Herpes simplex virus recombinant antigen and its uses

Abstract

This disclosure provides a fusion polypeptide comprising a soluble HSV viral surface antigen linked by in-frame fusion to a self-trimerizing protein trimerizing tag; and an immunogenic composition or vaccine comprising the fusion polypeptide. The fusion polypeptide, immunogenic composition, and vaccine provided herein can be used to generate an immune response, for example, for the treatment or prevention of HSV infection. The fusion polypeptide provided herein can be used to detect HSV antibodies, such as neutralizing antibodies.

Description

Cross-references to related applications

[0001] This application claims priority and benefit to International Patent Application No. PCT / CN2024 / 073279, filed on January 19, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure relates in some respects to recombinant peptides and fusion proteins for the prevention or treatment of herpes simplex virus (HSV) infection, the recombinant peptides and fusion proteins comprising herpes simplex virus antigens and immunogens in a trimer conformation, such as glycoproteins gB and / or gD, gD-gB and / or gB-gD or fragments thereof; and immunogenic compositions comprising these recombinant peptides and fusion proteins, with or without adjuvants. Background Technology

[0003] HSV (also known as herpes simplex virus) is a common infection that causes painful blisters or ulcers. It is treatable but not curable, and is a lifelong condition that can recur. There are two types of herpes simplex virus. It is estimated that about 4 billion people under the age of 50 worldwide (70%) have been infected with herpes simplex virus type 1 (HSV-1), which is the leading cause of oral herpes. It is estimated that about 491 million people aged 15 to 49 worldwide (13%) have been infected with herpes simplex virus type 2 (HSV-2), which is the leading cause of genital herpes. Type 1 (HSV-1) is primarily transmitted through oral contact and causes infection in or around the mouth (oral herpes or cold sores). It can also cause genital herpes. Most adults have been infected with HSV-1. Type 2 (HSV-2) is transmitted through sexual contact and causes genital herpes. Most people have no symptoms or only mild symptoms. However, the infection can cause painful blisters or ulcers, and these symptoms can recur over time. The blisters may rupture, ooze fluid, and then scab over. During the initial infection, people may experience fever, body aches, sore throat (oral herpes), headache, and swollen lymph nodes near the infection. Recurring symptoms of oral and genital herpes can be painful. Genital herpes can also cause stigma and negatively impact sexual relationships. If a pregnant woman is actively infected with HSV-2 during childbirth, she may transmit the virus to her newborn, potentially causing serious neurological disorders or neonatal death.

[0004] Currently available medications can alleviate symptoms but cannot cure the infection, thus there is an urgent need for effective vaccines and related treatments. Furthermore, there are currently no approved vaccines against HSV-2 infection. Chiron, GSK, Agenus, Genocea Biosciences Inc., and Sanofi have not successfully developed effective preventative and therapeutic vaccines against HSV-2 infection, although some vaccines have shown weak or transient efficacy. The key issues boil down to (1) the lack of use of viral antigens in their natural trimer conformation (e.g., gB and gD) in previous clinical trials; and (2) the need to select appropriate adjuvants to elicit the optimal immune response. Therefore, an effective HSV vaccine is urgently needed. This article presents the following findings: HSV gD, a key antigen that binds to host cell receptors, exists in a trimeric conformation; methods for generating gD and / or gB in homotrimeric form and gD-gB fusion antigens; and the use of these recombinant antigens, alone or in combination, and in combination with various adjuvants, as vaccines to induce strong humoral and T-cell-mediated immune responses and generate optimal immune protection against HSV infection. Summary of the Invention

[0005] In some embodiments, this document discloses a recombinant polypeptide comprising a soluble HSV protein surface antigen linked to a protein trimerization domain via in-frame fusion.

[0006] In some embodiments, this document discloses recombinant subunit vaccines comprising recombinant peptides. In some embodiments, the recombinant peptide comprises an extracellular domain (e.g., without transmembrane and cytoplasmic domains) of a glycoprotein or fragment or variant of an HSV (e.g., HSV-2 or HSV-1) fused within a trimerizing domain box capable of self-trimerization. In some embodiments, the protein trimerizing tag is selected from the group consisting of: a C-propeptide of procollagen capable of forming disulfide-linked homotrimers, a T4 foldon of fibritin from bacterial phage T4, and a leucine zipper from yeast GCN4. In some embodiments, the protein trimerizing tag is a C-peptide of human collagen (or procollagen) capable of forming disulfide-linked homotrimers. The resulting recombinant subunit vaccines (e.g., glycoprotein trimers (e.g., gD-trimer, gD-gB (wild-type (hereinafter: WT))-trimer, gD-gB (mutant)-trimer, gD (furin protease site (hereinafter: F))-gB (WT)-trimer, gD(F)-gB (mutant)-trimer)) can be expressed and purified from transfected cells and are expected to be in a trimer form with a native-like conformation. This will enhance the efficacy of HSV recombinant subunit vaccines in inducing neutralizing antibodies.

[0007] In some embodiments, HSV is a herpesvirus selected from the group consisting of: herpes simplex virus 1 and 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (HCMV), herpesvirus 6A and 6B (HHV-6A, HHV-6B), herpesvirus 7 (HHV-7), and Kaposi's sarcoma-associated herpesvirus (KSHV) and any combination thereof.

[0008] In some embodiments, HSV is human HSV-1 and / or HSV-2.

[0009] In any of the foregoing embodiments, the surface antigen may comprise an HSV glycoprotein (e.g., gD and / or gB) or a fragment or variant or epitope thereof, wherein the epitope is optionally a linear epitope or a conformational epitope, and wherein the protein comprises three recombinant polypeptides.

[0010] In any of the foregoing embodiments, the surface antigen may comprise gD, gD-gB (WT), gD-gB (mutant), gD(F)-gB (WT), gD(F)-gB (mutant) subpeptide, or any combination thereof.

[0011] In any of the foregoing embodiments, the surface antigen may comprise a signal peptide, a receptor-binding domain (RBD) peptide, a receptor-binding motif (RBM) peptide, a fusion peptide (FP), or any combination thereof.

[0012] In any of the foregoing embodiments, the surface antigen may comprise the receptor-binding domain (RBD) of the HSV glycoprotein.

[0013] In any of the foregoing embodiments, the surface antigen may comprise gD and / or gD-gB (WT) proteins.

[0014] In any of the foregoing embodiments, the surface antigen may be free of transmembrane (TM) domain peptides and / or cytoplasmic (CP) domain peptides.

[0015] In any of the foregoing embodiments, the surface antigen may be soluble or may not directly bind to the lipid bilayer (e.g., membrane or viral envelope).

[0016] In any of the foregoing embodiments, the surface antigens of the multiple recombinant polypeptides of the protein may be the same or different.

[0017] In any of the foregoing embodiments, the surface antigen may be fused directly to the C-terminal propeptide, or may be linked to the C-terminal propeptide via a linker, such as a linker comprising a glycine-XY repeat sequence, wherein X and Y are independently any amino acid, and optionally proline or hydroxyproline.

[0018] In any of the foregoing embodiments, the protein may be soluble.

[0019] In any of the foregoing embodiments, the protein may bind to a cell surface receptor of a subject, optionally wherein the subject is a mammal, such as a primate, for example, a human.

[0020] In any of the foregoing embodiments, the C-terminal propeptide may be derived from human collagen.

[0021] In any of the foregoing embodiments, the C-terminal propeptide may comprise a C-terminal propeptide of human procollagen selected from the group consisting of: proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI), or proα3(XI), or a fragment thereof.

[0022] In any of the foregoing embodiments, the C-terminal propeptides of the multiple recombinant peptides may be the same or different.

[0023] In any of the foregoing embodiments, the surface antigen in each recombinant polypeptide may be in a pre-fusion conformation.

[0024] In any of the foregoing embodiments, the surface antigen in each recombinant polypeptide may be in a fusion conformation.

[0025] In any of the foregoing embodiments, the recombinant polypeptide may comprise any one of SEQ ID NO: 1-28 or an amino acid sequence having at least 80% identity with it.

[0026] This article also provides an immunogen comprising the protein provided herein. This article provides a protein nanoparticle comprising the protein provided herein directly or indirectly linked to the nanoparticle. This article provides a virus-like particle (VLP) comprising the protein provided herein.

[0027] This document also provides an isolated nucleic acid that encodes one, two, three, or more recombinant polypeptides of the protein provided herein. In some embodiments, the polypeptide encoding an HSV glycoprotein peptide is fused within a polypeptide frame to a C-terminal propeptide encoding collagen. In some embodiments, the isolated nucleic acid provided herein is operatively linked to a promoter.

[0028] In some embodiments, the isolated nucleic acid provided herein is a DNA molecule. In some embodiments, the isolated nucleic acid provided herein is an RNA molecule, optionally an mRNA molecule, such as nucleoside-modified mRNA, non-amplified mRNA, self-amplified mRNA, or trans-amplified mRNA.

[0029] This document also provides a vector containing the isolated nucleic acid provided herein. In some embodiments, the vector is a viral vector.

[0030] In some aspects, this document provides a virus, a pseudovirus, or a cell comprising a vector provided herein, optionally wherein the virus or cell has a recombinant genome. In some aspects, this document provides an immunogenic composition comprising a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, or cell provided herein, and a pharmaceutically acceptable carrier.

[0031] This document also provides a vaccine comprising the immunogenic composition provided herein and optionally an adjuvant, wherein the vaccine is optionally a subunit vaccine. In some embodiments, the vaccine is a prophylactic and / or therapeutic vaccine.

[0032] In some respects, this article provides a method for producing a protein, the method comprising: expressing an isolated nucleic acid or vector, as provided herein, in a host cell to produce the protein provided herein; and purifying the protein. This article provides a protein produced by the method provided herein.

[0033] This document provides methods for generating an immune response against HSV glycoprotein peptides (e.g., gB and / or gD) or fragments or epitopes thereof in a subject, comprising administering to the subject an effective amount of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine as provided herein to generate an immune response. In some embodiments, this document provides proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines as provided herein for use in generating an immune response against HSV in a subject. In some embodiments, this document provides the use of proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines as provided herein in the preparation of a medicament for generating an immune response against HSV in a subject. In some embodiments, the methods provided herein are used for treating or preventing HSV infection. In some embodiments, this document provides for use of proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines as provided herein in the treatment or prevention of HSV infection in a subject. In some embodiments, this document provides for use of proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines as provided herein in the preparation of a medicament for the treatment or prevention of HSV infection in a subject. In some embodiments, the generation of an immune response inhibits or reduces HSV replication in the subject. In some embodiments, the immune response includes a cell-mediated response and / or a humoral response, optionally including the generation of one or more neutralizing antibodies, such as polyclonal or monoclonal antibodies. In some embodiments, the immune response targets an HSV glycoprotein peptide or a fragment or epitope thereof, but not a C-terminal propeptide. In some embodiments, administration to a subject does not result in antibody-dependent enhancement (ADE) in the subject due to prior exposure to one or more HSVs. In some embodiments, administration does not result in antibody-dependent enhancement (ADE) in the subject upon subsequent exposure to one or more HSVs. In some embodiments, the method further includes an initial immunization step and / or a booster step. In some embodiments, the administration step is performed via topical, transdermal, subcutaneous, intradermal, oral, intranasal (e.g., intranasal spray), intratracheal, sublingual, buccal, rectal, vaginal, inhalation, intravenous (e.g., intravenous injection), intra-arterial, intramuscular (e.g., intramuscular injection), intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intra-articular, periarticular, local, or epidermal application. In some embodiments, the effective amount is administered as a single dose or as a series of doses at one or more time intervals.In some embodiments, the effective amount is administered without adjuvant. In some embodiments, the effective amount is administered with adjuvant.

[0034] This document provides a method comprising administering an effective amount of the protein provided herein to a subject to generate neutralizing antibodies or neutralizing antiserum against HSV in the subject's body. In some embodiments, the subject is a mammal, optionally a human or a non-human primate. In some embodiments, the method further comprises isolating neutralizing antibodies or neutralizing antiserum from the subject's body. In some embodiments, the method further comprises administering an effective amount of the isolated neutralizing antibody or neutralizing antiserum to a human subject via passive immunization to prevent or treat HSV infection. In some embodiments, the neutralizing antibody or neutralizing antiserum against HSV comprises a polyclonal antibody against an HSV glycoprotein peptide or a fragment or epitope thereof, optionally wherein the neutralizing antibody or neutralizing antiserum contains or substantially contains no antibody against a C-terminal propeptide of collagen. In some embodiments, the neutralizing antibody comprises a monoclonal antibody against an HSV glycoprotein peptide or a fragment or epitope thereof, optionally wherein the neutralizing antibody contains or substantially contains no antibody against a C-terminal propeptide of collagen.

[0035] In some respects, the proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions or vaccines provided herein are used to induce an immune response against HSV (e.g., HSV-2 and / or HSV-1) in a subject and / or for use in the treatment or prevention of HSV (e.g., HSV-2 and / or HSV-1) infection.

[0036] In some aspects, this document provides for the use of the proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines provided herein for inducing an immune response against HSV in a subject and / or for treating or preventing HSV infection. In some aspects, this document provides for the use of the proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines provided herein for manufacturing a medicament or prophylactic agent for inducing an immune response against HSV in a subject and / or for treating or preventing HSV infection.

[0037] This document also provides methods for analyzing samples, including: contacting the sample with a protein provided herein, and detecting binding between the protein and an analyte capable of specifically binding to an HSV glycoprotein peptide (e.g., gB and / or gD) or a fragment or epitope thereof. In some embodiments, the analyte is an antibody, receptor, or cell that recognizes an HSV glycoprotein peptide (e.g., gB and / or gD) or a fragment or epitope thereof. In some embodiments, binding indicates the presence of the analyte in the sample and / or indicates HSV infection in the subject from whom the sample is derived.

[0038] This article provides kits that contain the protein described herein and a substrate, pad, or vial containing or immobilizing the protein, optionally wherein the kit is an ELISA or lateral chromatography assay kit.

[0039] This document provides a method for detecting HSV antibodies from mammalian samples, the method comprising the steps of: (1) contacting the sample with a fusion peptide provided herein, and (2) detecting antibodies binding to the HSV viral surface antigen in the fusion peptide. In some embodiments, the fusion peptide provided herein is provided for use in a method for detecting HSV antibodies from mammalian samples. In some embodiments, the fusion peptide provided herein is provided for use in the preparation of reagents for detecting HSV antibodies from mammalian samples. In some embodiments, the antibody is a neutralizing antibody.

[0040] In some embodiments, the method includes detecting the binding of an antibody to the HSV viral surface antigen in the fusion peptide with a secondary antibody. In some embodiments, the method includes detecting a neutralizing antibody, the resulting reading reflecting the inhibition of binding of the HSV viral surface antigen in the fusion peptide to the soluble HSV receptor.

[0041] In some embodiments, the soluble HSV receptor is fused with an Fc domain or a trimerization domain. In some embodiments, the soluble HSV receptor is a linker protein-1 or HVEM. In some embodiments, the soluble HSV receptor has the sequence shown in any of SEQ ID Nos: 29-30.

[0042] In some embodiments, the fusion peptide is labeled with a detection reagent. In some embodiments, prior to contact, the fusion peptide binds to an antibody that recognizes the C-terminal portion of collagen, wherein the antibody recognizing the C-terminal portion of collagen is labeled with a detection reagent. In some embodiments, the detection reagent is colloidal gold. In some embodiments, the HSV antibody is detected by lateral chromatography. Attached Figure Description

[0043] Figure 1Schematic diagrams of HSV (HSV-1 or HSV-2) fusion proteins disclosed in this article. (A) Schematic diagram of the complete sequence of HSV gB protein (including the signal sequence); (B) Schematic diagram of the complete sequence of HSV gD protein (with the signal sequence incorporated); (C) Schematic diagram of the sequence of HSV gB (H513P)-His fusion protein (with the additional signal sequence); (D) Schematic diagram of the sequence of HSV gD-trimeric fusion protein (characterized by the signal sequence); (E) Schematic diagram of the sequence of HSV gB protein with a His tag (including the signal sequence); (F) Schematic diagram of the sequence of HSV gB-trimeric protein (with the integrated signal sequence); (G) Schematic diagram of the complete sequence of HSV gD-gB-trimeric fusion protein (with the signal sequence); (H) Schematic diagram of the sequence of HSV gD(F)-gB-trimeric fusion protein (including the signal sequence and the furin cleavage site (F)). The construct was stably transfected into CHO cells for protein expression.

[0044] Figure 2 A schematic diagram illustrating the structure of the HSV-1 / HSV-2 fusion protein. (A) Schematic diagram of the structure of HSV gB(H513P)-His fusion protein in monomeric form; (B) Schematic diagram of the structure of HSV gD-trimeric fusion protein in trimeric form; (C) Schematic diagram of the structure of HSV gB(H513P)-His fusion protein (which can exist in monomeric or trimeric form); (D) Schematic diagram of the structure of HSV gB wild-type (WT)-trimeric fusion protein in trimeric arrangement; (E) Schematic diagram of the structure of HSV gB(H513P)-trimeric fusion protein in trimeric form; (F) Schematic diagram of the structure of HSV gD-gB(WT)-trimeric fusion protein in trimeric conformation; (G) Schematic diagram of the sequence and structure of HSV gD-gB(H513P)-trimeric fusion protein in trimeric form; (H) HSV in trimeric shape. (I) Schematic illustration of the sequence and structure of the gD(F)-gB(WT)-trimeric fusion protein; (I) Schematic illustration of the sequence and structure of the HSVgD(F)-gB(H513P)-trimeric fusion protein arranged in a trimeric configuration.

[0045] Figure 3SDS-PAGE and SEC-HPLC analyses of gD-His, gD-trimer, gB-trimer, and gD-gB-trimer. (A) gD-His, (B) gD-trimer, (C) gB-trimer, and (D) gD-gB-trimer were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue under non-reducing (-ME) and reducing (+ME) conditions. gD-trimer, gB-trimer, and gD-gB-trimer are homotrimeric proteins linked by disulfide bonds, while gD-His exists as a monomer. (E) SEC-HPLC analysis of the purity of gD-His, (F) gD-trimer, (G) gB-trimer, and (H) gD-gB-trimer.

[0046] Figure 4 Negative staining analysis of the structures of gD-His, gD-trimer, gB-trimer, and gD-gB-trimer. Negative staining electron microscopy (EM) and two-dimensional (2D) classification showed that (A) gD-His is a monomer, while (B) gD-trimer, (C) gB-trimer, and (D) gD-gB-trimer are in trimer form.

[0047] Figure 5 A schematic diagram of the cryo-electron microscopy (Cryo-EM) study workflow for HSV gD-gB trimers. (AJ) shows the merging of GO and Au nets for three-dimensional (3D) classification.

[0048] Figure 6 Structural analysis of gB in the HSV-2 gD-gB trimer. (A, B, E) show the analysis of the gB structure using cryo-electron microscopy (Cryo-EM) with non-uniform refinement (C1). (C, D, F) show the analysis of the gB structure using cryo-electron microscopy (Cryo-EM) with non-uniform refinement (C1).

[0049] Figure 7 Structural analysis of gD in HSV-2 gD-gB trimer. (A) Top view of the reconstructed structure of gD trimer; (B) Side view of the reconstructed structure of gD trimer.

[0050] Figure 8Assessment of binding affinity and functional activity of gD-His, gD-trimer, gB-trimer, and gD-gB-trimer. (A) ELISA was used to measure the binding affinity of gD-His (blue), gD-trimer (red), and gD-gB-trimer (green) to HSV gD receptor connexin 1-Fc, connexin 1-trimer, HVEM-Fc, and HVEM-trimer. gD-His showed weak binding to the gD receptor, while gD-trimer and gD-gB-trimer showed significantly stronger binding affinity to the receptor than monomeric gD-His. (B) Statistical analysis was performed on the EC50 values ​​of gD-His, gD-trimer, and gD-gB-trimer binding to gD receptor connexin 1-Fc, connexin 1-trimer, HVEM-Fc, and HVEM-trimer. The EC50 values ​​of gD-trimer and gD-gB-trimer were significantly lower than those of monomeric gD-His. (C) Comparison of the ability of gD-His, gD-trimer, and the natural HVEM ligand LIGHT to activate the HVEM signaling pathway showed that gD-His had almost no activity in HVEM receptor activation, while gD-trimer's ability to activate HVEM exceeded that of the natural ligand LIGHT. (D) The EC50 values ​​of gD-His, gD-trimer, and LIGHT for HVEM activation showed that monomeric gD-His failed to activate the HVEM pathway, while gD-trimer's activation of HVEM was 8 times that of LIGHT.

