. about 950 mg, about 1000 mg. or any amount therebetween or in a range of any high value and low value selected from these values. In some embodiments, the T-cell vaccine is administered to a subject in need thereof in an amount of about 10 pg to about 1 gram. For example, the T-cell vaccine can be administered to a subject in need thereof in an amount of about 10 pg, about 20 pg, about 30 pg. about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, about 100 pg, about 200 pg, about 300 pg, about 400 pg, about 500 pg, about 600 pg, about 700 pg, about 800 pg, about 900 pg, about 1000 pg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1 gram (1000 mg), or any amount therebetween or in a range of any high value and low value selected from these values.

[0120] In some embodiments, at least two doses of the T-cell vaccine are administered to the subject such as at least two doses, three doses, four doses, five doses, or more. In some embodiments, a second dose of the T-cell vaccine is administered to the subject at least a week after the first dose such as at least one week, two weeks, three weeks, four weeks, etc. In some 15

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[0122] embodiments, a second dose of the T-cell vaccine is administered to the subject at least one month after the first dose such as at one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, etc. In some embodiments, the T-cell vaccine is prepared using standard methods known in the art. For example, the T-cell vaccine can be produced in bacterial fermenters in a bioreactor along with purification processes. The bacterial fermenters can be E. coli.

[0123] II. T-cell Adjuvant

[0124] In some embodiments, the immunogenic composition described herein is used to enhance a second anti-viral therapeutic agent and thus functions as a T-cell adjuvant.

[0125] In some embodiments, the immunogenic composition described herein is coadministered with a second anti-viral therapeutic agent. In some embodiments, the immunogenic composition described herein is administered to a subject prior to administration of a second anti-viral therapeutic agent. In some embodiments, the immunogenic composition described herein is administered to a subject following administration of a second anti-viral therapeutic agent. In some embodiments, the immunogenic composition described herein is administered to a subject within 48 hours of diagnosis, which includes administering either within 24 hours of diagnosis or at the time of diagnosis. In some embodiments, the immunogenic composition described herein is administered to a subject within 48 hours of infection, which includes administering within 24 hours of infection.

[0126] In some embodiments, the second anti-viral therapeutic agent is selected from the group comprising a vaccine, an anti-viral, an antibody, and a small molecule. For example, the small molecule can include, but is not limited to a small molecule agonist, a small molecule antagonist, and antibiotics.

[0127] In some embodiments, the immunogenic composition as a T-cell adjuvant enhances the response to the second anti-viral therapeutic agent by at least 50% such as by about 50%, about 60%, about 70%. about 80%, or about 90%. As used herein, the term “enhances the response” or “enhanced response” refers to a measurable improvement in at least one symptom of a disease or condition.

[0128] In some embodiments, the T-cell adjuvant is administered at a dose of about 0.1 mg / kg of body weight to about 100 mg / kg of body weight of the subject in need thereof. For example, the T-cell vaccine can be administered to a subj ect in need thereof at a dose of about 0.1 mg / kg,

[0129] 16

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[0131] about 1.0 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg of body weight, or any dose in-between. In other embodiments, the T-cell adjuvant is administered in an amount of about 50 mg to about 1000 mg to the subject in need thereof. For example, the T-cell adjuvant can be administered to a subject in need thereof in an amount of about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg. about 250 mg, about 300 mg, about 350 mg, about 400 mg. about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, or any amount inbetween.

[0132] In some embodiments, the T-cell adjuvant is administered to a subject in need thereof in an amount of about 10 pg to about 1 gram. For example, the T-cell adjuvant can be administered to a subject in need thereof in an amount of about 10 pg, about 20 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, about 100 pg, about 200 pg, about 300 pg, about 400 pg, about 500 pg, about 600 pg, about 700 pg, about 800 pg, about 900 pg, about 1000 pg, about 100 mg, about 200 mg. about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1 gram (1,000 mg), or any amount in-between.

[0133] III. T-cell Diagnostics

[0134] In some embodiments, provided is a method of using the immunogenic composition for T-cell monitoring or diagnostics. For example, a sample (e.g., blood) can be combined with the immunogenic composition, incubated with said composition for a given period of time, and then cytokine production can be measured using an immunoassay such as flow cytometry and ELISpot assays. Increased cytokine levels correlate with an activated T-cell response.

[0135] In some embodiments, the immunogenic composition is combined with blood from a subject. After about 24-48 hours of incubation at 37 °C, the blood is spun down and the supernatant is analyzed for cytokine levels. The cytokine levels are quantified via ELISpot assays, ELISA assays, flow cytometry, and / or intracellular cytokine staining.

[0136] As used herein and in the appended claims, the singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise.

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[0139] As used herein, the terms “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted.