[0051] Figure 9 Comparison of the immunogenicity of gD-gB-trimer in the presence of different adjuvants. (A) Immunization regimen: BALB / c mice (n = 6 mice / group) were immunized with gD-gB-trimer mixed with various adjuvants (including CPG / CAS-1, CPG / alum, AS01B, and AS01E). The immunization schedule was on days 0, 14, and 28. (B) Comparison of gD / gB binding antibody levels. (C) Comparison of HSV-2 neutralizing antibody titers. (D) Comparison of neutralizing antibody / binding antibody ratios. (E) Comparison of ADCC activity in immune sera and (F) Corresponding fold changes.

[0052] Figure 10Immunogenicity of gD-trimer in mice. (A) Schematic diagram of immunization: BALB / c mice (n = 6 mice / group) were administered equal amounts of gD in the form of gD-His or gD-trimer (both mixed with 25 µL CAS-1 and 10 µg CpG) at three predetermined intervals on days 0, 14, and 28. (B) Comparison of gD-His and gD-trimer-induced gD-binding antibody levels showed no significant difference between the two groups. (C) Assessment of gD-His and gD-trimer-induced HSV-2 neutralizing antibody levels showed that gD-trimer-induced levels were significantly higher than those induced by monomeric gD-His. (D) Assessment of ADCC activity in the immune serum of mice immunized with gD-His and gD-trimer (measured as the RLU difference between the immune serum on day 42 and the pre-immunization serum on day 0). Compared with antibodies induced by monomeric gD-His, antibodies induced by gD-trimer exhibited significantly higher ADCC activity. (E) The fold change in ADCC activity was calculated by dividing the RLU of the immunized serum on day 42 by the RLU of the pre-immunized serum on day 0.

[0053] Figure 11 Immunogenicity of gD-gB-trimer in mice. (A) Schematic diagram of immunization: BALB / c mice (n = 6 mice / group) were immunized at three time points on days 0, 14, and 28 using the combination labeled in (B), with consistent gD loading. The complement-independent (B) and complement-dependent (C) activity levels of HSV-2 neutralizing antibodies were assessed on day 42. (D) Assessment of ADCC activity in immune sera from mice immunized with different combinations (measured as the RLU difference between immune sera on day 42 and pre-immunization sera on day 0). (E) Foldwise changes in ADCC activity were calculated by dividing the RLU of immune sera on day 42 by the RLU of pre-immunization sera on day 0.

[0054] Figure 12 Cell-mediated immune responses induced by gD and gB. (A) Detection of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-5) cytokines using ELISpot assay with gD-His as a stimulant. (B) Detection of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-5) cytokines using ELISpot assay with gB-His as a stimulant.

[0055] Figure 13Immunogenicity of gD-gB-trimer in guinea pigs. (A) Schematic diagram of immunization: Guinea pigs (n = 6 / group) were immunized with a combination of gD-His (9.8 µg) / gB-His (9.8 µg) with 50 µL adjuvant MF-59, 40 µg gD-gB-trimer with 100 µg CPG and 50 µL CAS-1 as adjuvant, or a saline-mediated control. Immunization was administered on days 0, 14, and 28, followed by intravaginal administration of 1 × 10⁻⁶ gD-gB-trimer on day 42. 6 HSV-2 G strain with TCID50 was challenged. Complement-independent (B) and complement-dependent (C) activity titers of HSV-2 neutralizing antibodies were assessed on day 42, with the gD-gB-trimer + CPG / CAS-1 group showing significantly higher levels than the gD-His / gB-His + MF-59 group. (D) Percentage change in body weight after challenge, and (E) Genital lesion score after challenge.

[0056] Figure 14 Principle of colloidal gold-binding antibody detection. (A) Main structure of the test strip. (B) Detection principle of colloidal gold lateral chromatography: negative versus positive (C). (D) Possible interpretations of the results. (E) Positive and negative controls.

[0057] Figure 15 Principle of Colloidal Gold Neutralizing Antibody Detection. (A) Main structure of the test strip. (B) Principle of neutralizing antibody detection: In the absence of any neutralizing antibodies, the binding of gD or gD-gB-trimers to host cell receptors (e.g., connexin-1 and HVEM) is uninhibited, resulting in a strong T-line signal. (C) When the test sample contains neutralizing antibodies, the antibodies bind to the gD-trimer or gD-gB-trimer, thereby inhibiting the binding of the trimer to connexin-1 and HVEM receptors, leading to a reduction or absence of the T-line signal, indicating a positive neutralizing antibody test. (D) Interpretation of results. (E) Positive and negative controls. Detailed Implementation

[0058] This paper discloses for the first time the surprising discovery that the HSV-2 gD protein may exist in a pre-fusion trimer conformation in its native state. Furthermore, it is now surprising for the first time that the pre-fusion trimer conformation can be effectively stabilized by a trimerizing domain linked to a soluble HSV-2 gD-containing glycoprotein (e.g., gD and gD-gB) via in-frame fusion. Therefore, this invention provides, for the first time, a method for producing recombinant HSV-2 gD-containing glycoproteins in a pre-fusion conformation, as well as vaccines containing these glycoproteins, which differ from other proposed vaccines containing HSV gD antigen monomers. Moreover, these vaccines disclosed herein exhibit remarkable efficacy in preventing HSV-2 infection and generating neutralizing antibody titers against HSV-2 infection.

[0059] This document provides a fusion polypeptide (also referred to as a fusion protein, recombinant polypeptide, recombinant protein, or recombinant antigen) comprising an HSV viral antigen or immunogen and a protein trimer tag. This document also provides a trimer of the fusion polypeptide, an immunogenic composition or vaccine comprising the fusion polypeptide trimer thereof, and methods or uses of the fusion polypeptide or its trimer, immunogenic composition, or vaccine for the prevention or treatment of HSV (e.g., HSV-1 and / or HSV-2) infection. In some embodiments, the viral antigen or immunogen may comprise a viral surface antigen (or glycoprotein) or a fragment or variant thereof from a DNA virus (e.g., HSV (e.g., HSV-1 and / or HSV-2)). In some embodiments, the viral antigen or immunogen may comprise one, two, or more viral surface antigens (or glycoproteins) or fragments or variants thereof from HSV (e.g., HSV-1 and / or HSV-2). In some embodiments, the viral antigen or immunogen may comprise an HSV (e.g., HSV-1 and / or HSV-2) gD protein or a fragment or variant thereof. In some embodiments, the viral antigen or immunogen may comprise an HSV (e.g., HSV-1 and / or HSV-2) gB protein or a fragment or variant thereof. In some embodiments, the viral antigen or immunogen may comprise: an HSV (e.g., HSV-1 and / or HSV-2) gD protein or a fragment or variant thereof, and an HSV (e.g., HSV-1 and / or HSV-2) gB protein or a fragment or variant thereof, e.g., a fusion of the two. In some embodiments, the viral antigen or immunogen may be soluble. In some embodiments, the soluble viral antigen or immunogen does not bind to a lipid bilayer (e.g., a membrane or viral envelope). In some embodiments, the viral antigen or immunogen may comprise a soluble portion of a viral surface antigen (or glycoprotein), e.g., an extracellular domain. In some embodiments, the viral antigen or immunogen may be linked to a protein trimerization tag capable of self-trimerization to form a fusion polypeptide via in-frame fusion. In some embodiments, the fusion polypeptide may be in the form of a covalently linked trimer. In some embodiments, the resulting fusion protein is secreted as a disulfide-linked homotrimer, which is structurally more stable while retaining the conformation of the natural-like trimeric viral antigen, thus serving as a more effective vaccine against these dangerous pathogens.

[0060] In some embodiments, the fusion peptide may comprise extracellular domains of HSV glycoproteins (e.g., gD and / or gB from HSV-1 and / or HSV-2). In some embodiments, the trimer of the fusion peptide may comprise promers of extracellular domains of recombinant HSV glycoproteins (e.g., gD and / or gB from HSV-11 and / or HSV-2).

[0061] In some embodiments, the fusion peptide or its trimer may be used as part of a vaccine or multivalent vaccine, optionally administered via intramuscular or intranasal injection, with or without an adjuvant, or with more than one adjuvant, to prevent or treat viral (e.g., HSV, such as HSV-1 and / or HSV-2) infection.

[0062] In some embodiments, this document discloses methods for using fusion peptides or their trimers to detect antibodies (e.g., neutralizing antibodies) against viruses (e.g., viral antigens) derived from mammalian serum, which can be used to diagnose viral infections. In some embodiments, the virus may be HSV (e.g., HSV-1 and / or HSV-2). In some embodiments, the viral antigen may be gB and / or gD.

[0063] In some embodiments, this document discloses methods for using fusion peptides or their trimers as antigens to generate polyclonal or monoclonal antibodies (e.g., neutralizing mAbs) that can be used for passive immunization, for example for treating HSV (e.g., HSV-1 and / or HSV-2) infections.

[0064] In some embodiments, the fusion polypeptide or its trimer may be used as part of a vaccine or a multivalent vaccine, wherein the vaccine may contain multiple fusion polypeptides or their trimers, the fusion polypeptides or their trimers containing viral antigens of the same protein of the same virus or viral antigens containing two or more different proteins of one or more viruses or one or more strains of the same virus.

[0065] The proteins (including fusion peptides or trimers thereof) containing HSV viral antigens or immunogens provided herein can be used to effectively and safely prevent or treat (e.g., therapeutically or prophylactically) HSV infection. For example, the proteins containing HSV viral antigens or immunogens provided herein treat HSV infection without mediating vaccine-induced disease enhancement (VED) and / or antibody-dependent enhancement (ADE). Furthermore, the proteins containing HSV viral antigens and immunogens provided herein are readily produced and exhibit stability under high stress conditions (e.g., high temperature, extreme pH, and high and low osmolarity). Therefore, the proteins and immunogenic compositions provided herein circumvent and address the production, stability, safety, and efficacy issues that hinder HSV vaccine development.

[0066] In some embodiments, a fusion polypeptide or trimer thereof comprising HSV viral antigens or immunogens (e.g., HSV gD, HSV gD-gB, HSV gD(F)-gB, HSV gB-gD, HSV gB(F)-gD protein peptides) can generate an immune response, such as an immune response against HSV-1 and / or HSV-2 protein peptides. In some embodiments, the immune response inhibits or reduces HSV replication in a subject (e.g., a patient). In some embodiments, the immune response inhibits or reduces HSV replication in a subject (e.g., a patient), wherein the HSV viral antigen or immunogen is HSV-2 and / or HSV-1 viral antigen or immunogen. In some embodiments, the immune response includes the production of one or more neutralizing antibodies, such as polyclonal and / or monoclonal antibodies. In some embodiments, the neutralizing antibodies inhibit or reduce HSV replication in a subject (e.g., a patient). In some embodiments, administration of the fusion polypeptide or trimer thereof to a subject (e.g., as an immunogenic composition) does not result in antibody-dependent enhancement (ADE) in the subject due to prior exposure to HSV. In some respects, fusion peptides or their trimers containing HSV viral antigens and immunogens are used as vaccines.

[0067] In some embodiments, HSV viral antigens and immunogens (e.g., HSV gD, HSV gD-gB, HSV gD(F)-gB, HSV gB-gD, or HSV gB(F)-gD protein peptides) are linked to proteins or peptides to form fusions or recombinant polypeptides or proteins. In some embodiments, gD and / or gB are derived from HSV-2 and / or HSV-1. In some embodiments, the protein or peptide linked to the HSV viral antigen or immunogen is capable of associating (e.g., covalently or non-covalently) with another protein or peptide (e.g., a protein or peptide contained in a fusion polypeptide). In some cases, the protein or peptide linked to the HSV viral antigen or immunogen is a polymerized domain.

[0068] In some embodiments, HSV viral antigens and immunogens (e.g., HSV glycoprotein peptides) are linked to a self-trimerizing protein trimerizing tag, which may be a C-terminal portion of collagen (e.g., a C-terminal propeptide of collagen), a GCN4 leucine zipper, or phage T4 fibrin or Foldon, to form a fusion peptide or recombinant polypeptide. In some embodiments, the C-terminal portion of collagen is derived from human collagen, such as the C-propeptide of α1 collagen, and is capable of self-trimerizing upon expression.

[0069] In some embodiments, linking HSV (e.g., HSV-2 and / or HSV-1) viral antigens and immunogens (e.g., gD, gD-gB (WT), gD-gB (mutant), gD(F)-gB (WT), gD(F)-gB (mutant) peptides) to protein trimerization tags (e.g., C-terminal propeptide of collagen) contributes to the ability of proteins to generate an immune response. For example, the generation of recombinant proteins may preserve the tertiary and quaternary structures of the HSV (e.g., HSV-2 and / or HSV-1) glycoprotein gD peptide, which may be important for the stability of the native conformation of the HSV (e.g., HSV-2 and / or HSV-1) gD peptide and, consequently, for the availability of antigenic sites on the protein surface capable of eliciting an immune response (e.g., neutralizing antibodies). Additionally, the linking of HSV (e.g., HSV-2 and / or HSV-1) gD peptides to proteins or peptides capable of self-trimerization causes the recombinant proteins to aggregate, thereby mimicking the native homotrimeric structure of HSV (e.g., HSV-2 and / or HSV-1) gD peptides on the viral envelope.

[0070] In some embodiments, HSV (e.g., HSV-2 and / or HSV-1) gD peptides are linked to protein trimerization tags (e.g., C-terminal propeptides of collagen) to generate self-trimerizing recombinant peptides. In some embodiments, the proteins provided herein comprise a variety of self-trimerizing glycoproteins (e.g., gD-trimers, gD-gB-trimers, or gB-gD-trimers) containing HSV (e.g., HSV-2 and / or HSV-1) gD. In some embodiments, the trimerizing properties of the recombinant protein contribute to protein stability. In some embodiments, the trimerizing properties of the recombinant protein contribute to the protein's ability to generate an immune response. In some embodiments, the trimerizing properties of the recombinant protein and / or the macroscopic structure of a variety of self-trimerizing recombinant proteins contribute to the protein's ability to generate an immune response.

[0071] This article also provides immunogenic compositions or vaccines comprising the fusion peptide or trimer thereof provided herein, methods for generating the fusion peptide or trimer thereof provided herein, methods for treating subjects with the fusion peptide or trimer thereof provided herein and compositions or vaccines thereof, and kits comprising the fusion peptide or trimer thereof provided herein.

[0072] For all purposes, all publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated herein by reference in their entirety as if each individual publication were individually incorporated by reference. Where the definitions shown herein contradict or otherwise are inconsistent with those shown in patents, applications, published applications, and other publications incorporated herein by reference, the definitions shown herein shall prevail. Some headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0073] I. Viral antigens and immunogens The fusion peptides or trimers thereof provided herein contain HSV viral antigens or immunogens. The HSV viral antigens or immunogens considered herein are capable of promoting or stimulating cell-mediated and / or humoral responses. In some embodiments, the response (e.g., a cell-mediated or humoral response) includes the production of antibodies (e.g., neutralizing antibodies). In some embodiments, the HSV viral antigens or immunogens are recognized by HSV reactive antibodies and / or T cells. In some embodiments, the HSV is human HSV. In some embodiments, the HSV is HSV-1 and / or HSV-2. In some embodiments, the HSV is human HSV-1 and / or human HSV-2.

[0074] In some embodiments, the HSV viral antigen or immunogen comprises structural proteins of HSV, fragments thereof, or variants thereof. In some embodiments, the HSV viral antigen or immunogen comprises HSV surface antigen (or glycoprotein) or fragments thereof or variants thereof. In some embodiments, the HSV viral antigen may comprise one, two, or more proteins selected from the group consisting of: gD, gB, membrane (M) proteins, nucleocapsid (N) proteins, and envelope (E) proteins. In some embodiments, the HSV viral antigen or immunogen comprises or further comprises non-structural proteins of HSV, fragments thereof, or variants thereof. A representative HSV-2 strain is derived from the G strain, ATCC ID: VR-734, and a representative HSV-1 strain is derived from the KOS strain, ATCC ID: VR-1493.

[0075] The HSV viral antigens or immunogens provided herein may comprise one, two, or more HSV viral surface antigens (or glycoproteins), fragments thereof, or variants thereof. The two or more HSV viral surface antigens (or glycoproteins), fragments thereof, and / or variants thereof may be identical or different, and may be derived from the same or different HSV species, subtypes, and / or strains, or from the same or different HSV viral surface antigens (or glycoproteins) of the same HSV species, subtypes, and / or strains. In some embodiments, two or more HSV viral surface antigens (or glycoproteins), fragments thereof, or variants may be fused directly or indirectly to each other via peptide linkers. In some embodiments, the linker may be a furin protease site (e.g., RRAR (SEQ ID NO: 31)).

[0076] In some embodiments, the HSV virus antigen is soluble. In some embodiments, the HSV virus antigen comprises a soluble portion (e.g., an extracellular domain) of an HSV virus surface antigen (or glycoprotein), or a fragment or variant thereof. In some embodiments, the soluble portion (e.g., an extracellular domain) of the HSV virus surface antigen (or glycoprotein) comprises its extracellular domain. In some embodiments, the HSV virus antigen comprises one, two, or more soluble portions (e.g., extracellular domains), fragments, or variants of an HSV virus surface antigen (or glycoprotein).

[0077] Herpesviruses are a large, double-stranded DNA, enveloped family of viruses that can cause lifelong infection and trigger a range of diseases. Nine human herpesviruses include herpes simplex virus 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), human cytomegalovirus (HCMV), human herpesvirus 6A (HHV6A), HHV6B, Epstein-Barr virus (EBV), HHV7, and Kaposi's sarcoma herpesvirus (KSHV). Viral entry into host cells requires viral binding to a specific receptor, followed by the synergistic action of multiple viral entry glycoproteins to trigger membrane fusion. The core fusion mechanism of all herpesviruses is conserved, but each species uses different receptors and receptor-binding glycoproteins.

[0078] dsDNA HSVs with genome sizes exceeding 150 kb (like phage T4) exhibit a lower mutation rate (10-10). -8 -10 -7In DNA viruses, there is a negative correlation between genome size and the mutation rate per base, while the mutation rate across the entire genome varies less. It is likely that a key differentiating feature between RNA and DNA viruses in terms of mutation rate is that most RNA viruses encode polymerases lack 3′-exonuclease proofreading activity, making them particularly prone to errors. This 3′-exonuclease activity confers a replication fidelity increase of approximately 10 to 100 times (R. Sanjuán, P. Domingo-Calap, in Genome Stability (2nd edition), 2021). DNA viruses with large genomes, particularly herpesviruses and poxviruses, encode numerous proteins that counteract host defenses. Viral factors are viral proteins that mimic host cytokines that stimulate cell proliferation and increase the number of viral targets. Viral receptors are viral proteins that mimic host defense cytokine receptors, thereby “luring” these cytokines away from their intended cellular receptors. For example, vaccinia virus encodes a complement control protein that blocks the complement cascade and a tumor necrosis factor viral receptor that binds to this host defense molecule. Herpes simplex virus (HSV) encodes two glycoproteins (gE and gI) that function as Fc receptors; these receptors bind to and inactivate antiviral antibodies (NealNathanson, Francisco González-Scarano, in Viral Pathogenesis (3rd ed.), 2016).

[0079] Similar to other enveloped viruses, HSV entry into target cells requires fusion of the viral and cell membranes. Of the 11 or more proteins present on the viral envelope, five (gC, gB, gD, gH, and gL) are involved in entry (Spear PG, Longnecker R (2003) Herpesvirus entry: an update [Herpesvirus entry: an update]. J VirolMethods [Journal of Virology Methods] 77: 10179-10185). Initially, gB and gC interact with the cell surface heparan sulfate (HS) proteoglycan, allowing virus-cell attachment, and then glycoprotein D (gD) binds to the cell surface receptor (Campadelli-Fiume G, Cocchi F, Menotti L, Lopez M, (2000) Rev Med Virol [Review of Medical Virology] 10: 305-319). The latter event is followed by membrane fusion mediated by gB and the heterodimer gH / gL (SpearPG, Longnecker R (2003) Herpesvirus entry: an update [Herpesvirus entry: latest developments]. J Virol Methods [Journal of Virology Methods] 77: 10179-10185). Binding of gD to functional cellular receptors (such as herpesvirus entry mediators (HVEM) (a member of the TNF receptor family) or connexin-1 (a member of the immunoglobulin superfamily)) is essential for HSV cell entry. gD consists of a V-like Ig domain with N-terminal and C-terminal extensions. The N-terminus of gD285 is flexible and extended, and when gD binds to HVEM, it folds into a hairpin structure containing all HVEM-binding residues. The C-terminus (residues 260-316) of the gD extracellular domain plays an important functional role in HSV entry.