[0140] As used herein, the phrases “in one embodiment,” “in various embodiments,” “in some embodiments,” and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may unless the context dictates otherwise.

[0141] As used herein, the terms “and / or” or “ / ” mean any one of the items, any combination of the items, or all of the items with which this term is associated.

[0142] As used herein, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise. All publications mentioned herein are incorporated herein by reference in their entireties.

[0143] The following examples serve to further illustrate the methods of the present disclosure.

[0144] EXAMPLES

[0145] Example 1. Materials and Methods.

[0146] This Example describes the materials and methods used in Examples 2-5.

[0147] 1 A. Infection of mice with LACV and neurological disease progression criteria

[0148] All animal studies were approved by the Rutgers IACUC. Wild-fype (C57B1 / 6) mice were purchased from Jackson Laboratories and maintained in a breeding colony at the Child Health Institute of New Jersey. LACV (La Crosse Virus, NR-540). a human isolate was purchased and obtained from BEI Resources. Mice at 3 (weanling) or 8 (adult) weeks of age were inoculated with 10’ PFU of LACV in phosphate-buffered saline (PBS) intraperitoneally in a volume of 200 pL / mouse. Mice were observed daily for signs of neurological disease that included hunched posture, seizures, reluctance, or inability to move normally, or paralysis. Animals with clear clinical signs of neurological disease were scored as clinical and euthanized immediately. As previously described, 100% of weanling mice were clinical at day 6 post infection (Alatrash, R et. al., (2024). Age-specific dynamics of neutralizing antibodies,

[0149] 18

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[0151] cytokines, and chemokines in response to La Crosse virus infection in mice. J Virol 98, e0176224).

[0152] IB, Bioinformatic analyses

[0153] All 9mer peptides encoded in the LACV proteome were predicted for their binding affinity7to MHC class I alleles A*0201, A*0101, A*1101, and B*0702. C57BL / 6 MHC I was also included for analyses (H2Kb). Binding predictions were performed using NetMHCpan 4.1 EL MHC class I prediction tool available on the Immune Epitope Database (IEDB) (iedb.org) website (Lundegaard C et al. 2008. NetMHC-3.0: accurate web-accessible predictions of human, mouse, and monkey MHC class I affinities for peptides of length 8-11. Nucleic Acids Res 36:W509-12). Peptides were selected if they were in the top 1% of binders for each MHC molecule analyzed for further analysis for MHC I immunogenicity available on the IEDB website. Immunogenic peptides were predicted as previously described (Calis JJ et al. 2013. Properties of MHC class I presented peptides that enhance immunogenicity. PLoS Comput Biol 9:el003266). Immunogenic peptides were assigned a positive immunogenic score. A total of 1769-mer peptides were identified by MHC class I predictions, as described above. MHC II immunogenicity score of peptides predicted to bind using 7-allele method.

[0154] IC. Bacillus cmthracis lethal factor (LFn) fusion protein design

[0155] Commercially synthesized gene fragments encoding the glycoprotein Gc, Gn, the nucleoprotein N, the nonstructural proteins NSm, NSs. and the RNA-dependent RNA polymerase (RdRp) of LACV were cloned into the LFn expression vector (pET-28 (b)+LFn). The amino acid sequences of Gc, Gn, N, NSm, NSs, and RdRp used are shown in Table 2. The pET-28 (b)+LFn containing the coding sequences of the LACV proteins were transformed into Escherichia coli BLR (DE3) (Millipore, Medford, MA). Selected clones were sequenced and used to verify the reading frame, and clones containing the correct sequence were used for protein expression. LFn used in the fusion proteins has the amino acid sequence of SEQ ID NO: 5

[0156] The LFn-LACV proteins were expressed upon induction of isopropyl-0-d-thiogalactopyranoside (IPTG; Sigma-Aldrich, St. Louis, MO, USA) in 5 liters of Luria broth containing kanamycin for 2 to 4 h. Cells were pelleted by centrifugation and resuspended in imidazole (1 mM) binding buffer (Novagen, Madison, WI) in the presence of a protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL). Cell pellets were sonicated and centrifuged at 4°C, and the supernatants were loaded in an equilibrated nickel-charged column for affinity purification. The bound proteins were eluted in 100 to 200 mM imidazole, desalted 19

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[0158] with a Sephadex G-25M column (Sigma-Aldrich, St. Louis, MO, USA), and eluted in phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA). The PBS-eluted proteins were passed through Detoxi-Gel (Thermo Fisher Scientific, Rockford, IL). Protein concentrations were determined, and samples were stored at -80°C.