[0080] HSV-1 gB is a structurally conserved class III membrane fusion protein composed of α- and β-secondary structural elements, combining structural features of class I and class II fusion proteins. The post-fusion conformation of gB has long been established, while the first description of its pre-fusion form was only recently reported (Vollmer et al., Sci. Adv. 2020; 6: eabc1726, Sep 25, 2020). Once triggered, the basic characteristics of the conformational change remain highly similar. gB ​​can fuse the HSV-1 envelope with the plasma membrane. For the gB fusion variant, triggering appears to be complex and regulated, and is only possible as part of a four-protein mechanism along with gD and gH / gL (38) (Vollmer et al., Sci. Adv. 2020; 6: eabc1726). Electron microscopy results of HSV-2 gD indicate that it is the native pre-fusion trimer (electron microscopy). HSV glycoproteins contain a signal peptide at the N-terminus, an extracellular domain, a transmembrane domain, and an intracellular domain.

[0081] In some embodiments, the HSV viral antigen or immunogen may comprise: (1) a soluble portion (e.g., an extracellular domain) of gD from HSV-2 and / or HSV-1, or a fragment or variant thereof; (2) a soluble portion (e.g., an extracellular domain) of gB from HSV-2 and / or HSV-1, or a fragment or variant thereof; or (3) a fusion of (1) and (2), wherein the fusion may comprise a soluble portion (e.g., an extracellular domain) of gD fused to the N-terminus or C-terminus of a soluble portion (e.g., an extracellular domain) of gB, or a fragment or variant thereof. In some embodiments, the soluble portion (e.g., an extracellular domain) of gD, or a fragment or variant thereof, is fused directly or indirectly to the N-terminus or C-terminus of a soluble portion (e.g., an extracellular domain) of gB, or a fragment or variant thereof, via a linker (e.g., a furin protease site). In some embodiments, the HSV virus antigen or immunogen may be a protein containing HSV (e.g., HSV-2 and / or HSV-1) gD, for example, an HSV virus antigen or immunogen containing a soluble portion (e.g., extracellular domain) of the gD protein or a fragment or variant thereof, such as a soluble portion (e.g., extracellular domain) of gD or a fragment or variant thereof, or a fusion of a soluble portion (e.g., extracellular domain) of gD or a fragment or variant thereof and a soluble portion (e.g., extracellular domain) of gB or a fragment or variant thereof.

[0082] In some embodiments, an HSV viral antigen or immunogen may comprise a soluble portion (e.g., an extracellular domain) of wild-type (WT) gD from HSV-2 and / or HSV-1, or a fragment or variant thereof. In some embodiments, an HSV viral antigen or immunogen may comprise a soluble portion (e.g., an extracellular domain) of wild-type (WT) gB from HSV-2 and / or HSV-1, or a fragment or variant thereof. In some embodiments, an HSV viral antigen or immunogen may comprise a soluble portion (e.g., an extracellular domain) of wild-type (WT) gD from HSV-2 and / or HSV-1, or a fragment or variant thereof, and a soluble portion (e.g., an extracellular domain) of wild-type (WT) gB from HSV-2 and / or HSV-1, or a fragment or variant thereof, such as a fusion thereof.

[0083] In some embodiments, the extracellular domain of gD from human HSV-2 comprises amino acids 26-331 of human HSV-2 gD (e.g., GenBank: QBH77835.1). In some embodiments, the extracellular domain of gB from human HSV-2 comprises amino acids 23-727 of human HSV-2 gB (e.g., YP_009137179.1).

[0084] In some cases, a soluble portion of an HSV viral antigen or a fragment or variant thereof contains an epitope of the HSV viral antigen. An epitope comprises an antigenic determinant chemical group or peptide sequence that is antigenic on the molecule, such that it elicits a specific immune response; for example, an epitope is an antigenic region that triggers a B cell and / or T cell response. Antibodies can bind to specific antigenic epitopes. Epitopes can be formed from sequential amino acids or from discontinuous amino acids juxtaposed through protein ternary folding. In some embodiments, an epitope is a linear epitope. In some embodiments, an epitope is a conformational epitope. In some embodiments, an epitope is a neutralizing epitope that can induce neutralizing antibodies. In some embodiments, an HSV viral antigen contains all epitopes of the HSV viral antigen (e.g., all neutralizing epitopes). In some embodiments, an HSV viral antigen contains one or more or all of the epitopes of the HSV gD protein (e.g., neutralizing epitopes). In some embodiments, an HSV viral antigen or immunogen contains one or more or all of the epitopes of the HSV gB protein (e.g., neutralizing epitopes). In some embodiments, the HSV viral antigen or immunogen contains one or more of the epitopes (e.g., neutralizing epitopes) of the HSV gD protein and one or more of the epitopes (e.g., neutralizing epitopes) of the HSV gB protein.

[0085] In some cases, the soluble portion of the HSV viral antigen does not contain transmembrane (TM) domain peptides and / or cytoplasmic (CP) domain peptides.

[0086] In some cases, variants of the HSV surface antigen (e.g., soluble portions of the HSV surface antigen, such as gD and gB) may contain one, two, three, four, five, or more amino acid substitutions, deletions, and / or insertions compared to the native HSV surface antigen. These substitutions, deletions, and / or insertions provide increased retention of the pre-fusion conformation compared to the trimer formed from the corresponding native HSV surface antigen (e.g., soluble portions of the HSV surface antigen, such as gD and gB). “Stabilization” of the pre-fusion conformation by one or more amino acid substitutions, deletions, and / or insertions can be, for example, energy stabilization (e.g., reducing the energy of the pre-fusion conformation relative to the post-fusion open conformation) and / or kinetic stabilization (e.g., reducing the rate of transition from the pre-fusion conformation to the post-fusion conformation). Additionally, stabilization of the HSV surface antigen trimer in the pre-fusion conformation can include increased resistance to denaturation compared to the corresponding native HSV surface antigen. This article provides methods for determining whether an HSV viral surface antigen trimer is in a pre-fusion conformation, including (but not limited to) negative staining electron microscopy and antibody binding assays using pre-fusion conformation-specific antibodies. In some embodiments, variants of the HSV viral surface antigen may be soluble portions (e.g., extracellular domains) or fragments thereof of HSV-2 gB containing an H513P substitution (e.g., H513P mutants of the HSV-2 gB extracellular domain), which can stabilize gB in its pre-fusion conformation. In some embodiments, variants of the HSV viral surface antigen may contain soluble portions (e.g., extracellular domains) or fragments thereof of HSV-1 gB containing an H516P substitution (e.g., H516P mutants of the HSV-1 gB extracellular domain), which helps to immobilize it in its pre-fusion form, thereby stabilizing gB in its pre-fusion conformation.

[0087] In some cases, fusion peptides are trimerized single subunits.

[0088] In some cases, HSV viral antigens or immunogens contain a soluble portion or fragment or variant of HSV (e.g., HSV-2 and HSV-1) gD in a pre-fusion conformation. This pre-fusion conformation is the structural conformation adopted after the extracellular domain of the HSV-gD protein can be processed into mature HSV (e.g., HSV-2 and HSV-1) gD proteins in the secretory system and before triggering a fusion event that causes HSV (e.g., HSV-2 and HSV-1) to transform into a post-fusion conformation. Figure 4 and Figure 7 The paper provides an exemplary three-dimensional structure of a gD protein in its pre-fusion conformation.

[0089] In some embodiments, the HSV viral antigen or immunogen may comprise gD, gB, gD-gB, gD-gB (WT), gD-gB (mutant), gD(F)-gB, gD(F)-gB (WT), or gD-gB (mutant). Referring to the HSV viral antigen that is part of the fusion polypeptide provided herein (e.g., as described throughout the specification, including the figures), it should be understood that, unless otherwise specifically stated: gD refers to the full-length gD protein or any fragment thereof, including wild-type gD protein or any fragment thereof (e.g., extracellular domain) or variants thereof; gB refers to the full-length gB protein or any fragment thereof, including wild-type gB protein or any fragment thereof (e.g., extracellular domain) or variant thereof; gB (WT) refers to wild-type full-length gB protein or any fragment thereof (e.g., extracellular domain). gB (mutant) refers to a variant of the wild-type full-length gB protein or any segment thereof (e.g., extracellular domain). For example, gB(H513P) refers to the H513P mutant of the wild-type full-length gB protein or any segment thereof (e.g., extracellular domain), and gB(H516P) refers to the H516P mutant of the wild-type full-length gB protein or any segment thereof (e.g., extracellular domain). gD-gB refers to a fusion of gD and gB directly connected; gD-gB(WT) refers to a fusion of gD and gB(WT) directly connected; gD-gB (mutant) refers to a fusion of gD and gB (mutant) directly linked; gD(F)-gB refers to a fusion of gD and gB linked by a furin protease site; gD(F)-gB(WT) refers to a fusion of gD and gB(WT) linked through a furin protease site; and gD(F)-gB (mutant) refers to a fusion of gD and gB (mutant) linked by the furin protease site.

[0090] In some embodiments, the HSV viral antigen does not contain a signal peptide, a transmembrane domain, and / or a cytoplasmic domain, and may contain truncated forms of full-length gD and / or gB proteins. In some embodiments, the HSV viral antigen contains a signal peptide.

[0091] In some embodiments, the HSV viral antigen or immunogen is generated from a codon-optimized nucleic acid sequence. In some embodiments, the HSV viral antigen or immunogen is generated from a non-codon-optimized nucleic acid sequence.

[0092] II. Recombinant peptides and proteins Considering the HSV viral antigens and immunogens provided herein, such as gD, gD-gB (WT), gD-gB (mutant), gD(F)-gB (WT), and gD(F)-gB (mutant) protein peptides (see, Part I), they can be combined (e.g., linked) with other proteins or peptides to form recombinant polypeptides (including fusion peptides), wherein the HSV can be HSV-1 and / or HSV-2, such as human HSV-1 and HSV-2. In some embodiments, individual recombinant polypeptides (e.g., monomers) provided herein associate to form multimers of the recombinant polypeptide, such as trimers. In some embodiments, association between individual recombinant polypeptide monomers occurs via covalent interactions. In some embodiments, association between individual recombinant polypeptide monomers occurs via non-covalent interactions. In some embodiments, the interaction (e.g., covalent or non-covalent) is achieved by a protein or peptide linked to an HSV viral antigen or immunogen (e.g., gD, gD-gB (WT), gD-gB (mutant), gD(F)-gB (WT), gD(F)-gB (mutant) protein peptide). In some embodiments, for example, when the HSV viral antigen or immunogen is a peptide as described herein, the protein or peptide linked to it can be selected such that the native homotrimeric structure of the glycoprotein is preserved. This can be advantageous for evoking a strong and effective immunogenic response against the HSV gB / gD protein peptide. For example, preserving and / or maintaining the native conformation of the HSV viral antigen or immunogen (e.g., gB / gD protein peptide) can improve or enable the exposure of antigenic sites that generate an immune response.

[0093] Furthermore, in some cases, recombinant peptides or polymerized recombinant peptides thereof aggregate or are aggregated to form multimeric proteins or complexes containing multiple recombinant peptides containing HSV viral antigens or immunogens. The formation of such proteins can be advantageous for generating strong and effective immunogenic responses against HSV viral antigens and / or immunogens. For example, the formation of proteins containing multiple recombinant peptides, and thus multiple HSV viral antigens, can preserve the tertiary and / or quaternary structures of the viral antigens, thereby allowing for immune responses against the native structures. In some cases, aggregation can confer structural stability to HSV viral antigens or immunogens, which in turn can expose potential antigenic sites that can promote immune responses.

[0094] 1. Fusion peptides and recombinant peptides In some embodiments, HSV viral antigens or immunogens may be linked to a trimerization domain (also known as a trimerization tag) to promote trimerization of monomers. In some embodiments, HSV (e.g., HSV-1 and / or HSV-2) viral antigens or immunogens may be linked to a trimerization domain at their C-terminus (C-terminal linker) or N-terminus (N-terminal linker). In some embodiments, trimerization stabilizes the proximal membrane region of HSV viral antigens or immunogens (e.g., gD, gD-gB (WT), gD-gB (mutant), gD(F)-gB (WT), gD(F)-gB (mutant) peptides) to a trimer configuration.

[0095] In some embodiments, HSV viral antigens or immunogens can be directly linked to the trimerizing domain or indirectly linked to the trimerizing domain via peptide linkers. In some embodiments, HSV viral antigens or immunogens can be linked to the multimerizing domain via in-frame fusion. In some embodiments, HSV viral antigens and multimerizing domains are expressed as a single polypeptide, which can trimerize to form a trimer through interactions between the trimerizing domains.

[0096] Non-limiting examples of exogenous polymerizing domains that promote stable trimers of soluble recombinant proteins include: the GCN4 leucine zipper from yeast GCN4 (Harbury et al. 1993 Science 262:1401-1407), the trimerizing motif from pulmonary surfactant (Hoppe et al. 1994 FEBS Lett 344:191-195), collagen (McAlinden et al. 2003 J Biol Chem 278:42200-42207), and phage T4 fibroin foldon (Miroshnikov et al. 1998 Protein Eng [Protein Engineering] 11:329-414), any of which can be linked to the HSV viral antigen or immunogen described herein (e.g., by linking to the C-terminus of the gD, gD-gB, or gD(F)-gB peptide) to promote the trimerization of the recombinant viral antigen or immunogen. See also U.S. Patent Nos. 7,268,116, 7,666,837, 7,691,815, 10,618,949, 10,906,944, and 10,960,070, and US2020 / 0009244, which are incorporated herein by reference in their entirety for all purposes.

[0097] In some embodiments, one or more peptide linkers (e.g., gly-serine linkers, such as a 10-amino acid glycine-serine peptide linker) may be used to link recombinant viral antigens or immunogens to a polymerizing domain. The trimer may include any stabilizing mutations (or combinations thereof) provided herein, provided that the recombinant viral antigen or immunogen trimer retains the desired properties (e.g., pre-fusion conformation).

[0098] For therapeutic feasibility, the required trimerized tag protein for biopharmaceutical design should meet the following criteria. Ideally, it should be part of a naturally secreted protein (like HSV gD / gB) that is also abundant in circulation (non-toxic); derived from humans (lacking immunogenicity); relatively stable (long half-life); and capable of efficient self-trimerization (reinforced by interchain covalent disulfide bonds), thus the trimerized HSV viral antigen or immunogen is structurally stable.

[0099] Collagen, a member of the fibrin family, is a major component of the extracellular matrix. It is the most abundant protein in mammals, accounting for nearly 25% of total body protein. Collagen plays a major structural role in the formation of bone, tendons, skin, cornea, cartilage, blood vessels, and teeth. Collagen types I, II, III, IV, V, and XI are synthesized into larger trimer precursors called procollagen, which consists of a central, unbroken triple helix domain composed of hundreds of "GXY" repeat sequences (or glycine repeat sequences) flanked by non-collagenous domains (NC), N-propeptide, and C-propeptide. During procollagen secretion, both the C-terminal and N-terminal extensions undergo proteolytic processing, triggering the assembly of mature proteins into collagen fibrils, thus forming the insoluble cellular matrix. BMP-1 is a protease that recognizes specific peptide sequences in procollagen near the junction between the glycine repeat sequence and the C-propeptide of collagen and is responsible for removing the propeptide. The trimer C-propeptide from type I collagen detachment is present in the serum of normal adults at concentrations ranging from 50 to 300 ng / mL, while levels are much higher in children, indicating active bone formation. In individuals with familial high serum concentrations of type I collagen C-propeptide, levels can reach as high as 1–6 μg / mL without significant abnormalities, suggesting that C-propeptide is non-toxic. Structural studies of the collagen trimer C-propeptide have revealed a trefoil structure, with all three subunits clustered together in a linker region near their N-terminus to attach to the rest of the procollagen molecule. This geometry, projecting the protein to be fused along one direction, is similar to the geometry of the Fc dimer.

[0100] Type I, IV, V, and XI collagens primarily assemble into heterotrimeric forms consisting of two α-1 chains and one α-2 chain (Type I, IV, and V) or three distinct chains (Type XI), which are highly homologous in sequence. Type II and III collagens are both homotrimers of the α-1 chain. For Type I collagen (the most abundant form of collagen), stable α(I) homotrimers also form, and their abundance varies across different tissues. When overexpressed alone in cells, most of the C-propeptide chains in these collagens can self-assemble into homotrimers. Although the N-propeptide domain is synthesized first, the assembly of the molecules into trimer collagen originates from the alignment association of the C-propeptide. The C-propeptide complex is believed to be stabilized by the formation of interchain disulfide bonds, but whether disulfide bond formation is necessary for proper chain alignment is unclear. The triple helix of the glycine repeat sequence then extends from the associated C-terminus to the N-terminus in a zipper-like manner. This knowledge has led to the use of recombinant DNA technology to create non-natural types of collagen matrices by exchanging C-propeptides from different collagen chains. Non-collagenous proteins (such as cytokines and growth factors) have also been fused to the N-terminus of procollagen or mature collagen to allow for the formation of new collagen matrices, which is designed to allow for the slow release of non-collagenous proteins from the cellular matrix. However, in both cases, the C-propeptide needs to be cleaved before the recombinant collagen fibrils assemble into the insoluble cellular matrix.

[0101] Therefore, the use of collagen in the recombinant peptides described herein has many advantages, including: (1) Collagen is the most abundant secreted protein in mammals, accounting for nearly 25% of the total protein in the body; (2) The main form of collagen is naturally present in the trimeric helix, and its globular C-propeptide is responsible for initiating trimerization; (3) The C-propeptide of collagen trimer, released from mature collagen via proteolytic hydrolysis, is naturally present in mammalian blood at sub-microgram / mL levels and is known to be non-toxic to the body; (4) The linear triple helix region of collagen can be included as a linker with a predicted spacing of 2.9 Å per residue, or excluded as part of the fusion protein, thereby allowing precise tuning of the distance between the protein to be trimerized and the C-propeptide of collagen to achieve optimal bioactivity; (5) The BMP1 recognition site that cleaves the C-propeptide from procollagen can be mutated or deleted to prevent the destruction of the trimer fusion protein; (6) The C-propeptide domain self-trimerizes via disulfide bonds, and it provides a universal affinity tag that can be used to purify any secreted fusion protein produced. In some embodiments, the C-propeptide of collagen linked to HSV viral antigens and immunogens (e.g., gD-containing protein peptides) can be recombined to generate soluble, covalently linked homotrimeric fusion proteins.

[0102] In some embodiments, an HSV viral antigen or immunogen is linked to the C-terminal portion of collagen (e.g., human collagen) to form a recombinant polypeptide. In some embodiments, the C-terminal portion of collagen comprises a C-propeptide of collagen. In some embodiments, the C-terminal portion of collagen is a C-propeptide without any triple-helix region of collagen. In some embodiments, the C-terminal portion of collagen comprises a triple-helix region of a glycine repeat sequence of collagen (e.g., amino acids 1156-1464 from human type I (α) collagen) linked to the C-propeptide, wherein the triple-helix region of the glycine repeat sequence can serve as a linker for linking to the HSV viral antigen. In some embodiments, the C-terminal portion of collagen comprises a truncated form of the C-propeptide, such as the trimer (truncated) contained in SEQ ID No: 15-28. In some embodiments, the C-terminal portion of collagen has a mutated or missing BMP-1 protease recognition site.

[0103] In some embodiments, the glycine repeat sequence comprises a glycine-XY repeat sequence, wherein X and Y are independently any amino acid or an amino acid sequence having at least 85%, 90%, 92%, 95%, or 97% identity with it, the amino acid sequence being capable of forming disulfide bonds between peptides and trimerizing the recombinant peptide. In some embodiments, X and Y are independently proline or hydroxyproline.

[0104] In some embodiments, the C-terminal portion of collagen in the recombinant polypeptide forms interpeptide disulfide bonds. In some embodiments, the recombinant protein forms a trimer (e.g., a homotrimer).

[0105] In some embodiments, the interpeptide disulfide bonds may comprise one or more of the following in any suitable combination: Cys15-136, Cys131-166, Cys291-301, Cys379-432, Cys336-361, Cys391-525, Cys480-488, Cys538-590, Cys617-649, Cys662-671, Cys743-749, Cys738-760, Cys840-851, Cys1032-1043, and Cys1082-1126. In some embodiments, the fusion polypeptide in the trimer may include one or more glycosylation sites (e.g., Asn-linked) in any suitable combination, for example, at one or all of the Asn residues at 17, 61, 122, 149, 165, 234, 282, 331, 343, 603, 616, 657, 709, 717, 801, 1074, 1098, and 1134.