[0159] ID. Splenic leukocytes harvest

[0160] Spleens from LACV-infected and uninfected mice were harvested at 6 and 14 days post-infection (dpi), as described previously (Lopez K et al. 2021. Novel murine models for studying Cache Valley virus pathogenesis and in utero transmission. Emerg Microbes Infect 10:1649-1659). Spleens were processed into single-cell suspensions by passing them through a 70 pM mesh filter, followed by red blood cell lysis using RBC Lysis Buffer (Thermo Fisher Scientific). Isolated cells were then seeded for subsequent ELISPOT and flow cytometry / ICS (intracellular cytokine staining) assays. Perfused whole brains were isolated at 6 dpi and a single-cell suspension made by homogenization and passage through a 70 pm filter. Cells w ere pelleted and resuspended in 70% Percoll / PBS and underlay ed on a 0-30% step Percoll gradient, which was centrifuged at 800g for 25 min at 4 °C. CNS immune cells were recovered at the 30-70% interface, rinsed in PBS, and placed on ice to await fixing or staining.

[0161] IF. ELISPOT test

[0162] Ex vivo ELISPOT assays w ere performed as previously described (Herrera BB et al.

[0163] 2018. T Cell Responses to Nonstructural Protein 3 Distinguish Infections by Dengue and Zika Viruses. mBio 9). Briefly, 96-well plates (Millipore) were coated with anti-mouse IFN-y antibodies (BD Biosciences) and incubated overnight at 4°C. Plates were blocked with RPMI 10% FBS and washed 2 times. Splenic leukocytes harvested from LACV-infected mice, as well as uninfected controls were seeded in triplicate at 2 x 105cells / well. LFn-LACV immunogens were added to each well (5 pg / ml) and plates were incubated at 37 °C with 5% CO2 for 18-24 hours. Cells stimulated with PMA (Phorbol 12-mynstate 13-acetate) served as a positive control and cells stimulated with LFn alone functioned as a negative control. After incubation and removal of cells, the detection antibody (BD Biosciences) was added and incubated at room temperature. Plates were washed and then incubated with streptavidin HRP (BD Biosciences). After washing, AEC substrate (BD Biosciences) was added before rinsing with water and air-drying. Digitized images were analyzed using a CTL ImmunoSpot® reader (Cellular Technology Limited). LFn-LACV immunogen-specific spots were calculated by subtracting the mean of the negative control values from the mean values of the specific stimulation. Positive responses were greater than four times the mean background, three 20

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[0165] standard deviations above the background, and >55 spot-forming cells (SFC) / 2 x 105total splenic leukocytes.

[0166] IG. Flow cytometry and Intracellular Cvtokine Staining (ICS) assay

[0167] Briefly, isolated cells were seeded at a density of 2 x 106cells per well in a 96-well Il-bottom plate, with LFn-LACV immunogens added to each well at a concentration of 10 pg / ml, followed by incubation at 37°C with 5% CO2for 18 hours. Cells stimulated with PMA served as a positive control and cells stimulated with LFn alone served as a negative control. Brefeldin A inhibitor (ER-Golgi protein trafficking inhibitor) was added during the last 4 hours of incubation. Post-incubation, cells were stained for viability using a viability' dye (LIVE / DEAD Fixable Aqua Dead Cell Stain Kit, Thermo Fisher Scientific) and Fc receptors were blocked using anti-mouse CD16 / CD32 Fey III / II (Thermo Fisher Sciences) and normal mouse serum. Cells were then stained for extracellular markers using a panel of antibodies (CD8a- Alexa Fluor 488, CD19- Brilliant Violet 650, CD4- APC-eFluor 780, CD3- Alexa Fluor 700, CD45.2-PE-Cy7 (only for CNS sample); all from Thermo Fisher Scientific), washed, and fixed and permeabilized using the FIX & PERM Cell Fixation and Permeabilization Kit (Thermo Fisher Scientific). Following permeabilization, cells were stained for intracellular cytokines (Granzyme B- PE-Cyanine 5.5, IL-2-PE, TNF-alpha- Brilliant Violet 421, IFN-y-eFluor 660; all from Thermo Fisher Scientific), washed, and resuspended in FACS buffer. Flow cytometry data was acquired using the Aurora system (Cytek® Biosciences) and analyzed using FlowJo software (version 10.2; TreeStar, Inc), retaining live cells and excluding doublets using SSC-A and FSC-A gating.

[0168] IH. In vivo cytotoxicity assay

[0169] Adult wild-type (C57B1 / 6) mice (recipients) were infected with 103LACV. Splenocytes (targets) were harvested from donor naive adult wild-type mice. Red blood cells (RBCs) were lysed, and the target cells were pulsed with 10 pg / ml of the irrelevant LFn-immunogen (ZIKV), LFn-Gc, LFn-N and LFn-NSm for 18 h at 37°C. The cells were then washed and labeled with Carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) in PBS / 0.1% BSA for 10 min at 37°C. LFn-LACV-pulsed cells were labeled with 5 pM CFSE (CFSEhlgh) and the irrelevant protein-pulsed cells with 0.5 pM CFSE (CFSElow). After washing, the two cell populations were mixed at a 1 : 1 ratio and 5 x 106cells from each population were injected intravenously (i.v.) into naive or infected recipient mice at 14 dpi. After 4 h, the mice were sacrificed and splenocytes were analyzed by flow cytometry, gating on lymphocyte cells using SSC-A and FSC-A gating. The percentage killing was calculated as follows: 100 - 21

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[0171] ((percentage LACV peptide-pulsed in infected mice / percentage irrelevant peptide-pulsed in infected mice) / (percentage LACV peptide-pulsed in naive mice / percentage irrelevant peptide-pulsed in naive mice)) x 100.

[0172] II. LFn-LACV immunizations

[0173] One week old mice were immunized s.c. with 50 pg of each LFn-Gc or LFn-N protein. Mock-immunized mice received LFn-ZIKV-NS3 protein. Mice were boosted with the same dose after 7 days following the first immunization. Mice were then challenged 7 days after the second dose immunization with 1000 PFU of LACV. Mice were monitored for neurological signs development.

[0174] 1 J. Quantification of viral RNA in brain tissue

[0175] Total RNA was extracted from homogenized mouse brains using the RNeasy Lipid Tissue Mini Kit (Qiagen), following the manufacturer’s protocol. Briefly, 20-30 mg of brain tissue was homogenized in QIAzol lysis reagent, followed by phase separation with chloroform and purification on silica-membrane spin columns. RNA concentration and purity were assessed using a NanoDrop One spectrophotometer (Thermo Fisher Scientific). One microgram of total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Cat# 4368814) with random primers, in accordance with the manufacturer's instructions.

[0176] Quantitative PCR (qPCR) was performed using the PowerTrack SYBR Green Master Mix (Thermo Fisher Scientific) on a QuantStudio 3Flex Real-Time PCR System (Applied Biosystems). The following LACV-specific primers targeting the S segment were used: forward 5’-ATTCTACCCGCTGACCATTG-3’ (SEQ ID NO: 48) and reverse 5’-GTGAGAGTGCCATAGCGTTG-3’ (SEQ ID NO: 49) (Hefti, H P. et al. (1999). Human MxA protein protects mice lacking a functional alpha / beta interferon system against La crosse virus and other lethal viral infections. J Virol 73, 6984-6991). GAPDH was used as the internal control gene. The ACt was determined by subtracting the average GAPDH Ct from the corresponding LACV Ct. To evaluate relative viral load, AACt values were computed using the vaccinated group as the calibrator. Fold changes in viral RNA abundance relative to the vaccinated group were calculated using the 2A(-AACt) method.

[0177] IK. Quantification and Statistical Analysis

[0178] All statistical analyses were performed using GraphPad Prism v7.01. Flow cytometry data were analyzed using FlowJo vl0.2, and ELISPOT data were quantified using ImmunoSpot software (Cellular Technology Limited). Statistical comparisons were made using one-way or two-way 22

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[0180] ANOVA with appropriate post hoc tests or log-rank (Mantel-Cox) test for survival analyses. For all analyses, p < 0.05 was considered statistically significant. Exact values of n (indicated in each figure legend) refer to the number of biological replicates, ty pically the number of individual mice per group. Where applicable, technical replicates (e.g., ELISPOT wells) were averaged per biological replicate. Data are presented as either mean ± standard deviation (SD) or individual data points with mean and range, as indicated.

[0181] Example 2. Adult mice mount a robust LACV-specific cellular response, while weanling mice lack these responses.