[0106] In some embodiments, the C-terminal portion is derived from human collagen. In some embodiments, the C-terminal portion comprises the C-terminal portion of proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI), or proα3(XI), or a fragment thereof. In some embodiments, the C-terminal portion is or comprises the C-terminal portion of proα1(I). In some embodiments, the C-terminal portion is or comprises amino acids 1156-1464 derived from human type I (α) collagen.

[0107] In some cases where HSV viral antigens are linked to their C-terminal portions to form recombinant peptides, the recombinant peptides form trimers, thereby producing homotrimers of the HSV viral antigens. In some embodiments, the HSV viral antigens of the trimerized recombinant peptides are in a pre-fusion or post-fusion conformation. In some embodiments, the conformational state exposes different antigenic sites on the HSV viral antigens. In some embodiments, the antigenic sites are epitopes, such as linear epitopes or conformational epitopes. An advantage of having trimerized recombinant peptides as described is that they can generate immune responses against a variety of potential and different antigenic sites.

[0108] In some embodiments, a trimerized recombinant polypeptide comprises a single recombinant polypeptide containing the same viral antigen or immunogen. In some embodiments, a trimerized recombinant polypeptide comprises a single recombinant polypeptide, each containing a viral antigen or immunogen different from the others. In some embodiments, a trimerized recombinant polypeptide comprises a single recombinant polypeptide, wherein one of the single recombinant polypeptides contains a viral antigen or immunogen different from the others. In some embodiments, a trimerized recombinant polypeptide comprises a single recombinant polypeptide, wherein two of these single recombinant polypeptides contain the same viral antigen or immunogen, and this viral antigen or immunogen is different from the viral antigen or immunogen contained in the remaining recombinant polypeptides.

[0109] In some embodiments, the recombinant polypeptide comprises any HSV viral antigen or immunogen described in Part I. In some embodiments, the recombinant polypeptide comprises any HSV viral antigen or immunogen described in Part I, linked to the C-terminal portion of the collagen described herein.

[0110] In some embodiments, the recombinant HSV (e.g., HSV-1 or HSV-2) glycoprotein extracellular domain trimer stabilized in the prefusion conformation comprises HSV glycoprotein extracellular domain (e.g., gD or gB) protomers containing mutations in HSV gD and / or protein gB.

[0111] In some embodiments, the promerum of the extracellular domain trimer of the recombinant HSV glycoprotein can be stabilized in the pre-fusion conformation by substitution, deletion or insertion of one or more amino acids.

[0112] In some embodiments, the recombinant polypeptide is or comprises an NTD peptide of a protein containing HSV-1 or HSV-2 gD. In some embodiments, the recombinant polypeptide is or comprises an RBD peptide of a protein containing HSV-1 or HSV-2 gD. In some embodiments, the recombinant polypeptide is or comprises both an NTD peptide and an RBD peptide of a protein containing HSV-1 or HSV-2 gD. In some embodiments, the recombinant polypeptide is or comprises a gB or gD-gB domain peptide of a protein containing HSV-1 or HSV-2 gD.

[0113] In some embodiments, the recombinant polypeptide may or may not contain a signal peptide. In some embodiments, the signal peptide is located at the N-terminus of the recombinant polypeptide. In some embodiments, the signal peptide may be a native signal peptide of gD or gB from HSV-2 or HSV-1 (e.g., human HSV-2 or human HSV-1). In some embodiments, the signal peptide may comprise the sequence of amino acids 1-25 of the HSV-2 gD protein (e.g., GenBank: QBH77835.1) or amino acids 1-22 of the HSV-2 gB protein (e.g., GenBank: YP_009137179.1).

[0114] An exemplary HSV-2 recombinant polypeptide that does not contain a signal peptide is provided in SEQ ID NO: 4.

[0115] Regarding protein trimer tags that are part of the recombinant polypeptides provided herein, for example, as described throughout the specification including the figures, unless otherwise specified, they may be abbreviated as "-trimer," for example, "gD-trimer" refers to gD linked to a protein trimer tag at its C-terminus; "gD-gB-trimer" refers to gD-gB linked to a protein trimer tag at its C-terminus; and "gD(F)-gB-trimer" refers to gD(F)-gB linked to a protein trimer tag at its C-terminus.

[0116] In some embodiments, the recombinant polypeptide gD-trimer is or comprises the amino acid sequence shown in any one of SEQ ID NO: 1, 8, 15, and 22, or any antigenic fragment thereof. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 1, 8, 15, and 22. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having substitutions, deletions, and / or insertions at one or more amino acid positions compared to any one of SEQ ID NO: 1, 8, 15, and 22.

[0117] In some embodiments, the recombinant polypeptide gB-trimer is or comprises the amino acid sequence shown in any one of SEQ ID NO: 2, 3, 9, 10, 16, 17, 23, and 24, or any antigenic fragment thereof. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 2, 3, 9, 10, 16, 17, 23, and 24. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having substitutions, deletions, and / or insertions at one or more amino acid positions compared to any one of SEQ ID NO: 2, 3, 9, 10, 16, 17, 23, and 24.

[0118] In some embodiments, the recombinant polypeptide gD-gB-trimer is or comprises the amino acid sequence shown in any one of SEQ ID NO: 4, 5, 11, 12, 18, 19, 25, and 26, or any antigenic fragment thereof. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 4, 5, 11, 12, 18, 19, 25, and 26. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having substitutions, deletions, and / or insertions at one or more amino acid positions compared to any one of SEQ ID NO: 4, 5, 11, 12, 18, 19, 25, and 26.

[0119] In some embodiments, the recombinant polypeptide gD(F)-gB-trimer is or comprises the amino acid sequence shown in SEQ ID NO: 6, 7, 13, 14, 20, 21, 27 and 28 or any antigenic fragment thereof. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NO: 6, 7, 13, 14, 20, 21, 27 and 28. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having substitutions, deletions and / or insertions at one or more amino acid positions compared to any one of SEQ ID NO: 6, 7, 13, 14, 20, 21, 27 and 28.

[0120] As shown above, in some embodiments, the recombinant peptides provided herein can not only associate to form trimers, but also aggregate or be aggregated to generate proteins comprising multiple recombinant peptides. In some embodiments, the formed protein has a macroscopic structure. In some cases, the macroscopic structure can confer structural stability to the HSV viral antigen or immunogenic recombinant peptides, thereby enabling the exposure of potential antigenic sites that can promote an immune response.

[0121] In some embodiments, trimerized recombinant peptides aggregate to form a protein containing multiple trimerized recombinant peptides. In some embodiments, multiple trimerized recombinant peptides form a protein having a macroscopic structure.

[0122] In some embodiments, the protein comprising multiple recombinant peptides described herein is an immunogen. In some embodiments, the protein comprising multiple recombinant peptides described herein is contained in nanoparticles. For example, in some embodiments, the protein is directly linked to the nanoparticles (e.g., protein nanoparticles). In some embodiments, the protein is indirectly linked to the nanoparticles. In some embodiments, the protein comprising multiple recombinant peptides described herein is contained in virus-like particles (VLPs).

[0123] In some embodiments, this document provides complexes of recombinant peptides provided herein. In some embodiments, the complex is a trimeric fusion protein (i.e., a trimer). In some embodiments, this document provides a complex comprising, in any suitable combination, a recombinant peptide selected from the group consisting of SEQ ID NO: 1-28, or a fragment, variant, or mutant thereof. In some embodiments, this document provides a complex comprising a trimer of a recombinant peptide selected from the group consisting of SEQ ID NO: 1-28, or a fragment, variant, or mutant thereof, wherein the recombinant peptides trimerize via interpeptide disulfide bonds to form a trimer.

[0124] In some embodiments, this document provides a trimer fusion protein comprising three recombinant peptides, each recombinant peptide comprising, from the amino terminus to the carboxyl terminus: a) a first region comprising gD of HSV-2 and / or HSV-1; b) a second region comprising gB of HSV-1 and / or HSV-2; and c) a C-terminal portion of collagen, wherein the C-terminal portion of collagen in the recombinant peptide forms interpeptide disulfide bonds.

[0125] In some embodiments, this document provides a trimer fusion protein comprising three recombinant peptides, each recombinant peptide comprising, from the amino terminus to the carboxyl terminus: a) a first region comprising gD of HSV-2 and / or HSV-1; and b) a C-terminal portion of collagen, wherein the C-terminal portion of collagen in the recombinant peptide forms interpeptide disulfide bonds.

[0126] In some embodiments, this document provides a method for preventing or treating HSV infection in a subject (e.g., a mammal), the method comprising immunizing the subject with an effective amount of a fusion protein or trimer thereof disclosed herein. In some embodiments, the method comprises immunizing the subject with two or more fusion proteins or trimers thereof disclosed herein. The two or more fusion proteins may comprise HSV viral antigens derived from the same or different HSV species, subtypes, and / or strains, or the same or different HSV viral surface antigens (or glycoproteins) derived from the same HSV species, subtypes, and / or strains. In some embodiments, the two or more fusion proteins may comprise HSV viral antigens derived from a first HSV and a second HSV (e.g., HSV-1 and HSV-2), respectively. In some embodiments, the two or more fusion proteins may comprise HSV (e.g., HSV-1 and / or HSV-2) gD and HSV (e.g., HSV-1 and / or HSV-2) gB, respectively. In some embodiments, neutralizing antibodies against the first and second HSVs are generated in the subject. In some embodiments, the first HSV and the second HSV are different variants of HSV, and neutralizing antibodies generated in the subject neutralize two or more HSV-1 and / or HSV-2 and their mutants.

[0127] In some embodiments, the method includes immunizing a subject (e.g., a mammal) with two or more doses of the fusion protein. In some embodiments, the fusion protein is administered as a booster dose after one or more doses of an immunogen comprising an HSV glycoprotein peptide containing gD, gD-gB (WT), gD-gB (mutant), gD(F)-gB (WT), or gD(F)-gB (mutant).

[0128] In some embodiments, this document provides engineered fusion peptides containing gD glycoproteins derived from or modified from HSVs (e.g., HSV-1 and / or HSV-2). In some embodiments, the fusion peptides disclosed herein are stable in their pre-fusion conformation compared to the wild-type protein sequence of HSV.

[0129] 2. Polynucleotides and carriers Also provided are polynucleotides (nucleic acid molecules) encoding the HSV viral antigen or immunogen and recombinant peptides provided herein, and vectors for genetically engineered cells to express such HSV viral antigens or immunogens and recombinant peptides.

[0130] In some embodiments, a polynucleotide encoding the recombinant polypeptide provided herein is provided. In some aspects, the polynucleotide contains a single nucleic acid sequence, such as a nucleic acid sequence encoding the recombinant polypeptide. In other cases, the polynucleotide contains a first nucleic acid sequence encoding a recombinant polypeptide comprising a specific HSV viral antigen or immunogen and a second nucleic acid sequence encoding a recombinant polypeptide comprising a different HSV viral antigen or immunogen.

[0131] In some embodiments, the polynucleotide encoding the recombinant polypeptide contains at least one promoter operatively linked to the recombinant polypeptide to control its expression. In some embodiments, the polynucleotide contains two, three, or more promoters operatively linked to the recombinant polypeptide to control its expression.

[0132] In some embodiments, for example, when the polynucleotide contains two or more nucleic acid coding sequences, such as sequences encoding recombinant polypeptides containing different HSV viral antigens or immunogens, at least one promoter is operatively linked to the two or more nucleic acid sequences to control the expression of the two or more nucleic acid sequences. In some embodiments, the polynucleotide contains two, three or more promoters operatively linked to the recombinant polypeptide to control the expression of the recombinant polypeptide.

[0133] In some embodiments, the expression of one or more recombinant peptides is inducible or conditional. Thus, in some aspects, the polynucleotide encoding one or more recombinant peptides contains a conditional promoter, enhancer, or trans-activator. In some such aspects, the conditional promoter, enhancer, or trans-activator is an inducible promoter, enhancer, or trans-activator or a repressive promoter, enhancer, or trans-activator. For example, in some embodiments, an inducible promoter or conditional promoter can be used to restrict the expression of the recombinant peptide to a specific microenvironment. In some embodiments, expression driven by an inducible promoter or conditional promoter is regulated by exposure to exogenous factors (e.g., heat, radiation, or drugs).

[0134] In cases where a polynucleotide contains more than one nucleic acid sequence encoding a recombinant polypeptide, the polynucleotide may further include a nucleic acid sequence encoding a peptide that is a translation product of the nucleic acid sequence during or after translation, encoding a self-cleaving peptide, or encoding a peptide that causes ribosome jumping (e.g., the T2A peptide), or encoding an internal ribosome entry site (IRES) between more than one nucleic acid sequence.

[0135] In some embodiments, a polynucleotide encoding one or more recombinant polypeptides is introduced into a composition containing cultured cells (e.g., host cells), for example by retroviral transduction, transfection, or transformation. In some embodiments, this may allow the expression (e.g., generation) of the recombinant polypeptide. In some embodiments, the expressed recombinant polypeptide is purified.

[0136] In some embodiments, the polynucleotides (nucleic acid molecules) provided herein encode HSV viral antigens or immunogens as described herein. In some embodiments, the polynucleotides (nucleic acid molecules) provided herein encode recombinant polypeptides containing HSV viral antigens or immunogens, such as protein peptides containing gD, as described herein.

[0137] Vectors or constructs containing nucleic acid molecules as described herein are also provided. In some embodiments, the vector or construct contains one or more promoters operatively linked to a nucleic acid molecule encoding a recombinant polypeptide to drive its expression. In some embodiments, the promoter is operatively linked to one or more nucleic acid molecules (e.g., nucleic acid molecules encoding recombinant polypeptides containing different HSV viral antigens or immunogens).

[0138] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector, an adenovirus, or an adeno-associated virus. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the retroviral vector is a gamma retroviral vector.

[0139] In some embodiments, the vector or construct includes a single promoter that drives the expression of one or more nucleic acid molecules carrying a polynucleotide. In some embodiments, such a promoter may be polycistronic (bicistronic or tricistronic, see, for example, U.S. Patent No. 6,060,273). For example, in some embodiments, the transcription unit may be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), thereby allowing co-expression of gene products (e.g., encoding different recombinant polypeptides) via information from a single promoter. In some embodiments, the vectors provided herein are bicistronic, allowing the vector to contain and express two nucleic acid sequences. In some embodiments, the vectors provided herein are tricistronic, allowing the vector to contain and express three nucleic acid sequences.

[0140] In some embodiments, a single promoter directs the expression of RNA containing two or three genes (e.g., encoding a chimeric signaling receptor and a recombinant receptor) within a single open reading frame (ORF), separated from each other by sequences encoding self-cleaving peptides (e.g., the 2A sequence) or protease recognition sites (e.g., furin protease). Thus, the ORF encodes a single polypeptide that is processed into a single protein during (in the case of 2A) or post-translational. In some cases, the peptide (e.g., T2A) can induce ribosome skipping (ribosome jumping) synthesis of the peptide bond at the C-terminus of the 2A element, resulting in separation between the end of the 2A sequence and the next downstream peptide (see, e.g., de Felipe). Genetic Vaccines and Ther. [Gene Vaccines and Therapies] 2:13 (2004) and deFelipe et al. Traffic [Transportation] 5:616-626 (2004). Many 2A elements are known in the art. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein include, but are not limited to, 2A sequences from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), tilapia tussock moth virus (T2A), and porcine swine cyclovir-1 (P2A), as described in U.S. Patent Publication No. 20070116690.

[0141] In some embodiments, the vector is contained within a virus. In some embodiments, the virus is a pseudovirus. In some embodiments, the virus is a virus-like particle. In some embodiments, the vector is contained within a cell. In some embodiments, the virus or cell containing the vector contains a recombinant genome.

[0142] In some embodiments, cells expressing the fusion peptides provided herein or containing the fusion peptides, polynucleotides, or vectors provided herein are provided. Cells can be used to produce the fusion peptides or their trimers. Cells can be mammalian cells, such as human cells, or non-human mammalian cells, such as CHO cells, HEK293 cells, or cells derived therefrom.

[0143] III. Immunogenic compositions and formulations In some embodiments, this document provides immunogenic compositions or vaccines comprising recombinant peptides or trimers thereof. In some embodiments, the immunogenic compositions or vaccines comprise two or more recombinant peptides or trimers thereof disclosed herein. The two or more recombinant peptides or trimers thereof may each comprise the same or different HSV viral surface antigens (or glycoproteins) from different HSV species, subtypes, and / or strains, or the same or different HSV viral surface antigens (or glycoproteins) from the same HSV species, subtypes, and / or strains. In some embodiments, the two or more recombinant peptides or trimers thereof may comprise HSV viral antigens derived from a first HSV and a second HSV (e.g., HSV-1 and HSV-2), respectively. In some embodiments, the two or more recombinant peptides or trimers thereof may each comprise HSV (e.g., HSV-1 and / or HSV-2) gD or fragments or variants thereof and HSV (e.g., HSV-1 and / or HSV-2) gB or fragments or variants thereof. In some embodiments, the two or more recombinant peptides or trimers thereof may each comprise gD-trimers and gB-trimers or trimers thereof.

[0144] In some embodiments, this document discloses monovalent or multivalent (e.g., bivalent, trivalent, or quadrivalent) vaccines comprising the fusion peptides or trimers disclosed herein.

[0145] In some embodiments, this document discloses a monovalent vaccine comprising an HSV (e.g., HSV-1 and / or HSV-2) viral antigen trimer disclosed herein, such as gD-trimer, gD-gB-trimer, gD-gB(WT)-trimer, gD-gB(mutant)-trimer, gD(F)-gB-trimer, gD(F)-gB(WT)-trimer, gD(F)-gB(mutant)-trimer, or a trimer thereof. In some embodiments, this document discloses a bivalent vaccine comprising HSV (e.g., HSV-1 and / or HSV-2) viral antigen trimers disclosed herein, wherein these HSV viral antigen trimers are selected from gD-trimer, gD-gB(WT)-trimer, gD-gB(mutant)-trimer, gD(F)-gB(WT)-trimer, and gD(F)-gB(mutant)-trimer. In some embodiments, this document discloses a bivalent vaccine comprising at least one HSV glycoprotein trimer containing a first HSV viral antigen and at least one HSV glycoprotein trimer containing a second HSV viral antigen. In some embodiments, the first HSV virus antigen and the second HSV virus antigen are derived from the same or different HSV virus surface antigens (or glycoproteins) of different HSV species, subtypes and / or strains (e.g., HSV-2 and HSV-1), or from the same or different HSV virus surface antigens (or glycoproteins) of the same HSV species, subtypes and / or strains (e.g., HSV-2 or HSV-1).

[0146] In some embodiments, this document discloses a trivalent vaccine comprising the HSV (e.g., HSV-1 and / or HSV-2) viral antigen trimer disclosed herein. In some embodiments, this document discloses a trivalent vaccine comprising at least one HSV viral antigen trimer containing a first HSV viral antigen, at least one HSV viral antigen trimer containing a second HSV viral antigen, and at least one HSV viral antigen trimer containing a third HSV viral antigen. In some embodiments, the first HSV glycoprotein antigen, the second HSV glycoprotein antigen, and the third HSV glycoprotein antigen are derived from the same HSV viral antigen of one or more viral species or strains / subtypes, or from two, three, or more different HSV viral antigens of one or more viral species or one or more strains / subtypes of the same viral species. In some embodiments, this document discloses a quadrivalent vaccine comprising the HSV (e.g., HSV-1 and / or HSV-2) viral antigen trimer disclosed herein. In some embodiments, this document discloses a quadrivalent vaccine comprising at least one HSV virus antigen trimer containing a first HSV virus antigen, at least one HSV virus antigen trimer containing a second HSV virus antigen, at least one HSV virus antigen trimer containing a third HSV virus antigen, and at least one HSV virus antigen trimer containing a fourth HSV virus antigen. In some embodiments, the first HSV glycoprotein antigen, the second HSV glycoprotein antigen, the third HSV glycoprotein antigen, and the fourth HSV glycoprotein antigen are derived from the same HSV virus antigen of one or more virus species or strains / subtypes, or from two, three, four, or more different HSV virus antigens of one or more virus species or one or more strains / subtypes of the same virus species.