[0182] Previous studies have documented high immunogenicity of structural antigens in bunyaviruses, but the immunogenicity of non-structural proteins has not been thoroughly investigated. To identify LACV T cell targets, a study was performed using in silico peptide analysis of the entire LACV proteome, evaluating potential epitopes restricted by both human and mouse MHC Class I and Class II molecules. The analysis highlighted multiple immunogenic peptides in structural and non-structural proteins. Out of 176 total peptides identified as binders to MHC Class I molecules, an average of 12, 18, and 7 peptides were associated with Gn, Gc, and N, respectively. For the non-structural proteins, 5, 6, and 59 peptides were identified binding to NSs, NSm, and RdRp, respectively, suggesting these proteins as dominant targets for T cell-mediated immunity suggesting that these proteins may serve as dominant targets for T cell-mediated immunity (Table 1). Among the peptides identified, Gc and RdRp contained the highest number of highly immunogenic sequences. Notably, 12 peptides were predicted to bind to both human and mouse MHC Class I molecules, underscoring their potential as cross-species immunogenic epitopes (Table 1). Based on these predictions, ex vivo analyses were performed using a panel of immunogens, consisting of a modified Bacillus anthracis lethal factor (LFn) fused to each of the LACV structural or non-structural proteins. LFn has been shown to transport full-length antigens into the cytosol to induce virus-specific T cell responses via the MHC Class I and Class II pathways (Herrera BB et al. 2018. Sustained Specific and Cross-Reactive T Cell Responses to Zika and Dengue Virus NS3 in West Africa. J Virol 92; Herrera BB et al. 2018. T Cell Responses to Nonstructural Protein 3 Distinguish Infections by Dengue and Zika Viruses. mBio 9; Herrera BB et al. 2018. A modified anthrax toxin-based enzyme-linked immunospot assay reveals robust T cell responses in symptomatic and asymptomatic Ebola virus exposed individuals. PLoS Negl Trop

[0183] 23

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[0185] Dis 12:e0006530; Akanmu S et al. 2023. High SARS-CoV-2 seroprevalence in Lagos, Nigeria with robust antibody and cellular immune responses. J Clin Virol Plus 3: 100156).

[0186] To quantify the LACV-specific cellular responses, IFN-y production by splenic leukocytes from LACV-infected and uninfected C57BL / 6 wild-tjpe mice was measured in enzyme-linked immunospot (ELISPOT) assays at 6 and 14 days post-infection. Adult mice mounted a robust cellular response to multiple LACV proteins by 6 dpi, which persisted by 14 dpi (FIG. 1A). Structural proteins Gc and N induced the strongest responses, followed by NSm as compared to the negligible responses observed in uninfected adult controls (FIG. IB). In contrast, weanling mice at 6 dpi showed minimal IFN-y responses to any LACV proteins, which were similar to the uninfected controls (FIG. 1A). Because all weanling mice succumbed to infection by 7 dpi, data at 14 dpi could not be obtained. These data indicate that the age-dependent immunological disparity in LACV infection extends to both the magnitude and breadth of T cell responses.

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[0189] Table 4. In silico prediction of immunogenic peptides within LACV proteome with HLA-restriction. Predicted immunogenic LACV-derived epitopes with human and / or MHC restriction.

[0190]

[0191] Example 3. Adult mice exhibit a significantly higher percentage of both CD4+and CD8+splenic T cells following LACV infection compared to weanlings.

[0192] It was then evaluated whether the limited LACV-specific cellular responses in weanlings reflect a smaller pool of available T cells. Splenocytes from adult and weanling mice were analyzed by flow cytometry at 6 and 14 dpi for the frequency of CD4+and CD8+T cells. In adult mice, LACV infection triggered a significant increase in CD4+T cell numbers at 6 dpi compared to age-matched uninfected controls at 6 dpi (FIG. 2A). This population expansion 25

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[0194] persisted through 14 dpi. Similarly, CD8+T cells in adults exhibited a more pronounced expansion, reaching statistical significance at both time points (FIG. 2B). By contrast, the percentage of CD4+and CD8+T cells in weanlings at 6 dpi remained unchanged from uninfected age-matched controls and was significantly lower than the percentages observed in infected adult mice at 6 dpi, corresponding with their poor ex vivo IFN-y response. These findings indicate that both CD4+and CD8+T cell numbers expand in adults, but not in weanlings post LACV-challenge.

[0195] Example 4. Adult mice mount functional LACV-specific CD4+ and CD8+ T cell responses, whereas weanling mice do not.

[0196] To determine the functional capacity of virus-specific T cells in adult and weanling mice following LACV challenge, intracellular cytokine staining was performed on splenic leukocytes pulsed with LFn-LACV immunogens. The production of IFN-y, granzyme B, interleukin-2 (IL-2), and TNF-a in CD4+ and CD8+ T cells was simultaneously measured (FIGS.3A-3D). Adult mice displayed robust IFN-y responses across structural and most non-structural proteins by 6 dpi. In adult mice, CD4+and CD8+T cells displayed a robust IFN-y response to all structural and most non-structural LACV proteins as early as 6 dpi, with levels significantly higher than in age-matched uninfected controls (FIGS. 3A and 3B). CD4+T cell responses began to decline by 14 dpi, while CD8+T cell responses increased at 14 dpi against most LACV proteins, indicating sustained effector activity of CD8 T cells (FIGS. 3A and 3B). Importantly, weanling mice exhibited negligible cytokine responses by CD4+and CD8+T cells, mirroring their poor splenic T cell expansion and heightened mortality (FIGS.