[0147] In some embodiments, this document provides an immunogenic composition or vaccine comprising a recombinant polypeptide or a trimer thereof, the recombinant polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 1-28 and an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 1-28. In some embodiments, this document provides an immunogenic composition or vaccine comprising any combination of two or more recombinant polypeptides or their trimers, the recombinant polypeptides comprising a sequence selected from the group consisting of SEQ ID NO: 1-28 and an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 1-28. In some embodiments, this document provides an immunogenic composition comprising one or two recombinant polypeptides or their trimers having the sequences shown in SEQ ID NO: 1 and / or 4.

[0148] In some embodiments, a unit dose of the immunogenic composition may contain about 10 µg to about 100 µg of HSV-2 antigen, preferably about 25 µg to about 75 µg of HSV antigen, preferably about 40 µg to about 60 µg of HSV antigen, or about 50 µg of HSV antigen. In some embodiments, the dose contains 3 µg of HSV antigen. In other embodiments, the dose contains 9 µg of HSV antigen. In still other embodiments, the dose contains 30 µg of HSV antigen.

[0149] In some cases, it may be desirable to combine the disclosed viral immunogen or fusion protein or trimer with other pharmaceutical products (e.g., vaccines) that induce a protective response against other pathogens (e.g., pathogens). For example, a composition comprising a recombinant HSV antigen as described herein (e.g., a fusion protein or its trimer) can be administered simultaneously (typically separately) or sequentially with other vaccines (e.g., influenza or varicella-zoster vaccines) recommended by the Advisory Committee on Immunization Practices (ACIP); cdc.gov / vaccines / acip / index.html) for a target age group (e.g., infants approximately one to six months old). Therefore, a disclosed viral immunogen comprising the recombinant HSV viral antigen described herein can be administered simultaneously or sequentially with vaccines against, for example, hepatitis B (HepB), diphtheria, tetanus and pertussis (DTaP), pneumococcal bacteria (PCV), Haemophilus influenzae type b (Hib), polio, influenza, and rotavirus.

[0150] Multivalent or combination vaccines provide protection against multiple pathogens. In some respects, multivalent vaccines can prevent multiple strains and / or subtypes of the same pathogen. In others, multivalent vaccines prevent multiple pathogens, such as the combination vaccine Tdap (which prevents tetanus, pertussis, and diphtheria strains). Multivalent vaccines are highly desirable because they minimize the number of immunizations required to produce protection against multiple pathogens or causative strains, thus reducing administration costs and increasing coverage. This is especially useful, for example, when vaccinating infants or children.

[0151] In some embodiments, the vaccines described herein, such as those comprising immunogenic compositions, are multivalent vaccines. In some embodiments, the antigenic material used to incorporate into the multivalent vaccine composition is derived from an HSV strain or type, as described herein (see, for example, Part I). The antigen used to incorporate into the multivalent vaccine composition may be derived from one or more strains or subtypes of HSV, such as two to five strains or subtypes, to provide broader-spectrum protection. In one embodiment, the antigen used to incorporate into the multivalent vaccine composition is derived from multiple strains or subtypes of HSV. Other useful antigens include live, attenuated, and inactivated viruses, such as inactivated poliovirus (Jiang et al., J. Biol. Stand. [Journal of Biostandardization], (1986) 14:103-9), attenuated strains of hepatitis A virus (Bradley et al., J. Med. Virol. [Journal of Medical Virology], (1984) 14:373-86), attenuated measles virus (James et al., N. Engl. J. Med. [New England Journal of Medicine], (1995) 332:1262-6), and epitopes of pertussis virus (e.g., ACEL-IMUNE acellular DTP, Wyeth-Lederle Vaccines and Pediatrics).

[0152] In some respects, the vaccines provided herein are universal vaccines. In some embodiments, a universal vaccine is a vaccine that prevents multiple strains or subtypes of the same virus (e.g., multiple strains or subtypes of HSV, such as HSV-1 and HSV-2). Developing an effective universal HSV vaccine would reduce costs and save manpower.

[0153] In some respects, universal vaccines consist of multiple epitopes derived from different viral strains or subtypes (e.g., containing HSV-2 gD protein peptide epitopes and HSV-1 gB protein peptide epitopes). In other respects, universal vaccines consist of a single epitope that is conserved across different viral strains or subtypes. For example, universal vaccines may be based on one or more relatively conserved domains of the HSV protein.

[0154] Immunogenic compositions comprising a disclosed viral immunogen (e.g., a disclosed recombinant polypeptide or its trimer, or a nucleic acid molecule encoding a protopolymer of the disclosed recombinant polypeptide or its trimer) and a pharmaceutically acceptable carrier are also provided. In some embodiments, the immunogenic composition comprises a trimerized recombinant polypeptide provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a protein complex and optionally a pharmaceutically acceptable carrier, the protein complex comprising one of the trimerized recombinant polypeptides provided herein. In some embodiments, the immunogenic composition comprises protein nanoparticles provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a VLP provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises isolated nucleic acids provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a vector provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a virus provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a pseudovirus provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises the cells provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition (as described herein) is a vaccine. In some embodiments, the vaccine is a prophylactic vaccine. In some embodiments, the vaccine is a therapeutic vaccine. In some embodiments, the vaccine is both a prophylactic and a therapeutic vaccine. Such pharmaceutical compositions can be administered to subjects via a variety of routes of administration known to those skilled in the art, such as intramuscular, intradermal, subcutaneous, intravenous, intraarticular, intra-articular, intraperitoneal, intranasal, sublingual, tonsillar, oropharyngeal, or other parenteral and mucosal routes. In several embodiments, a pharmaceutical composition comprising one or more of the disclosed recombinant immunogens is an immunogenic composition. The actual methods for preparing the administerable composition will be known or obvious to those skilled in the art and are described in more detail in publications such as Remingtons Pharmaceutical Sciences, 19th edition, Mack Publishing Company, Easton, Pennsylvania, 1995.

[0155] Therefore, the fusion proteins or their trimers described herein can be formulated with pharmaceutically acceptable carriers to help maintain biological activity while also promoting increased stability during storage within an acceptable temperature range. Potential carriers include, but are not limited to, physiologically balanced culture media, phosphate-buffered saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers (e.g., proteins, peptides, or hydrolysates (e.g., albumin, gelatin)), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., monosodium glutamate), or other protectants. The resulting aqueous solutions can be used as is or lyophilized. The lyophilized formulation is combined with a sterile solution prior to single or multiple administrations.

[0156] Formulated compositions (especially liquid formulations) may contain antimicrobial agents to prevent or minimize degradation during storage, including but not limited to effective concentrations (typically 1% w / v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben. Antimicrobial agents may be contraindicated for some patients; therefore, lyophilized formulations may be reconstituted in solutions with or without such components.

[0157] The immunogenic compositions or vaccines disclosed herein may contain pharmaceutically acceptable mediators that approximate physiological conditions, such as pH adjusters and buffers, tension modifiers, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

[0158] Immunogenic compositions or vaccines may be excluding adjuvants or optionally including adjuvants to enhance the host's immune response. Suitable adjuvants include, for example, toll-like receptor (TLR) agonists, alum (aluminum hydroxide), AlPO4, aluminum hydroxide gel, lipid A and its derivatives or variants, oil emulsions, saponins, neutral liposomes, liposomes containing vaccines and cytokines, nonionic block copolymers, and chemokines. Nonionic block polymers containing polyoxyethylene (POE) and polyoxypropylene (POP), such as POE-POP-POE block copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Indiana) and IL-12 (Genetics Institute, Cambridge, Massachusetts), as well as many other suitable adjuvants well known in the art, can be used as adjuvants (Newman et al., 1998). , Critical Reviews in Therapeutic Drug Carrier Systems[Review of Therapeutic Drug Delivery Systems] 15:89-142). The advantage of these adjuvants is that they help to stimulate the immune system in a non-specific manner, thereby enhancing the immune response to the drug product. In some embodiments, the immunogenic compositions or vaccines disclosed herein may include one, two, or more adjuvants, or be administered together with one, two, or more adjuvants.

[0159] Examples of suitable adjuvants for immunogenic compositions or vaccine compositions include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, MPL™, and IL-12. In some embodiments, the vaccine compositions or nanoparticle immunogens disclosed herein can be formulated into controlled-release or delayed-release formulations. This can be achieved in compositions containing sustained-release polymers or via microencapsulation delivery systems or bioadhesive gels. Various pharmaceutical compositions can be prepared according to standard procedures well known in the art.

[0160] In some embodiments, the adjuvant may comprise a water-in-oil adjuvant or an oil-in-water adjuvant, such as a water-in-oil emulsion or an oil-in-water emulsion. In some embodiments, the adjuvant may comprise a metabolizable oil (e.g., squalene) and α-tocopherol in the form of an oil-in-water emulsion, and polyoxyethylene sorbitan monooleate (Tween-80). In some embodiments, the adjuvant formulation may comprise about 2% to about 10% squalene, about 2% to about 10% α-tocopherol (e.g., D-α-tocopherol), and about 0.3% to about 3% polyoxyethylene sorbitan monooleate. In some embodiments, the adjuvant formulation may comprise about 5% squalene, about 5% tocopherol, and about 0.4% polyoxyethylene sorbitan monooleate. In some embodiments, the immunogenic compositions disclosed herein may contain 3-de- O -Acyl monophosphoryl lipid A (3D-MPL) and an adjuvant in the form of an oil-in-water emulsion, the adjuvant containing a metabolizable oil, α-tocopherol, and polyoxyethylene sorbitan monooleate. In some embodiments, the immunogenic compositions or vaccines disclosed herein may contain QS21 (soapberry ( Quillaja saponaria Molina The extract of QS21, 3D-MPL, and an oil-in-water emulsion, wherein the oil-in-water emulsion comprises a metabolizable oil, α-tocopherol, and polyoxyethylene sorbitan monooleate. In some embodiments, the immunogenic compositions or vaccines disclosed herein may contain QS21, 3D-MPL, and an oil-in-water emulsion, wherein the oil-in-water emulsion has the following composition: a metabolizable oil (e.g., squalene), α-tocopherol, and Tween-80. In some embodiments, the immunogenic compositions or vaccines disclosed herein may contain an adjuvant in the form of a liposomal composition.

[0161] In some embodiments, the adjuvant may comprise a metabolizable oil (e.g., squalene), polyoxyethylene sorbitan monooleate (Tween-80), and Span 85. In some embodiments, the adjuvant formulation may comprise about 5% (w / v) squalene, about 0.5% (w / v) polyoxyethylene sorbitan monooleate, and about 0.5% (w / v) Span 85.

[0162] In some embodiments, the adjuvant may comprise saponins, cholesterol, and phospholipids, for example, in the form of a nanoparticle composition. In some embodiments, the immunogenic compositions or vaccines disclosed herein may contain a mixture of separately purified fractions of saponin, which are subsequently formulated with cholesterol and phospholipids.

[0163] In some embodiments, the immunogenic compositions or vaccines disclosed herein may contain an adjuvant selected from the group consisting of: MF59 ™ Matrix-A ™ Matrix-C ™ Matrix-M ™ AS01, AS02, AS03 and AS04.

[0164] In some embodiments, the immunogenic compositions or vaccines disclosed herein may contain a Toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide of 8 to 35 nucleotides in length containing an unmethylated cytidine-phosphate-guanosine (also known as CpG or cytosine-phosphate-guanosine) motif, and the amount of recombinant HSV antigen and oligonucleotide present in the immunogenic composition to effectively stimulate an immune response against the HSV antigen in mammalian subjects (e.g., human subjects in need). TLR9 (CD289) recognizes unmethylated cytidine-phosphate-guanosine (CpG) motifs found in microbial DNA, which can be mimicked using synthetic CpG-containing oligodeoxynucleotides (CpG-ODNs). CpG-ODNs are known to enhance antibody production and stimulate T helper 1 (Th1) cell responses (Coffman et al., Immunity, 33:492-503, 2010). Optimal oligonucleotide TLR9 agonists typically contain a palindromic sequence following the general formula: 5'-purine-purine-CG-pyrimidine-pyrimidine-3' or 5'-purine-purine-CG-pyrimidine-pyrimidine-CG-3'. U.S. Patent No. 6,589,940 is incorporated herein by reference in its entirety. In some embodiments, the CpG oligonucleotide is linear. In other embodiments, the CpG oligonucleotide is cyclic or comprises one or more hairpin rings. The CpG oligonucleotide can be single-stranded or double-stranded. In some embodiments, the CpG oligonucleotide may contain modifications. Modifications include, but are not limited to, modifications of 3'OH or 5'OH groups, modifications of nucleotide bases, modifications of sugar components, and modifications of phosphate groups. Modified bases may be included in the palindromic sequence of the CpG oligonucleotide as long as one or more modified bases maintain the same specificity to their natural complementary bases via Watson-Crick base pairing (e.g., the palindromic portion remains self-complementary). In some embodiments, the CpG oligonucleotide contains non-classical bases. In some embodiments, the CpG oligonucleotide comprises a modified nucleoside. In some embodiments, the modified nucleoside is selected from the group consisting of 2'-deoxy-7-dezoguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted arabinoguanosine, and 2'-O-substituted arabinoguanosine. The CpG oligonucleotide may contain phosphate group modifications. For example, in addition to phosphodiester bonds, phosphate ester modifications include, but are not limited to, methyl phosphonate, thiophosphate, aminophosphate (bridged or non-bridged), triphosphate, and dithiophosphate, and may be used in any combination. Other non-phosphate ester bonds may also be used. In some embodiments, the oligonucleotide comprises only a thiophosphate backbone. In some embodiments, the oligonucleotide comprises only a phosphodiester backbone. In some embodiments, the oligonucleotide comprises a combination of phosphate ester bonds in the phosphate ester backbone, such as a combination of phosphodiester and thiophosphate bonds.Oligonucleotides with a phosphate thioester backbone can be more immunogenic than oligonucleotides with a phosphodiester backbone and appear to be less susceptible to degradation after injection into the host (Braun et al., J Immunol, 141:2084-2089, 1988; and Latimer et al., Mol Immunol, 32:1057-1064, 1995). The CpG oligonucleotides disclosed herein comprise at least one, two, or three internucleotide phosphate thioester bonds. In some embodiments, when multiple CpG oligonucleotide molecules are present in a pharmaceutical composition comprising at least one excipient, both stereoisomers of the phosphate thioester bonds are present in the multiple CpG oligonucleotide molecules. In some embodiments, all internucleotide bonds in the CpG oligonucleotide are phosphate thioester bonds, or in other words, the CpG oligonucleotide has a phosphate thioester backbone. Exemplary CpG oligonucleotides (e.g., 5'-TGACTGTGAACGTTCGAGATGA-3') are disclosed in U.S. Patent Nos. 7,255,868, 7,479,285, 7,785,610, 8,003,115, 8,114,418, 8,222,398, 8,333,980, 8,597,665, 8,669,237, and 9,028,845, all of which are incorporated herein by reference in their entirety for all purposes.

[0165] Adjuvants included in or administered with immunogenic compositions or vaccines may comprise one or more adjuvants, which may be used in combination, and may include, but are not limited to, alum (aluminum salts), oil-in-water emulsions, CpG oligonucleotides, liposomes, and microparticles, such as poly(lactide-co-glycolic acid) microparticles (Shah et al., MethodsMol Biol [Molecular Biology Methods], 1494:1-14, 2017). In some embodiments, the immunogenic composition or vaccine further comprises an aluminum salt adjuvant containing adsorbed recombinant HSV antigen. In some embodiments, the aluminum salt adjuvant comprises one or more of the group consisting of amorphous hydroxyphosphate aluminum sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate. In some embodiments, the aluminum salt adjuvant comprises one or both of aluminum hydroxide and aluminum phosphate. In some embodiments, the aluminum salt adjuvant comprises aluminum hydroxide. In some embodiments, a unit dose of the immunogenic composition or vaccine comprises about 0.25 to about 0.50 mg Al. 3+ Or approximately 0.35 mg Al 3+In some embodiments, the immunogenic composition further comprises additional adjuvants. Other suitable adjuvants include, but are not limited to, water-encapsulated squalene emulsions (e.g., MF59 or AS03), TLR3 agonists (e.g., poly-IC or poly-ICLC), TLR4 agonists (e.g., bacterial lipopolysaccharide derivatives such as monophospholipid A (MPL) and / or saponins (e.g., Quil A or QS-21, such as those in AS01 or AS02), TLR5 agonists (bacterial flagellin), and TLR7, TLR8, and / or TLR9 agonists (imidazoquinoline derivatives, such as imiquimod and resimimod) (Coffman et al., Immunity [Immunology] 33:492-503, 2010). In some embodiments, additional adjuvants comprise MPL and alum (e.g., AS04). For veterinary use and for generating antibodies in nonhuman animals, the mitogenic component of Freund's adjuvant (full Freund's adjuvant and incomplete Freund's adjuvant) may be used.

[0166] In some embodiments, one or more adjuvants may comprise CpG oligonucleotides or alum, or both. In some embodiments, one or more adjuvants may comprise CpG oligonucleotides or oil-in-water adjuvants, or both. In some embodiments, the oil-in-water adjuvant is a squalene-in-water emulsion comprising squalene, α-tocopherol, and polysorbate 80 (i.e., CAS-1). In some embodiments, the oil-in-water adjuvant is a squalene-in-water emulsion comprising squalene, Span 85, and polysorbate 80.

[0167] In some embodiments, the immunogenic composition or vaccine comprises pharmaceutically acceptable excipients (including, for example, solvents, swelling agents, buffers, tension modifiers, and preservatives) (Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the immunogenic composition or vaccine may comprise excipients that function as one or more of solvents, swelling agents, buffers, and tension modifiers (e.g., sodium chloride in saline can be used as both an aqueous medium and a tension modifier).

[0168] In some embodiments, the immunogenic composition or vaccine comprises an aqueous medium as a solvent. Suitable media include, for example, sterile water, saline solution, phosphate-buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0169] Immunogenic compositions or vaccines may contain buffers. Buffers control pH to inhibit degradation of the active agent during processing, storage, and optionally reconstitution. Suitable buffers include, for example, salts comprising acetate, citrate, phosphate, or sulfate. Other suitable buffers include, for example, amino acids such as arginine, glycine, histidine, and lysine. Buffers may further contain hydrochloric acid or sodium hydroxide. In some embodiments, the buffer maintains the pH of the composition in the range of 6 to 9. In some embodiments, the pH is greater than (lower limit) 6, 7, or 8. In some embodiments, the pH is less than (upper limit) 9, 8, or 7. That is, the pH is in the range of about 6 to 9, where the lower limit is lower than the upper limit.

[0170] Immunogenic compositions or vaccines may contain tonic modulators. Suitable tonic modulators include, for example, dextran, glycerol, sodium chloride, glycerol, and mannitol.

[0171] Immunogenic compositions or vaccines may contain swelling agents. Swelling agents are particularly useful when pharmaceutical compositions are lyophilized prior to administration. In some embodiments, swelling agents are protective agents that help stabilize and prevent degradation of the active agent during freeze-drying or spray-drying and / or storage. Suitable swelling agents are sugars (monosaccharides, disaccharides, and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, and raffinose.

[0172] Immunogenic compositions or vaccines may contain preservatives. Suitable preservatives include, for example, antioxidants and antimicrobial agents. However, in preferred embodiments, the immunogenic compositions or vaccines are prepared under sterile conditions and packaged in single-use containers, thus eliminating the need for preservatives.

[0173] In some embodiments, the composition or vaccine may be provided as a sterile composition. The pharmaceutical composition typically contains an effective amount of the disclosed recombinant immunogen and can be prepared using conventional techniques. Typically, the amount of immunogen in each dose of the immunogenic composition is selected to induce an immune response without significant adverse side effects. In some embodiments, the composition may be provided in a unit dosage form for inducing an immune response in a subject. The unit dosage form contains a suitable single preselected dose for administration to a subject, or a suitable label or measurement multiple of two or more preselected unit doses, and / or a metering device for administering the unit dose or multiples thereof. In other embodiments, the composition further includes an adjuvant.

[0174] IV. Methods for inducing immune responses In some embodiments, this document provides a method for generating an immune response against an HSV protein in a subject, the method comprising administering to the subject an effective amount of a recombinant peptide, trimer, complex, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus, VLP, cell, immunogenic composition, or vaccine provided herein. In some embodiments, the method is used to generate an immune response in a subject against an HSV surface antigen (e.g., a protein containing gD or an antigenic fragment thereof, and optionally one or more sequences shown in SEQ ID NO: 1-28 or antigenic fragment thereof), wherein the surface antigen comprises a protein containing gD or an antigenic fragment thereof.