[0197] 3C and 3D)

[0198] A similar trend was observed for other cytokines. Granzyme B expression was significantly elevated in adult mice, but was minimal in weanlings. IL-2 and TNF-a responses were generally weak across both age groups, though adult mice consistently showed higher levels than weanlings. TNF-a expression was detectable in adults and weanlings but remained low overall compared to other analyzed cytokines. These results indicate that virus-specific T cells in adult mice mount functional effector responses to LACV infection, whereas such responses are notably weak or absent in weanling mice.

[0199] Polyfunctionality was further examined by assessing the capacity of T cells to concurrently produce multiple cytokines. In adult mice, -20-34% of CD8+T cells co-produced IFN-y and granzyme B at 6 dpi, rising to 54-69% by 14 dpi (FIGS. 4A and 4B). A smaller 26

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[0201] proportion of CD8+T cells demonstrated dual functionality involving granzyme B with IL-2 or IFN-y with IL-2 (FIGS.4A and 4B). These polyfunctional responses were most pronounced following pulsing cells with the LFn-LACV Gc, Gn, or RdRp immunogens, underscoring the immunogenicity of these proteins (FIGS. 4A and 4B). CD4+T cells also exhibited multicytokine functionality, although IFN-y was the dominant effector molecule (FIGS. 4C and 4D). These CD4+T cell effector functions were sustained at 14 dpi, underscoring their role in orchestrating immune responses during LACV infection. Thus, in contrast to weanlings, adult mice generated a robust, polyfunctional T cell response that likely contributes to effective viral clearance and survival.

[0202] Example 5. CD8+T cells against LACV Gc andN exhibit potent in vivo cytotoxicity and confer protection of weanlings upon vaccination.

[0203] Given the prominent ex vivo cytotoxic profile of CD81T cells responding to Gc and N proteins, in vivo cytotoxicity assays were performed based on carboxyfluorescein succinimidyl ester (CFSE). CFSE-labeled splenocytes pulsed with individual LFn-LACV proteins (LFn-LACV-Gc or -N), were transferred into LACV-infected adult mice at 14 dpi, and LACV-infected weanlings at 6 dpi, and the percentage of target cell killing was calculated (FIG. 5A).

[0204] In adult mice, target cells pulsed with LFn-LACV-Gc or -N were eliminated at rates of -53% and -40%, respectively (FIGS. 5A and 5D). In contrast, weanling mice exhibited minimal killing of target cells pulsed with LFn-LACV-Gc (-5%) or LFn-LACV-N (-7%) at 6 dpi, the latest timepoint that could be assessed (FIGS. 5A and 5D). These results show that LACV-specific T cells mediate cytotoxicity in LACV-infected adult mice, but not in weanlings.

[0205] To translate these findings into a vaccination strategy for the susceptible weanling mice, immunized neonatal mice (1 week old) were immunized with either LFn-LACV Gc or LFn-LACV N and subsequently challenged with LACV when they reached weanling age (FIGS.

[0206] 5B and 5C). A single dose of Gc or N significantly improved survival compared to control groups, whereas a second dose substantially increased survival rates to -70-80%. Immunized mice exhibited measurable in vivo cytotoxic activity of 19% with a single dose, confirming that vaccine-elicited T cell responses mediate partial protection against LACV (FIG.5B). The two-dose immunization regimen significantly improved survival compared to control groups, increasing survival rates to -70-80% (FIG.5C). To assess whether immunization impacted T cell cytotoxicity in weanlings, the CFSE-based in vivo cytotoxicity assay was repeated postimmunization. CFSE-labeled splenocytes pulsed with individual LFn-LACV proteins were transferred into their respective LFn-LACV-Gc or -N immunized recipient mice, and 27

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[0208] cytotoxiciW was measured. Strikingly, LFn-LACV-Gc immunization enhanced target cell killing to -21% in weanling mice, a significant increase compared to the negligible cytotoxic response observed post-infection. Similarly, LFn-LACV-N immunization improved target cell elimination to -19%, demonstrating a clear enhancement in cytotoxic capacity relative to unvaccinated weanling mice (FIG. 5D). These results confirm that vaccine-elicited T cell responses contribute to protection against LACV-induced disease in weanling mice by enhancing T cell cytotoxic activity. Finally, to directly evaluate the impact of vaccination on viral burden, LACV RNA levels in the brains of vaccinated and control weanling mice were measured at 4 days post-challenge. Both LFn-LACV-Gc and LFn-LACV-N immunized mice exhibited significantly reduced viral loads compared to saline-injected age-matched controls, indicating that the immunization strategy effectively limits viral replication in the brain (FIG.5E). Together with the enhanced cytotoxicity and increased survival observed in vaccinated mice, these results suggest that vaccine-elicited immune responses mediate protection against LACV-induced disease in weanlings by promoting effector function and restricting viral burden in the CNS.