[0175] In some embodiments, this document provides a method for preventing or treating HSV (e.g., HSV-2 and / or HSV-1) infection, the method comprising immunizing a subject with an effective amount of a recombinant peptide, trimer, complex, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus, VLP, cell, immunogenic composition or vaccine provided herein.

[0176] In some embodiments, the recombinant peptides, trimers, complexes, immunogenic compositions, vaccines, protein nanoparticles, isolated nucleic acids, vectors, viruses, pseudoviruses, VLPs, or cells contained in, encoding, or expressing recombinant peptides selected from the group consisting of SEQ ID NO: 1-28 are described herein.

[0177] In some embodiments, a subject may be administered an effective amount of a combination of two or more recombinant peptides, trimers, complexes, protein nanoparticles, isolated nucleic acids, vectors, viruses, pseudoviruses, VLPs, or cells provided herein, or may be immunized with an effective amount of a combination of two or more recombinant peptides, trimers, complexes, protein nanoparticles, isolated nucleic acids, vectors, viruses, pseudoviruses, VLPs, or cells provided herein. In some embodiments, the recombinant peptides, trimers, complexes, immunogenic compositions, vaccines, protein nanoparticles, isolated nucleic acids, vectors, viruses, pseudoviruses, VLPs, or cells provided herein contain, encode, or express a recombinant peptide selected from the group consisting of SEQ ID NO: 1-28. In some embodiments, the subject is administered an effective amount of a recombinant polypeptide, trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus, VLP, or cell (containing, encoding, or expressing the recombinant polypeptide shown in SEQ ID NO: 1, SEQ ID NO: 4, and / or SEQ ID NO: 5), or the subject is immunized with an effective amount of the recombinant polypeptide, trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus, VLP, or cell.

[0178] The disclosed recombinant immunogen (e.g., a glycoprotein antigen containing recombinant HSV gD, such as gD-trimer, gD-gB(WT)-trimer, gD-gB(mutant)-trimer, gD(F)-gB(WT)-trimer, gD(F)-gB(mutant)-trimer, or the glycoprotein described herein), trimer, complex, immunogenic composition, vaccine, nucleic acid molecule (e.g., DNA molecule) or vector encoding a protopolymer of the disclosed recombinant HSV glycoprotein antigen, or protein nanoparticles or virus-like particles, virus, pseudovirus, or cells containing the disclosed recombinant polypeptide (e.g., antigen containing recombinant HSV gD) may be administered to a subject to induce an immune response against the corresponding HSV G antigen in the subject. In a specific instance, the subject is a human. The immune response may be a protective immune response, such as a response that inhibits subsequent corresponding HSV infection. The initiation of an immune response may also be used to treat or inhibit infections and diseases associated with the corresponding HSV.

[0179] Subjects who may be selected for prevention or treatment, for example, those who have HSV infection due to exposure to or potential exposure to HSV, or those at risk of HSV infection. Subjects may be monitored for HSV-related infection or symptoms, or both, after administration or immunization with the disclosed recombinant immunogen.

[0180] Typical subjects intended for prevention or treatment using the therapeutic agents and methods disclosed herein include humans, as well as non-human primates and other animals. To identify subjects for prevention or treatment according to the methods disclosed herein, recognized screening methods are employed to determine risk factors associated with the targeted or suspected disease or condition, or to determine the subject's existing disease or condition status. These screening methods include, for example, routine examinations to identify environmental, family, occupational, and other risk factors that may be associated with the targeted or suspected disease or condition, and diagnostic methods, such as various ELISA and other immunoassays, to detect and / or characterize HSV infection. These and other routine methods enable clinicians to select patients who require therapy using the methods and pharmaceutical compositions disclosed herein. Based on these methods and principles, the compositions may be administered according to the teachings herein or other routine methods, as a stand-alone prevention or treatment program, or as a follow-up, adjunctive, or coordinating treatment regimen to other therapies.

[0181] The recombinant immunogens or their trimers, complexes, immunogenic compositions, vaccines, protein nanoparticles, isolated nucleic acids, vectors, viruses, pseudoviruses, or cells disclosed herein may be used for prophylactic or therapeutic purposes. When provided prophylactically, the disclosed therapeutic agents are provided prior to the onset of any symptoms, such as before infection. Prophylactic administration of the disclosed therapeutic agents is intended to prevent or improve any subsequent infection. When provided therapeutically, the disclosed therapeutic agents are provided at or after the onset of symptoms of illness or infection, such as after the onset of HSV infection symptoms corresponding to HSV glycoprotein antigens, or after a diagnosis of HSV infection. Therefore, therapeutic agents may be provided before anticipated exposure to HSV, after exposure to or suspected exposure to the virus, or after actual onset of infection in order to reduce the anticipated severity, duration, or extent of infection and / or associated disease symptoms.

[0182] The recombinant immunogen and its immunogenic composition described herein are provided to subjects in an amount that effectively induces or enhances an immune response against a glycoprotein antigen in a subject (preferably human). The actual dose of the disclosed recombinant immunogen will vary depending on factors such as the subject's disease indication and specific condition (e.g., age, body size, fitness, symptom severity, susceptibility factors, etc.), the time and route of administration, any concurrent medications or treatments, and the specific pharmacology of the composition used to elicit the desired activity or biological response in the subject. Dosing regimens may be adjusted to provide optimal prophylactic or therapeutic responses.

[0183] In some embodiments, the recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus or cell disclosed herein may be administered or immunized via topical application, percutaneous, subcutaneous (e.g., subcutaneous injection), intradermal, oral, intranasal (e.g., intranasal spray), intratracheal, sublingual, buccal, rectal, vaginal, inhalation, intravenous (e.g., intravenous injection), intra-arterial, intramuscular (e.g., intramuscular injection), intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intra-articular, periarticular, local or epidermal application.

[0184] Immunogenic compositions or vaccines are administered as a single dose or in a series of doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses) at intervals of several weeks (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months). Immunogenic compositions or vaccines comprising one or more of the disclosed recombinant immunogens can be used in coordinated (or prime-boost) vaccination regimens or combination formulations. In some embodiments, novel combination immunogenic compositions and coordinated immunization regimens employ individual immunogens or formulations, each designed to elicit an antiviral immune response, such as an immune response against HSV glycoprotein antigens. Individual immunogenic compositions or vaccines that elicit an antiviral immune response may be combined in a multivalent immunogenic composition (administered to a subject in a single immunization step), or they may be administered alone (in a monovalent immunogenic composition or vaccine) in a coordinated (or prime-boost) immunization regimen. In some embodiments, an immunogenic composition or vaccine comprising one or more of the disclosed recombinant immunogens may be used for vaccine revaccination. In some embodiments, an immunogenic composition or vaccine comprising one or more of the disclosed recombinant immunogens may be used as a booster vaccination, wherein the prime vaccination was performed with an immunogen other than the recombinant immunogens provided herein.

[0185] Multiple booster immunizations can be performed, and each booster can use a different recombinant immunogen disclosed. In some instances, a booster can use the same immunogen as another booster or primary immunization. Primary and booster immunizations can be administered as a single dose or multiple doses (e.g., two, three, four, five, six, or more doses may be given to the subject over several days, weeks, or months). Multiple boosters can also be given, such as one to five (e.g., 1, 2, 3, 4, or 5 boosters) or more. Different doses can be used in a series of sequential immunizations. For example, a relatively large dose may be used in the primary immunization, followed by a relatively small booster.

[0186] In some embodiments, booster doses may be administered approximately two weeks, three to eight weeks, or four weeks after primary immunization, or approximately several months after primary immunization. In some embodiments, booster doses may be administered approximately 5 months, 6 months, 7 months, 8 months, 10 months, 12 months, 18 months, or 24 months after primary immunization, or at longer or shorter intervals after primary immunization. Additional periodic booster doses may also be used at appropriate times to enhance the subject's "immune memory." The appropriateness of the selected vaccination parameters (e.g., formulation, dosage, regimen, etc.) can be determined by collecting aliquots of serum from the subject during the immunization program and measuring antibody titers. Additionally, the subject's clinical condition can be monitored to achieve the desired effect, such as prevention of infection or improvement of disease status (e.g., reduction of viral load). If such monitoring indicates that vaccination has not been optimal, the subject may be boosted with an additional dose of the immunogenic composition, and vaccination parameters may be adjusted in a manner intended to enhance the immune response.

[0187] In some embodiments, a primary-boost approach may include a DNA-primary and protein-boost vaccination regimen for the subject. This approach may include two or more administrations of nucleic acid molecules or proteins.

[0188] For protein therapy agents, typically, each dose will contain 1-200 μg of protein.

[0189] The amount used in the immunogenic composition is selected based on the subject population (e.g., infants or elderly individuals, who may be over 60 years of age). The optimal amount of a particular composition can be determined through standard studies involving observation of antibody titers and other responses in subjects. It should be understood that a therapeutically effective amount of the disclosed recombinant immunogen (e.g., the disclosed recombinant HSV glycoprotein antigen, such as a trimer, protein, viral vector, or nucleic acid molecule in the immunogenic composition) may include an amount that does not effectively elicit an immune response by administration of a single dose but effectively elicits an immune response after administration of multiple doses (e.g., in a prime-boost regimen).

[0190] Following administration of the recombinant immunogen disclosed herein, the subject's immune system typically responds to the immunogenic composition by producing antibodies specific to the HSV glycoprotein peptides included in the immunogen. Such a response indicates that an immunogenic dose has been delivered to the subject.

[0191] In some embodiments, the subject's antibody response is determined in the context of evaluating an effective dose / immunization regimen. In most cases, it is sufficient to assess the antibody titer in the serum or plasma obtained from the subject. Decisions regarding whether to administer a booster vaccination and / or to change the amount of therapeutic agent administered to an individual can be based at least in part on antibody titer levels. Antibody titer levels can be based, for example, on an immunobinding assay, which measures the concentration of antibodies in serum that bind to antigens, including, for example, recombinant HSV glycoprotein antigens, such as gD-trimer, gD-gB(WT)-trimer, gD-gB(mutant)-trimer, gD(F)-gB(WT)-trimer, and gD(F)-gB(mutant)-trimer.

[0192] For the effectiveness of these methods, complete elimination, reduction, or prevention of HSV infection is not required. For example, compared to HSV infection in the absence of an immunogen, inducing an immune response against HSV with one or more of the disclosed recombinant immunogens can reduce or suppress HSV infection by a desired amount, such as at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable infected cells). In another instance, HSV replication can be reduced or suppressed by the disclosed methods. For the effectiveness of these methods, complete elimination of HSV replication is not required. For example, compared to HSV replication in the absence of an immune response, an immune response induced using one or more of the disclosed recombinant immunogens can reduce the corresponding HSV replication by a desired amount, such as at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable HSV replication).

[0193] In some embodiments, the disclosed recombinant immunogen is administered to the subject simultaneously with the adjuvant. In other embodiments, the disclosed recombinant immunogen is administered to the subject after the adjuvant and for a period sufficient to induce an immune response.

[0194] One method of administering nucleic acids is direct immunization with plasmid DNA (e.g., mammalian expression plasmids). Immunization via nucleic acid constructs is well known in the art and has been taught, for example, in U.S. Patent No. 5,643,578 (describing a method of immunizing vertebrates by introducing DNA encoding a desired antigen to elicit a cell-mediated or humoral response) and U.S. Patent Nos. 5,593,972 and 5,817,637 (describing the operative linking of a nucleic acid sequence encoding an antigen to a regulatory sequence capable of expression). U.S. Patent No. 5,880,103 describes several methods for delivering nucleic acids encoding immunogenic peptides or other antigens to an organism. These methods include liposome delivery of nucleic acids (or the synthetic peptide itself) and immunostimulatory constructs or ISCMS™ (which spontaneously form negatively charged cage-like structures of 30–40 nm in size when cholesterol and Quil A™ (saponins) are mixed). Using ISCOMS™ as an antigen delivery medium, protective immunity has been generated in various experimental infection models, including toxoplasmosis and EBV-induced tumors (Mowat and Donachie). Immunol. Today [Immunology Today] 12:383, 1991). It was found that antigen doses as low as 1 μg encapsulated in ISCOMS™ could induce class I-mediated CTL responses (Takahashi et al., Nature [Nature] 344:873, 1990.

[0195] In some embodiments, the plasmid DNA vaccine is used to express the disclosed recombinant immunogen in a subject. For example, a nucleic acid molecule encoding the disclosed recombinant immunogen may be administered to a subject to induce an immune response against an HSV glycoprotein antigen. In some embodiments, the nucleic acid molecule may be contained on a plasmid vector (e.g., the pVRC8400 vector) for DNA immunization (described in Barouch et al., ...). J. Virol [Journal of Virology] , (As cited in 79, 8828-8834, 2005, which is incorporated herein by reference).

[0196] In another method of immunization using nucleic acids, the disclosed glycoprotein antigen (e.g., trimer, protein) containing recombinant HSV gD can be expressed by an attenuated viral host or vector or bacterial vector. Recombinant vaccinia virus, adeno-associated virus (AAV), herpesvirus, retrovirus, cytomegalovirus, or other viral vectors can be used to express peptides or proteins, thereby evoking a CTL response. For example, vaccinia vectors and methods for use in immunization protocols are described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette-Guérin) provides another vector for expressing peptides (see Stover, Nature[Nature] 351:456-460, 1991.

[0197] In one embodiment, a nucleic acid encoding the disclosed recombinant HSV glycoprotein antigen is introduced directly into cells. For example, the nucleic acid can be loaded onto gold microspheres using standard methods and introduced into the skin using a device such as Bio-Rad's HELIOS™ gene gun. The nucleic acid can be a "naked" nucleic acid, consisting of plasmids controlled by a strong promoter. Typically, the DNA is injected into muscle, but it can also be injected directly into other sites. Injection doses are typically from about 0.5 μg / kg to about 50 mg / kg, and typically from about 0.005 mg / kg to about 5 mg / kg (see, for example, U.S. Patent No. 5,589,466).

[0198] For example, nucleic acids can be loaded onto gold microspheres using standard methods and introduced into the skin using devices such as Bio-Rad Laboratories' HELIOS™ gene gun. The nucleic acids can be "naked" nucleic acids, consisting of plasmids controlled by strong promoters. Typically, the DNA is injected into muscle, but it can also be injected directly into other sites. Injection doses are typically from about 0.5 μg / kg to about 50 mg / kg, and typically from about 0.005 mg / kg to about 5 mg / kg (see, for example, U.S. Patent No. 5,589,466).

[0199] In another embodiment, an mRNA-based immunization protocol can be used to directly deliver nucleic acids encoding the disclosed recombinant HSV glycoprotein antigen into cells. In some embodiments, mRNA-based nucleic acid-based vaccines can provide an effective alternative to the aforementioned methods. mRNA vaccines avoid the safety issues associated with DNA integration into the host genome and can be translated directly in the cytoplasm of host cells. Furthermore, cell-free in vitro RNA synthesis is simple and avoids the complexities associated with viral vector manufacturing. Two exemplary forms of RNA-based vaccination that can be used to deliver nucleic acids encoding the disclosed antigen containing recombinant HSV gD include conventional non-amplifying mRNA immunization (see, e.g., Petsch et al., “Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection,” Nature biotechnology, 30(12):1210-6, 2012) and self-amplifying mRNA immunization (see, e.g., Geall et al., “Nonviral delivery of self-amplifying RNA vaccines,” PNAS, 109(36): 14604-14609, 2012; Magini et al., “Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic)). Viral Challenge [Self-amplifying mRNA vaccines expressing multiple conserved influenza antigens provide protection against homologous and heterologous viral challenges], PLoS One, 11(8):e0161193, 2016; and Brito et al., "Self-amplifying mRNA vaccines," Adv Genet. 89:179-233, 2015).

[0200] In some embodiments, administration of a prophylactic or therapeutically effective amount of one or more of the disclosed recombinant immunogens to a subject induces a neutralizing immune response in the subject. To assess neutralizing activity, serum may be collected from the subject at appropriate time points after immunization, frozen, and stored for neutralization testing. Methods for determining neutralizing activity are known to those skilled in the art and further described herein, and include, but are not limited to, plaque reduction neutralization (PRNT) assays, micro-neutralization assays, flow cytometry-based assays, and single-cycle infection assays. In some embodiments, a panel of HSV pseudoviruses may be used to determine serum neutralizing activity.

[0201] In some embodiments, administration of a prophylactic or therapeutically effective amount of one or more of the disclosed recombinant immunogens to a subject induces a neutralizing immune response in the subject. To assess neutralizing activity, serum may be collected from the subject at appropriate time points after immunization, frozen, and stored for neutralization testing. Methods for determining neutralizing activity are known to those skilled in the art and further described herein, and include, but are not limited to, plaque reduction neutralization (PRNT) assays, micro-neutralization assays, flow cytometry-based assays, and single-cycle infection assays. In some embodiments, a panel of HSV pseudoviruses may be used to determine serum neutralizing activity.

[0202] In some embodiments, a neutralizing immune response induced by the recombinant immunogen disclosed herein generates neutralizing antibodies against HSV. In some embodiments, the neutralizing antibodies described herein bind to a cellular receptor or co-receptor of HSV or a component thereof. In some embodiments, the viral receptor or co-receptor is an HSV receptor or co-receptor, preferably a human HSV receptor, such as an HSV receptor or co-receptor. In some embodiments, the neutralizing antibodies described herein regulate, reduce, antagonize, alleviate, block, inhibit, eliminate, and / or interfere with at least one HSV activity or binding, or HSV receptor activity or binding, such as HSV release, HSV receptor signaling, membrane HSV cleavage, HSV activity, HSV production, and / or synthesis, in vitro, in situ, and / or in vivo. In some embodiments, the recombinant immunogen disclosed herein induces neutralizing antibodies against HSV that regulate, reduce, antagonize, alleviate, block, inhibit, eliminate, and / or interfere with the binding of HSV to an HSV receptor or co-receptor (e.g., a functional cellular receptor, such as herpesvirus invasion mediator (HVEM) (a member of the TNF receptor family) or connexin-1 (a member of the immunoglobulin superfamily)).

[0203] V. Detection and Diagnostic Methods Methods for detecting HSV antibodies in a subject using the recombinant immunogen or its trimer or complex disclosed herein are also provided. In some embodiments, the HSV comprises HSV-2 and / or HSV-1. In some embodiments, HSV-2 is mammalian HSV-2, such as human HSV-2. In some embodiments, HSV-1 is mammalian HSV-1, such as human HSV-1. In some embodiments, the antibody comprises a polyclonal antibody and / or a monoclonal antibody. In some embodiments, the antibody is a neutralizing antibody.

[0204] In some embodiments, the subject is an individual to be tested for the presence of HSV antibodies. The subject may or may not have an HSV infection. The subject may be suspected of having an HSV infection or suspected of having been exposed to HSV, or may not be suspected. In some embodiments, the subject may be a mammal, such as a human.

[0205] In some embodiments, the method may include detecting HSV antibodies from a subject sample. In some embodiments, the sample may be a bodily secretion sample (e.g., a liquid), such as blood, serum, plasma, whole blood, saliva, blister fluid, or breast milk; or a sample from a subject's tissue or organ. The sample may be refrigerated or frozen prior to the assay.

[0206] In some embodiments, the method includes: (1) contacting a sample with an HSV viral surface antigen linked to a protein trimer tag provided herein via in-frame fusion; and (2) detecting the binding of an antibody to the HSV viral antigen.

[0207] Any entity that contains, encodes, or expresses HSV viral surface antigen linked to a protein trimer tag via in-frame fusion (e.g., the recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus, or cell of the present invention) can be used to perform the above detection method, as long as it can bind to an antibody against HSV.

[0208] As used herein, the term contact refers to introducing a sample with HSV surface antigen linked to a protein trimer tag via in-frame fusion (e.g., by combining or mixing them), such that the HSV surface antigen linked to the protein trimer tag via in-frame fusion can physically contact antibodies in the sample (if present). When antibodies against HSV are present in the sample, an antibody / antigen complex is then formed, which can be detected. The binding of antibodies in the sample to the HSV surface antigen is accomplished under conditions suitable for complex formation. Such conditions (e.g., appropriate concentration, buffer, temperature, reaction time) and methods for optimizing such conditions are known to those skilled in the art.

[0209] Binding can be measured using a variety of standard methods in the art, including but not limited to agglutination assays, precipitation assays, enzyme immunoassays (e.g., ELISA), immunoprecipitation assays, immunoblotting assays, and other immunoassays, such as those described in, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual* (Cold Spring Harbor Labs Press, 1989) and Harlow et al., *Antibodies, a Laboratory Manual* (Cold Spring Harbor Labs Press, 1988), both of which are incorporated herein by reference in their entirety. These references also provide examples of complex formation conditions. Binding can also be measured using competitive binding assays, such as competitive enzyme-linked immunosorbent assays or competitive ligand-receptor binding assays.