[0209] These findings demonstrate significant differences in T cell responses between adult and weanling mice. Adult mice mounted robust LACV-specific T cell responses, characterized by strong IFN-y production against both structural and nonstructural antigens, with the highest responses targeting the structural proteins Gc and N. In contrast, weanling mice displayed significantly weaker T cell responses at 6 dpi, underscoring the inability of their immature immune systems to mount effective cellular immunity. This disparity contributes to the heightened susceptibility of weanlings to severe LACV infection.

[0210] Further analysis revealed significant expansion of CD4+and CD8+T cell populations in adult mice following LACV infection, accompanied by robust cytokine production, including IFN-y and granzyme B. Polyfunctional CD8+T cells, which co-produced IFN-y and granzyme B, were prominent in adults, supporting effective viral control and clearance. By contrast, weanlings exhibited limited T cell expansion and functional responses, suggesting an immature adaptive immune system unable to mount sufficient antiviral defenses. Importantly, at 6 dpi, weanling mice are symptomatic and show signs of neurological disease, suggesting that T cell responses may be partially compartmentalized to the CNS at this stage. In support of this, a significant increase in CD4+T cell frequency in the brains of LACV-infected w eanling mice was observed compared to uninfected controls, while adult mice exhibited only a modest,

[0211] 28

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[0213] non-significant increase. CD8+T cell frequencies remained unchanged in both age groups. These findings are consistent with prior studies demonstrating T cell infiltration into the CNS of weanlings during LACV infection. While CNS infiltration may contribute to the reduced peripheral T cell frequencies observed in weanlings, it does not fully account for their overall impaired T cell function, which includes reduced cytokine production and cytotoxicity. This supports the idea that the weanling adaptive immune compartment is fundamentally limited in its antiviral potential.

[0214] Consistent with prior studies, these findings reaffirm the essential role of CD4+and CD8+T cells in mediating protection in adult mice. Depletion or knockout of these subsets led to disease progression and neurological complications at later stages of LACV infection in adult mice, emphasizing the necessity of adaptive immunity in sustaining the protective effects initially provided by innate responses. On the other hand, the poor immune outcomes in eanlings may stem from deficiencies in both innate and adaptive immunity in younger mice. Evidence suggests that dendritic cells in young mice exhibit impaired functionality, due to reduced maturity or lower expression of co-stimulatory molecules (Dakic A et al. 2004. Development of the Dendritic Cell System during Mouse Ontogeny. The Journal of Immunology 172: 1018-1027). These findings are supported by a recent study of age-dependent susceptibility to LACV in cynomolgus macaques, which reported that w eanl ing animals exhibit lower baseline dendritic cell frequencies and w eaker IFN-stimulated gene responses compared to adults. Despite this, infected macaques — including weanlings — were asymptomatic and cleared infection without signs of encephalitis, suggesting that adaptive responses, particularly activated CD4+T cells, may compensate for weaker early innate responses in younger hosts. Additional evidence from a recent study of rhesus macaques infected intracranially with LACV demonstrated robust innate and adaptive responses within the CNS that resolved infection without causing neurological disease. These findings support the hypothesis that age and species-specific immune factors, including glial cell-mediated control, may significantly influence neuropathogenesis and disease outcomes in LACV infections. Understanding these mechanisms is critical for informing vaccine strategies aimed at enhancing immunity in vulnerable populations.

[0215] The ex vivo analysis, which included both structural and nonstructural proteins, represents a significant advancement over previous studies on bunyaviruses, which focused primarily on structural antigens (Alatrash R et al. 2024. The Adaptive Immune Response 29

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[0217] against Bunyavirales. Viruses 16:483). Immunogenic epitopes were identified across the proteome, providing a broad array of potential T cell targets. The strong immunogenicity of the structural proteins Gc and N was validated through their ability to elicit robust CD8+T cell responses and significant in vivo cytotoxicity. Immunization with these proteins conferred protection in weanling mice upon LACV challenge, demonstrating their potential as vaccine candidates. Consistent with this protection, significantly reduced viral loads in the brains of vaccinated mice were observed compared to saline-injected controls, indicating that immunization with LFn-LACV-Gc or -N not only improves survival, but also limits CNS viral replication. These findings align with studies on genetically related viruses within the Peribunyaviridae family, such as Jamestown Canyon Virus (JCV), Oropouche Virus (OROV), and Bunyamwera Virus (BUNV), where glycoproteins (Gn / Gc) and nucleocapsid (N) proteins were identified as key targets for T cell responses based solely on immunoinformatic analysis. Similarly, comprehensive analyses of Rift Valley Fever Vims (RVFV) and Severe Fever with Thrombocytopenia Syndrome Vims (SFTSV) have highlighted the immunogenicity7of structural and nonstmctural proteins. Notably, cross-reactive N-specific CD8+T cells have been observed across hantaviruses, and T cell responses targeting Gn and Gc have been identified in patients infected with Hantaan vims (HTNV) and Andes Virus (ANDV).

[0218] These findings served as a validation of the high immunogenicity7of identified viral proteins in this study and their protective potential in weanling mice. Notably, this work develops a vaccine against LACV. Mice vaccinated with DNA encoding the glycoproteins Gn and Gc produced neutralizing antibodies and exhibited a high degree of protection against challenge with high doses of LACV. Depletion of CD4+T cells in mice vaccinated with DNA encoding Gn / Gc reduced their capacity7to control the infection.

[0219] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0220] All references cited herein are incorporated herein by reference in their entireties.

[0221] 30

[0222] 182402162.2

Claims

Docket No. 070439.01934CLAIMSWhat is claimed is:

1. An immunogenic composition comprising an antigen fused to a cytotoxin, wherein the cytotoxin is a bacterial cytotoxin with a catalytic infectious domain removed, and the antigen induces a T-cell response to La Crosse virus (LACV).

2. The composition of claim 1, wherein the bacterial cytotoxin is Bacillus anthracis lethal factor (LFn).

3. The composition of claim 1, wherein the cytotoxin comprises the amino acid sequence of any one of SEQ ID NOs: 4-6.

4. The composition of claim 1, wherein the antigen fused is fused to the cytotoxin via a protein linker that comprises the amino acid sequence of SEQ ID NO: 7.

5. The composition of claim 1, wherein the antigen is derived from a structural protein of LACV.

6. The composition of claim 5, wherein the antigen is a Gc protein or a nucleocapsid (N) protein.

7. The composition of claim 1, wherein the antigen is derived from a nonstructural protein of LACV.

8. The composition of claim 7, wherein the antigen is NSm, NSs, or RNA-dependent RNA polymerase (RdRp).

9. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier.

10. A kit comprising the composition of claim 1, wherein the kit comprises:31182402162.2Docket No. 070439.01934a) an immunoassay to quantify cytokine levels from a sample of blood for detecting a T-cell-mediated immune response; andb) instructions for use.

11. A method of monitoring T-cells, comprising the following steps:a) mixing blood from a subject with the composition of claim 1;b) incubating the mixture of a) for at least 24 hours at about 37 °C;c) centrifuging the mixture of b) following the incubation period;d) collecting the supernatant from c); ande) quantifying cytokine levels with an immunoassay.

12. The method of claim 11, wherein the cytokine levels are quantified with an ELIspot assay, an ELISA assay, or flow cytometry.

13. A T-cell vaccine comprising the composition of claim 1, wherein the T-cell vaccine comprises an aqueous or liposomal formulation.

14. The T-cell vaccine of claim 13, wherein the T-cell vaccine further comprises an adjuvant.

15. An isolated polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

16. The amino acid sequence of claim 15, wherein said sequence is fused to an antigen.

17. The amino acid sequence of claim 15, wherein said sequence is fused to the antigen via a protein linker that comprises the amino acid sequence of SEQ ID NO: 7.

18. The amino acid sequence of claim 17, wherein the antigen is derived from a structural protein of LAC V.

19. The amino acid sequence of claim 18, wherein the antigen is a Gc protein or a nucleocapsid (N) protein.32182402162.2Docket No. 070439.0193420. A pharmaceutical composition comprising the antigen-fused amino acid sequence of claim 17 and a pharmaceutically acceptable carrier.

21. A method of eliciting a T-cell response in a subject comprising administering to the subject a composition comprising an antigen fused to a cytotoxin, wherein the antigen induces a T-cell response to La Crosse virus (LACV).

22. The method of claim 21, wherein the cytotoxin is a bacterial cytotoxin.

23. The method of claim 22, wherein the bacterial cytotoxin is Bacillus anthracis lethal factor (LFn).

24. The method of claim 23, wherein the cytotoxin comprises the amino acid sequence of SEQ ID NO: 5.

25. The method of claim 21, wherein the antigen is derived from a structural protein of LACV.

26. The method of claim 25, wherein the antigen is a Gc protein or a nucleocapsid (N) protein.

27. The method of claim 21, wherein the subject is a human.

28. The method of claim 21, comprising administering at least two doses of the composition to the subject.

29. The method of claim 21, wherein the T-cell response:a) enhances a second anti-viral therapeutic agent; andb) serves as a T-cell vaccine; and / or enables T-cell diagnostics.33182402162.2

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