[0210] In some embodiments, the HSV surface antigen may comprise a viral antigen containing gD. In some embodiments, the antibody may comprise an antibody against HSV gD (e.g., HSV-2 gD and / or HSV-1 gD).

[0211] In some embodiments, HSV viral surface antigen or recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticles, isolated nucleic acid, vector, virus, pseudovirus or cell may be labeled with a detection reagent.

[0212] Examples of detection reagents include, but are not limited to, radiolabeled, fluorescent, chemiluminescent, chromophore, enzyme, phosphorescent, and electronically labeled substances; metal sol labels, colored beads, physical labels, magnetic agents, or ligands, such as colloidal gold, fluorescein, radioisotopes, phosphatases (e.g., alkaline phosphatase), biotin, avidin, peroxidases (e.g., horseradish peroxidase), β-galactosidase, and biotin-related or avidin-related compounds (e.g., streptavidin or ImmunoPure7 NeutrAvidin). In some embodiments, the detection agent is an enzyme, such as β-D-galactosidase, glucose oxidase, horseradish peroxidase, alkaline phosphatase, β-lactamase, glucose-6-phosphate dehydrogenase, urease, uricase, superoxide dismutase, luciferase, pyruvate kinase, lactate dehydrogenase, galactose oxidase, acetylcholinesterase, enterokinase, tyrosinase, and xanthine oxidase; or a detection particle, such as enzymatic particles (e.g., nanoparticles), polystyrene particles (e.g., microspheres), latex particles, gold-containing particles (e.g., gold nanoparticles), colloidal gold particles, metal particles (e.g., iron oxide nanoparticles), magnetic particles, fluorescently detectable particles, or semiconductor particles (e.g., nanocrystals).

[0213] In some embodiments, HSV viral surface antigen (or recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus or cell) can bind to a colloidal gold-labeled antibody that recognizes a trimerized domain (e.g., the C-terminal portion of collagen) for colloidal gold labeling.

[0214] In some embodiments, HSV viral surface antigen or recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticles, isolated nucleic acid, vector, virus, pseudovirus or cell can be immobilized on, for example, a substrate or solid surface.

[0215] Suitable materials for substrates or solid surfaces include, but are not limited to, plastics, glass, gels, celluloid, fabrics, paper, and particulate materials. Examples of materials for substrates or solid surfaces include, but are not limited to, latex, polystyrene, nylon, nitrocellulose, agarose, cotton, PVDF (polyvinylidene fluoride), and magnetic resins. Suitable shapes for substrate or solid surface materials include, but are not limited to, pores (e.g., microtiter plate wells), microtiter plates, test strips, strips, beads, lateral chromatography devices, membranes, filters, tubes, petri dishes, celluloid-type matrices, magnetic particles, and other particulates. Exemplary substrates or solid surfaces include, for example, test strips (e.g., lateral chromatography devices), ELISA plates, test strips, immunospot strips, radioimmunoassay plates, agarose beads, plastic beads, latex beads, cotton thread, plastic chips, immunoblotting membranes, immunoblotting paper, and flow-through membranes. For examples of substrate or solid surface materials, see, for instance, Kemeny, DM (1991) A Practical Guide to ELISA, Pergamon Press, Elmsford, NY, pp. 33-44, and Price, C. and Newman, D., eds. Principles and Practice of Immunoassay, 2nd ed. (1997) Stockton Press, NY, both of which are incorporated herein by reference in their entirety.

[0216] In some embodiments, binding can be measured using a secondary antibody that specifically binds to an antibody against HSV. In some embodiments, the secondary antibody may be an antibody from a species different from the species from which the anti-HSV antibody to be tested is derived. In some embodiments, when the antibody against HSV to be tested is a human antibody, the secondary antibody may be an anti-IgG antibody, for example, an anti-human IgG antibody from a non-human mammal (e.g., an anti-human IgG antibody from a mouse, rabbit, goat, sheep, pig, dog, cat, etc.). In some embodiments, the method includes contacting the sample to be tested with an HSV viral surface antigen (or a recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus, or cell) and a secondary antibody, and detecting the complex of the HSV viral surface antigen, the antibody against HSV, and the secondary antibody. Detection of the complex of the HSV viral surface antigen, the antibody against HSV, and the secondary antibody indicates the presence of an antibody against HSV.

[0217] In some embodiments, any one or both of the HSV surface antigen (or recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus or cell) and secondary antibody may be labeled with a detection reagent that can be used to detect the complex of HSV surface antigen, antibody against HSV and secondary antibody.

[0218] In some embodiments, any one or both of the HSV viral surface antigen (or recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus or cell) and secondary antibody may be immobilized on, for example, a substrate or solid surface.

[0219] In some embodiments, the HSV surface antigen (or recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus or cell) is labeled with a detection reagent (e.g., colloidal gold), and a secondary antibody is immobilized to capture the complex of the HSV surface antigen and the antibody to be tested.

[0220] In some embodiments, the secondary antibody is labeled with a detection reagent (e.g., colloidal gold), and the HSV viral surface antigen (or recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus, or cell) is immobilized to capture the complex of the antibody and secondary antibody to be tested.

[0221] In some embodiments, the combination can be measured using lateral chromatography. Lateral chromatography immunoassays are widely used in many different areas of analytical chemistry and medicine, such as for clinical diagnostics to determine the presence of a target analyte in a sample (e.g., body fluids). Previous work on lateral chromatography immunoassays is illustrated by the following U.S. patents and patent applications: U.S. Patent Nos. 5,602,040, 5,622,871, 5,656,503, 6,187,598, 6,228,660, 6,818,455, 2001 / 0008774, 2005 / 0244986, U.S. Patent Nos. 6,352,862, 2003 / 0207465, 2003 / Patent Nos. 0143755, 2003 / 0219908, US Patent Nos. 5,714,389, 5,989,921, 6,485,982, Serial No. 11 / 035,047, US Patent Nos. 5,656,448, 5,559,041, 5,252,496, 5,728,587, 6,027,943, 6,506,612, 6,541,277, 6,737,277 B1, 5,073,484, 5,654,162, 6,020,147, 4,956,302, 5,120,643, 6,534,320, 4,942,522, 4,703,017, 4,743,560, 5,591,645 and RE 38,430 E.

[0222] Lateral chromatography can detect functional properties of analytes, such as interaction-blocking characteristics. In some embodiments, the analyte is an antibody, such as a neutralizing (or blocking) antibody, for example, an antibody that interrupts the interaction of two or more molecular components in the host (e.g., viral proteins and cell surface proteins). In some embodiments, the antibody is an anti-HSV (e.g., HSV-2 and / or HSV-1) antibody. In some embodiments, the antibody is an anti-gB and / or anti-gD antibody, wherein the gD and / or gB are derived from HSV (e.g., HSV-2 and / or HSV-1).

[0223] Lateral chromatography apparatus may be a chromatography strip comprising one or more test zones and optionally one or more control zones. In some embodiments, the chromatography strip is a membrane. In some embodiments, the chromatography strip is a porous membrane. The pore size of the chromatography strip can vary considerably. In some embodiments, the chromatography strip contains pores of about 1 μm to about 20 μm (e.g., any one of about 1 μm to about 10 μm, about 5 μm to about 15 μm, or about 10 μm to about 20 μm). In some embodiments, the chromatography strip contains an absorbent material. In some embodiments, the chromatography strip contains a non-absorbent material. In some embodiments, the chromatography strip contains a material selected from the group consisting of: cellulose, cellulose blends, nitrocellulose, cellulose esters, mixed nitrocellulose esters, polyesters, acrylonitrile copolymers, rayon, glass fibers, polyethylene terephthalate fibers, polypropylene, and combinations thereof. In some embodiments, the membrane is a nitrocellulose membrane.

[0224] In some embodiments, the chromatography strip or a portion thereof is treated with a blocking agent, for example, to increase the specificity of any binding interactions. In some embodiments, the blocking agent comprises casein, bovine serum albumin (BSA), methylated BSA, whole animal serum, skim milk powder, or combinations thereof. When the chromatography strip is blocked, the charge of the chromatography strip (e.g., nitrocellulose) is neutralized, and therefore no other protein or component thereof can bind to the blocked chromatography strip. Additionally, the chromatographic structure of the chromatography strip is altered, and the flow can be more like a sliding or slip flow than the flow of conventional chromatography.

[0225] Certain components of the test strips described herein contain detection reagents to facilitate (qualitative and / or quantitative) identification of said components at certain regions of the test strip (e.g., test area, control area). In some embodiments, molecular components of a molecular binding system are labeled with detection reagents. In some embodiments, other components, such as antibodies, antigens, ligands, or receptors in the sample binding region, are labeled with detection reagents. In some embodiments, two or more components of the test strip are labeled with detection reagents, each component being labeled with a unique detection reagent that can be distinguished from other detection reagents on the test strip (e.g., based on color).

[0226] In some embodiments, the detection reagent comprises an enzyme. In some embodiments, the detection reagent comprises a polymerase containing multiple enzymes. In some embodiments, the enzyme is selected from the group consisting of: β-D-galactosidase, glucose oxidase, horseradish peroxidase, alkaline phosphatase, β-lactamase, glucose-6-phosphate dehydrogenase, urease, uricase, superoxide dismutase, luciferase, pyruvate kinase, lactate dehydrogenase, galactose oxidase, acetylcholinesterase, enterokinase, tyrosinase, and xanthine oxidase.

[0227] In some embodiments, the detection reagent comprises detection particles. In some embodiments, the detection particles comprise enzymatic particles (e.g., nanoparticles), polystyrene particles (e.g., microspheres), latex particles, gold-containing particles (e.g., gold nanoparticles), colloidal gold particles, metal particles (e.g., iron oxide nanoparticles), magnetic particles, fluorescently detectable particles, or semiconductor particles (e.g., nanocrystals).

[0228] In some embodiments, the test strip further includes an absorption zone. Typically, the absorption zone is configured to remove excess liquid from the chromatography strip, for example, in a reversible or irreversible manner. In some embodiments, the absorption zone is configured as a reversible desiccant (allowing liquid to flow back from the absorption zone). In some embodiments, the absorption zone is configured as an irreversible desiccant. In some embodiments, the absorption zone includes a absorbent pad. In some embodiments, the absorbent pad includes an absorbent material. In some embodiments, the absorbent pad includes filter paper, a glass fiber filter, etc.

[0229] In some embodiments, the absorption region is located downstream of the chromatography strip. In some embodiments, the absorption region and the chromatography strip are connected by a capillary tube.

[0230] In some embodiments, the test strip further includes a sample loading region comprising a sample pad. In some embodiments, the sample pad is in capillary communication with one or more downstream components of the test strip.

[0231] In some embodiments, the sample loading area is configured to receive samples.

[0232] In some embodiments, one of the following—HSV viral surface antigen (or recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticles, isolated nucleic acid, vector, virus, pseudovirus, VLP, or cell) and secondary antibody—is immobilized on the capture zone, and the other is labeled with an detection reagent (e.g., colloidal gold) and contained in the sampling pad. A subject's sample loaded into the sample loading zone may flow through the device simultaneously or sequentially with a reagent (which may specifically bind to the antibody to be tested and is pre-prepared in the sample loading zone) to the absorption zone. Color development in the capture zone indicates the presence of the antibody to be tested.

[0233] In some embodiments, the lateral chromatography apparatus further includes a control region on which a first control binding pair (e.g., antigen, antibody, ligand, or receptor) is immobilized, and a sample loading region further includes a second control binding pair (e.g., antigen, antibody, ligand, or receptor) that can specifically bind to the first control binding pair and is labeled with a detection reagent (e.g., colloidal gold). In some embodiments, the first and second control binding pairs are respectively an antigen and an antibody (e.g., IgG from a non-human species and an antibody against said IgG). In some embodiments, IgG from a non-human species is labeled with a detection reagent (e.g., colloidal gold) and included in the sample loading region, and an antibody against said IgG is immobilized on the control region. In some embodiments, IgG from a non-human species is labeled with a detection reagent (e.g., colloidal gold) and included in the sample loading region, and an antibody against said IgG is immobilized on the control region. In some embodiments, IgG from a non-human species is immobilized on the control region, and an antibody against said IgG is labeled with a detection reagent (e.g., colloidal gold) and included in the sample loading region. In some embodiments, the IgG from a non-human species is IgG from a non-human mammal (e.g., mouse, rabbit, goat, sheep, pig, dog, cat, etc.). In some embodiments, the antibody against said IgG is an antibody against another non-human mammal (e.g., mouse, rabbit, goat, sheep, pig, dog, cat, etc.).

[0234] In some embodiments, binding can be measured using an HSV receptor that specifically binds to the HSV surface antigen (e.g., HSV gD). In some embodiments, the method includes contacting the sample to be tested with the HSV surface antigen (or recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus, VLP, or cell) and the HSV receptor, and detecting the HSV surface antigen and HSV receptor complex. Neutralizing antibodies can competitively bind to the HSV surface antigen against the HSV receptor, thereby allowing the detection of neutralizing antibodies, with the resulting reading reflecting the inhibition of HSV surface antigen binding to the soluble HSV receptor. Therefore, if the binding complex of the HSV receptor and HSV surface antigen is detected, the neutralizing antibody to be tested is not present in the sample. If the binding complex of the HSV receptor and HSV surface antigen is not detected, the neutralizing antibody to be tested is present in the sample.

[0235] In some embodiments, the HSV receptor may be a soluble HSV receptor. In some embodiments, the HSV receptor may be a herpesvirus invasion mediator (HVEM) or connexin-1, or a fragment or variant thereof, or a fusion containing thereof, provided that it can specifically bind to an HSV surface antigen (e.g., HSV gD). In some embodiments, the HSV receptor is a mammalian HSV receptor, such as a human HSV receptor, such as human HVEM or human connexin-1. In some embodiments, the HSV receptor may be a soluble portion (e.g., an extracellular domain) of an HSV receptor that specifically binds to an HSV surface antigen (e.g., HSV gD). In some embodiments, the HSV receptor may be a soluble portion (e.g., an extracellular domain) of HVEM or connexin-1, or a fragment or variant thereof, or a fusion containing thereof. In some embodiments, the HSV receptor may be fused with another polypeptide (e.g., an Fc domain (e.g., the Fc domain of IgG (e.g., human IgG)) or a trimerization domain (e.g., as described herein)). In some embodiments, the HSV receptor may be a fusion of the HVEM extracellular domain or the connexin-1 extracellular domain and the Fc domain (e.g., the Fc domain of IgG (e.g., human IgG)), wherein the HVEM extracellular domain or the connexin-1 extracellular domain may be fused to the Fc (e.g., HVEM-Fc or connexin-1-Fc) at its N-terminus or C-terminus. In some embodiments, the HSV receptor may be a fusion of the HVEM extracellular domain or the connexin-1 extracellular domain and a trimerizing tag (e.g., the C-terminal portion of collagen), wherein the HVEM extracellular domain or the connexin-1 extracellular domain may be fused to the trimerizing tag (e.g., HVEM-trimer or connexin-1-trimer) at its N-terminus or C-terminus. In some embodiments, the HVEM-Fc has the sequence shown in SEQ ID NO: 30. In some embodiments, the connexin-1-Fc has the sequence shown in SEQ ID NO: 29.

[0236] In some embodiments, any one or both of the HSV surface antigen (or recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus, VLP or cell) and HSV receptor can be labeled with a detection agent that can be used to detect the complex of HSV surface antigen and HSV receptor.

[0237] In some embodiments, any one or both of the HSV viral surface antigen (or recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus, VLP or cell) and HSV receptor can be immobilized on, for example, a substrate or solid surface.

[0238] In some embodiments, HSV viral surface antigen (or recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus, VLP or cell) is labeled with a detection agent (e.g., colloidal gold), and HSV receptors are immobilized to capture HSV viral surface antigen.

[0239] In some embodiments, the HSV receptor is labeled with a detection agent (e.g., colloidal gold), and the HSV surface antigen (or recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus, VLP, or cell) is immobilized to capture the antibody or HSV receptor to be tested.

[0240] In some embodiments, binding to HSV receptors can be measured using a lateral chromatography apparatus (e.g., the strip described herein).

[0241] In some embodiments, one of the following—the HSV surface antigen (or recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticles, isolated nucleic acid, vector, virus, pseudovirus, VLP, or cell) and the HSV receptor—is immobilized on the capture region, and the other is labeled with an detection reagent (e.g., colloidal gold) and prepared in the sample loading region. A subject's sample loaded into the sample loading region can flow through the device simultaneously or sequentially with the reagent pre-prepared in the sample loading region to the absorption region. No color development or only a faint color development in the capture region indicates the presence of the antibody to be tested.

[0242] In some embodiments, a lateral chromatography apparatus for detecting binding involving HSV receptors may further include a control region on which a first control binding pair (e.g., antigen, antibody, ligand, or receptor) is immobilized, and a sample loading region further includes a second control binding pair (e.g., antigen, antibody, ligand, or receptor) that can specifically bind to the first control binding pair and is labeled with a detection reagent (e.g., colloidal gold). In some embodiments, the first and second control binding pairs are respectively an antigen and an antibody (e.g., IgG from a non-human species and an antibody against said IgG). In some embodiments, IgG from a non-human species is labeled with a detection reagent (e.g., colloidal gold) and prepared in the sample loading region, and an antibody against said IgG is immobilized in the control region. In some embodiments, IgG from a non-human species is labeled with a detection reagent (e.g., colloidal gold) and prepared in the sample loading region, and an antibody against said IgG is immobilized in the control region. In some embodiments, IgG from a non-human species is immobilized in the control region, and an antibody against said IgG is immobilized in the control region. In some embodiments, IgG from a non-human species is labeled with a detection reagent (e.g., colloidal gold) and prepared in the sample loading region, and an antibody against the IgG is labeled with a detection reagent (e.g., colloidal gold) and prepared in the sample loading region. In some embodiments, the IgG from the non-human species is IgG from a non-human mammal (e.g., mouse, rabbit, goat, sheep, pig, dog, cat, etc.). In some embodiments, the antibody against the IgG is an antibody from another non-human mammal (e.g., mouse, rabbit, goat, sheep, pig, dog, cat, etc.).

[0243] VI. Products or reagent kits Articles or kits containing the recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus, VLP, or cell disclosed herein are also provided. Articles may include containers and labels or instructions for use on or associated with such containers. Suitable containers include, for example, bottles, vials, syringes, test tubes, IV solution bags, etc. Containers may be formed from a variety of materials, such as glass or plastic. In some embodiments, the container has a sterile inlet. Exemplary containers include intravenous solution bags, vials, including those with stoppers that can be punctured by an injection needle. Articles or kits may further include instructions for use indicating that the composition can be used to treat a specific condition, such as the condition described herein (e.g., HSV infection). Alternatively or additionally, articles or kits may further include another or the same container containing a pharmaceutically acceptable buffer. It may further include other materials, such as other buffers, diluents, filters, needles, and / or syringes.

[0244] Labels or package inserts may indicate that the composition is intended for the treatment of an individual's HSV infection. Labels or package inserts on or associated with the container may indicate instructions for reconstitution and / or use of the formulation. Labels or package inserts may further indicate that the formulation may be used or is intended for use subcutaneously, intravenously, or by other routes of administration to treat or prevent an individual's HSV infection.

[0245] In some embodiments, the container contains a composition that, alone or in combination with another composition, is effective in treating, preventing, and / or diagnosing a condition. The article or kit may include (a) a first container containing a composition (i.e., a first drug) comprising the recombinant immunogen or its trimer, complex, immunogenic composition, vaccine, protein nanoparticle, isolated nucleic acid, vector, virus, pseudovirus, VLP, or cell disclosed herein; and (b) a second container containing a composition (i.e., a second drug) comprising additional ingredients, such as adjuvants (e.g., CpG, alum, and / or CAS-1) or additional therapeutic agents, and the article or kit further includes instructions on a label or package insert to guide treatment of a subject with an effective amount of the second drug. CAS-1 comprises α-tocopherol, squalene, and polysorbate 80 in an oil-in-water emulsion.

[0246] In some embodiments, the container contains a composition that is effective in treating, preventing, and / or diagnosing a condition. The composition comprises an HSV antigen fusion protein disclosed herein (e.g., in Part III) and one or more adjuvants.

[0247] the term Unless otherwise defined, all technical terms, symbols, and other technical and scientific vocabulary or terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some instances, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and such definitions herein should not necessarily be construed as indicating a significant difference from the meanings commonly understood in the art.

[0248] The terms “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Peptides (including the provided receptors and other peptides, such as linkers or peptides) can include amino acid residues, including native and / or non-native amino acid residues. These terms also include post-expression modifications of peptides, such as glycosylation, sialylation, acetylation, and phosphorylation. In some respects, peptides may contain modifications relative to the native or natural sequence, as long as the protein maintains the desired activity. These modifications may be intentional (e.g., through site-directed mutagenesis) or accidental (e.g., through mutations in the host producing the protein or errors due to PCR amplification).

[0249] The term "intra-frame fusion" generally refers to the joining of two or more ORFs in a manner that preserves the correct reading frame of the original open reading frame (ORF) to form a continuous, longer ORF. This means the expression of the fused nucleotide sequence, resulting in a single polypeptide chain without any transposition.

[0250] The term "furin protease site," also known as a furin cleavage site or furin cleavage sequence, refers to the amino acid sequence in a polypeptide or protein that serves as a recognition sequence for enzymatic protease cleavage by furin or furin-like proteases. Typically, furin cleavage sites have a common sequence Arg-XX-Arg, where X is any amino acid. The cleavage site is located after the carboxyl-terminal arginine (Arg) residue in the sequence.

[0251] The term "variant" or "mutant," also known as a functional variant, and in the context of proteins or peptides, refers to a peptide having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a parent peptide and having the same or substantially the same function as the parent peptide. A functional variant of the parent peptide can also refer to a peptide that has the addition, deletion, and / or substitution of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) compared to the parent peptide and has the same or substantially the same function as the parent peptide. In some embodiments, variants or mutants of HSV viral antigens (e.g., gD, gB, or combinations thereof) contain at least one epitope of the HSV viral antigen, such as at least one neutralizing epitope.

[0252] The term "C-propeptide of collagen" refers to the C-terminal globular and non-triple-helix domain of collagen, which is capable of self-assembling into trimers. Unlike the triple-helix region of collagen, C-propeptide does not contain any glycine repeat sequences and is typically removed from the procollagen precursor via proteolytic hydrolysis during procollagen secretion prior to collagen fibrillation.

[0253] The term "glycine repeat sequence" refers to the central linear triple helix forming region of collagen, which contains hundreds of (Gly-XY)n repeat sequences in its amino acid sequence. These repeat sequences are also rich in proline at the X and / or Y positions. After removing the N- and C-propeptides, the collagen triple helix containing the glycine repeat sequences can assemble into higher-order insoluble collagen fibrils, which constitute the major components of the cellular matrix.

[0254] As used herein, “subject” is a mammal, such as a human or other animal, and typically a human. In some embodiments, a subject (e.g., a patient) who is administered one or more agents, cells, cell populations, or compositions is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject can be male or female and can be of any suitable age, including infants, adolescents, teenagers, adults, older adults (e.g., over 60 years of age), and elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[0255] As used herein, “treatment” (and its grammatical variations, such as “treat” or “treating”) means the complete or partial improvement or reduction of a disease or symptom or disorder, or its associated symptoms, adverse reactions, or outcomes or phenotypes. The desired effects of treatment include, but are not limited to, prevention of the onset or recurrence of disease, relief of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, slowing of disease progression, improvement or reduction of disease state, and relief or improvement of prognosis. These terms do not imply a complete cure of disease or the complete elimination of any symptoms or one or more effects on all symptoms or outcomes.

[0256] As used herein, “delaying disease development” means postponing, hindering, slowing, stabilizing, inhibiting, and / or delaying the development of a disease (e.g., cancer). Such delay can vary in length, depending on the history of the disease and / or the individual being treated. In some embodiments, sufficient or significant delay can actually cover prevention because the individual has not developed the disease. For example, it may delay the development of advanced cancer (e.g., metastasis).

[0257] As used herein, “prevention” includes providing prevention of the onset or recurrence of the disease in subjects who may be susceptible to the disease but have not yet been diagnosed with it. In some embodiments, the cells and compositions provided are used to delay the development of the disease or slow its progression.

[0258] As used herein, "inhibition" function or activity refers to a reduction in function or activity compared to other identical conditions other than the target condition or parameter, or alternatively, compared to another condition. For example, cells that inhibit tumor growth reduce the tumor growth rate compared to the tumor growth rate in the absence of cells.

[0259] In the context of administration, the “effective amount” of a pharmaceutical agent (e.g., a pharmaceutical formulation, cell, or composition) refers to the amount that effectively achieves the desired outcome (e.g., a therapeutic or preventative outcome) at the necessary dose / amount and for the necessary duration.

[0260] A “therapeutic effective amount” of a pharmaceutical preparation (e.g., a drug formulation or cells) refers to the amount that effectively achieves the desired therapeutic outcome (e.g., for treating a disease, condition, or disorder) and / or the pharmacokinetic or pharmacodynamic effect of treatment at the necessary dose and for the necessary duration. Therapeutic effective amounts can vary depending on factors such as the subject’s disease state, age, sex, weight, and the cell population administered. In some embodiments, the provided method involves administering cells and / or the composition at an effective amount (e.g., a therapeutically effective amount).

[0261] "Prophylactic effective dose" refers to the amount that effectively achieves the desired preventive outcome at the necessary dose and for the required time period. Typically, but not necessarily, because the preventive dose is administered to the subject before or early in the course of the disease, this preventive effective dose will be less than the therapeutic effective dose. In the context of a low tumor burden, in some respects, the preventive effective dose will be higher than the therapeutic effective dose. An effective dose of a vaccine or other agent is sufficient to generate the desired response, such as reducing or eliminating signs or symptoms of a symptom or disease (e.g., pneumonia). For example, this could be the amount required to inhibit viral replication or to measurably alter the external symptoms of viral infection. Generally, this amount will be sufficient to measurably inhibit viral (e.g., HSV-1 and / or 2) replication or infectivity. When administered to a subject, a dose that will reach target tissue concentrations that have been shown to achieve inhibition of viral replication in vitro will typically be used. In some embodiments, "effective dose" is the amount used to treat (including prevent) one or more symptoms and / or underlying causes (e.g., treating HSV infection) of any disorder or disease. In some embodiments, the effective dose is the therapeutic effective dose. In some embodiments, an effective amount is an amount that prevents the development of one or more signs or symptoms of a particular disease or condition (such as one or more signs or symptoms associated with coronavirus infection).

[0262] As used herein, the terms “antigen” or “immunogen” are used interchangeably and refer to a substance, typically a protein, capable of inducing an immune response in a subject. The term also refers to an immunologically active protein, meaning that once administered to a subject (either directly or by administering to the subject a nucleotide sequence or carrier encoding the protein), it can elicit a humoral and / or cellular immune response against that protein. Unless otherwise stated, the terms “vaccine immunogen” are used interchangeably with “protein antigen” or “immunogenic peptide.”

[0263] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. For a specific nucleic acid sequence, a conservatively modified variant refers to those nucleic acids that encode the same or substantially the same amino acid sequence, or, where the nucleic acid does not encode an amino acid sequence, substantially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For polypeptide sequences, a "conservatively modified variant" refers to a variant with conserved amino acid substitutions, where the amino acid residues are replaced by other amino acid residues with side chains having similar charges. Families of amino acid residues with side chains having similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, aspartic acid, glutamic acid, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0264] Epitopes are antigenic determinants. These are specific chemical groups or peptide sequences on a molecule that possess antigenicity, enabling them to elicit a specific immune response. For example, epitopes are antigenic regions that elicit responses from B cells and / or T cells. Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through protein ternary folding.

[0265] Unless otherwise stated, a fusion protein is a recombinant protein containing amino acid sequences from at least two unrelated proteins linked together via peptide bonds to prepare a single protein. Therefore, it does not cover naturally occurring HSV surface antigens referred to herein as fusion (F) proteins. Unrelated amino acid sequences may be directly linked to each other or linked using adapter sequences. As used herein, proteins whose amino acid sequences are not typically linked together via peptide bonds in their native environment (e.g., intracellularly) are unrelated. For example, the amino acid sequences of viral antigens and collagen or procollagen are typically not linked together via peptide bonds.

[0266] An immunogen is a protein or part thereof that can induce an immune response in a mammal (e.g., a mammal infected with or at risk of infection with a pathogen). Administration of an immunogen can trigger protective and / or active immunity against a target pathogen.

[0267] An immunogenic composition is a composition containing an immunogenic peptide that induces a measurable CTL response against a virus expressing the immunogenic peptide, or induces a measurable B cell response (e.g., antibody production) against the immunogenic peptide.

[0268] Sequence identity or similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed as identity or similarity between these sequences. Sequence identity can be measured as a percentage of identity; the higher the percentage, the more identical the sequences. Two sequences are “substantially identical” if, when compared and aligned within a comparison window or a specified region to obtain maximum correspondence (e.g., using one of the following sequence comparison algorithms or measured by manual alignment and visual inspection), they have a specified percentage of identical amino acid residues or nucleotides (i.e., 60% identity in a specified region, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity, or, when not specified, across the entire sequence). Optionally, identity is present in regions of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably in regions of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0269] As used herein, the term "sequence identity" refers to the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when they are aligned to maximize sequence matching (i.e., taking into account gaps and insertions). Sequence alignment and the calculation of sequence identity percentages can be performed using suitable computer programs known in the art. Such programs include, but are not limited to, BLAST, ALIGN, ClustalW, EMBOSS Needle, etc. An example of a local alignment program is BLAST (Basic Tool for Local Sequence Alignment Retrieval) with default parameters, which is available from the website of the National Center for Biotechnology Information (NCBI), currently available at http: / / www.ncbi.nlm.nih.gov / / , and was originally described in Altschul et al. (1990) J. Mol. Biol. [Journal of Molecular Biology] 215; 403-410. Examples of global alignment programs (which optimize alignment on full-length sequences) are the EMBOSS Needle and EMBOSS Stretcher programs based on the Needleman-Wunsch algorithm with default parameters (Needleman, Saul B.; and Wunsch, Christian D. (1970), “A general method applicable to the search for similarities in the amino acid sequence of two proteins”, Journal of Molecular Biology 48 (3): 443-53), which are available at http: / / www.ebi.ac.uk / Tools / psa / .

[0270] A vaccine is a pharmaceutical composition that elicits a preventive or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine elicits an antigen-specific immune response against an antigen of a pathogen (e.g., a viral pathogen) or against an antigen-specific cellular component associated with a pathological condition. A vaccine may include polynucleotides (e.g., nucleic acids encoding a disclosed antigen), peptides or polypeptides (e.g., the disclosed antigen), viruses, cells, or one or more cellular components. In some embodiments, the vaccine or vaccine immunogen or vaccine composition is expressed by a fusion construct and self-assembled into nanoparticles displaying an immunogenic polypeptide or protein on its surface.

[0271] Virus-like particles (VLPs) are non-replicating viral capsids derived from any of several viruses. VLPs typically consist of one or more viral proteins or particle-forming polypeptides derived from these proteins, such as, but not limited to, those called capsid proteins, outer shell proteins, shell proteins, surface proteins, and / or envelope proteins. In suitable expression systems, VLPs can spontaneously form when proteins are recombinantly expressed. Methods for producing specific VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as electron microscopy, biophysical characterization, etc. See, for example, Baker et al. (1991) Biophys. J. [Journal of Biophysics] 60:1445-1456; and Hagensee et al. (1994) J. Virol. [Journal of Virology] 68:4503-4505. For example, VLPs can be separated by density gradient centrifugation and / or identified by characteristic density banding. Alternatively, cryo-electron microscopy can be performed on the vitrified aqueous samples of the VLP preparations under discussion, and images can be recorded under appropriate exposure conditions.

[0272] As used herein, the term "about" refers to a typical range of error for a given value that is readily known to those skilled in the art. References to "about" values ​​or parameters herein include (and describe) embodiments relating to that value or parameter itself.

[0273] As used herein, the singular forms “a / an” and “the” include plural indicators unless the context clearly indicates otherwise. For example, “a / an” means “at least one / type” or “one / type or more / types”.

[0274] The term "comprise" and its variations (such as "comprises" and "comprising"), as well as "contain," "have," and "including"), mean that the listed steps or elements are included, but other steps or elements are not excluded. "Constitutes of" means that any unspecified steps or elements are excluded. "Substantially constitutes of" means that steps or elements that do not substantially affect the essential and novel features of the claimed invention are not excluded. The term "comprise" and its variations also include cases of "consisting of" and "substantially constitutes of".

[0275] Unless otherwise stated, nucleic acid sequences are written from left to right in the 5' to 3' direction; while amino acid sequences are written from left to right in the N-terminus to C-terminus direction.

[0276] Throughout this disclosure, all aspects of the claimed subject matter are presented in scope. It should be understood that this scope designation is for convenience and brevity only and should not be construed as a rigid limitation on the scope of the claimed subject matter. Therefore, the scope should be considered as explicitly disclosing all possible sub-scopes and individual values ​​within those scopes. For example, in the case of providing a range of values, it should be understood that every intermediate value between the upper and lower limits of that range, as well as any other value or intermediate value within that range, is covered within the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included within that smaller range and also covered within the claimed subject matter, subject to any limit explicitly excluded within the stated scope. When a stated scope includes one or both of these limits, the scope excluding any one or both of the included limits is also included within the claimed subject matter. This applies regardless of the width of the scope.

[0277] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It can be a solution, suspension, liquid, powder, paste (aqueous, non-aqueous), or any combination thereof.

[0278] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures, as well as vectors incorporated into the host cell genome into which they have been introduced. Some vectors are capable of directing the expression of the nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0279] Exemplary embodiments Example 1. A protein comprising a plurality of recombinant polypeptides, each recombinant polypeptide containing an HSV surface antigen linked to a C-terminal propeptide of collagen, wherein the C-terminal propeptides of these recombinant polypeptides form interpeptide disulfide bonds.

[0280] Example 2. The protein as described in Example 1, wherein the HSV is HSV-1 or HSV-2.

[0281] Example 3. A protein as described in Example 1 or 2, wherein the surface antigen comprises a protein peptide containing HSV gD or a fragment thereof or an epitope, wherein the epitope is optionally a linear epitope or a conformational epitope, and wherein the protein comprises three recombinant polypeptides.

[0282] Example 4. The protein as described in Example 3, wherein the surface antigen comprises a signal peptide, gD protein, gD-gB (WT) protein, gD-gB (mutant) protein, gD(F)-gB (WT protein), gD(F)-gB (mutant) protein, or any combination thereof.

[0283] Example 5. The protein as described in Example 3, wherein the surface antigen comprises a signal peptide, a receptor-binding domain (RBD) peptide, a receptor-binding motif (RBM) peptide, a fusion peptide (FP), or any combination thereof.

[0284] Example 6. The protein as described in any one of Examples 3-5, wherein the surface antigen comprises the receptor-binding domain (RBD) of the gD and / or gB proteins.

[0285] Example 7. The protein as described in any one of Examples 3-7, wherein the surface antigen does not contain transmembrane (TM) domain peptides and / or cytoplasmic (CP) domain peptides.

[0286] Example 8. The protein as described in any one of Examples 1-7, wherein the surface antigen is soluble or does not directly bind to a lipid bilayer, such as a membrane or viral envelope.

[0287] Example 9. The protein as described in any one of Examples 1-8, wherein the surface antigens of the plurality of recombinant polypeptides of the protein are the same or different.

[0288] Example 10. A protein as described in any one of Examples 1-9, wherein the surface antigen is fused directly to the C-terminal propeptide, or linked to the C-terminal propeptide via a linker, for example, a linker comprising a glycine-XY repeat sequence, wherein X and Y are independently any amino acid, and optionally proline or hydroxyproline.

[0289] Example 11. A protein as described in any one of Examples 1-10, wherein the protein is soluble or does not directly bind to a lipid bilayer, such as a membrane or viral envelope.

[0290] Example 12. A protein as described in any one of Examples 1-11, wherein the protein is capable of binding to a cell surface receptor of a subject, optionally wherein the subject is a mammal, such as a primate, for example, a human.

[0291] Example 13. The protein as described in any one of Examples 1-12, wherein the C-terminal propeptide is derived from human collagen.

[0292] Example 14. The protein as described in any one of Examples 1-13, wherein the C-terminal propeptide comprises a C-terminal polypeptide of proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI) or proα3(XI), or a fragment thereof.

[0293] Example 15. The protein as described in any one of Examples 1-14, wherein the C-terminal propeptides of the plurality of recombinant polypeptides are the same or different.

[0294] Example 16. The protein as described in any one of Examples 1-15, having an amino acid sequence that is at least 90% identical to that protein, is capable of forming disulfide bonds between polypeptides and trimerizing the recombinant polypeptide.

[0295] Example 17. The protein as described in any one of Examples 1-16, wherein the surface antigen in each recombinant polypeptide is in a pre-fusion conformation or a post-fusion conformation.

[0296] Example 18. The protein as described in any one of Examples 1-17, wherein the recombinant polypeptide comprises any one of SEQ ID NO: 1-28 or has at least 80% identity with the amino acid sequence thereon.

[0297] Example 19. An immunogen comprising the protein as described in any one of Examples 1-30.

[0298] Example 20. A protein nanoparticle comprising a protein as described in any one of Examples 1-33, which is directly or indirectly connected to the nanoparticle.

[0299] Example 21. A virus-like particle (VLP) comprising the protein as described in any one of Examples 1-30.

[0300] Example 22. An isolated nucleic acid encoding one, two, three or more recombinant polypeptides of the protein as described in any one of Examples 1-30, wherein the polypeptide encoding the surface antigen is fused within a polypeptide frame to the C-terminal propeptide encoding collagen.

[0301] Example 23. An isolated nucleic acid as described in Example 21 or 22, which is operatively linked to a promoter.

[0302] Example 24. The isolated nucleic acid as described in any one of Examples 21-23, wherein the isolated nucleic acid is a DNA molecule.

[0303] Example 25. The isolated nucleic acid as described in any one of Examples 21-24, wherein the isolated nucleic acid is an RNA molecule, optionally an mRNA molecule, such as nucleoside-modified mRNA, non-amplified mRNA, self-amplified mRNA, or trans-amplified mRNA.

[0304] Example 26. A vector comprising the isolated nucleic acid as described in any one of Examples 25-29.

[0305] Example 27. The vector as described in Example 26 is a viral vector.

[0306] Example 28. A virus, pseudovirus, or cell comprising a vector as described in Example 30 or 31, optionally wherein the virus or cell has a recombinant genome.

[0307] Example 29. An immunogenic composition comprising a protein, immunogen, protein nanoparticles, VLP, isolated nucleic acid, vector, virus, pseudovirus or cell, as described in any one of Examples 1-28, and a pharmaceutically acceptable carrier.

[0308] Example 30. A vaccine comprising the immunogenic composition as described in Example 41 and optionally an adjuvant, wherein the vaccine is optionally a subunit vaccine, and / or optionally a prophylactic and / or therapeutic vaccine.

[0309] Example 31. A vaccine as described in Example 30, wherein the vaccine contains a variety of different adjuvants.

[0310] Example 32. A method for producing a protein, the method comprising: expressing an isolated nucleic acid or vector as described in any one of Examples 21-27 in a host cell to produce a protein as described in any one of Examples 1-18; and purifying the protein.

[0311] Example 33. A protein produced by the method described in Example 32.

[0312] Example 34. A method for generating an immune response to HSV surface antigen in a subject, the method comprising administering to the subject an effective amount of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine as described in any one of Examples 1-31 to generate the immune response.

[0313] Example 35. The method described in Example 34, for treating or preventing HSV infection.

[0314] Example 36. The method as described in Example 34 or 35, wherein the immune response generates an inhibition or reduction of HSV replication in the subject.

[0315] Example 37. The method of any one of Examples 34-36, wherein the immune response includes a cell-mediated response and / or a humoral response, optionally including the production of one or more neutralizing antibodies, such as polyclonal antibodies or monoclonal antibodies.

[0316] Example 38. The method as described in any one of Examples 34-38, wherein the immune response is directed against the HSV surface antigen but not against the C-terminal propeptide.

[0317] Example 39. The method as described in any one of Examples 34-38, wherein the administration does not result in antibody-dependent enhancement (ADE) in the subject due to prior exposure to one or more HSVs.

[0318] Example 40. The method as described in any one of Examples 34-39, wherein the administration does not cause antibody-dependent enhancement (ADE) in the subject upon subsequent exposure to one or more HSVs.

[0319] Example 41. The method as described in any one of Examples 34-40, further comprising an initial exemption step and / or a strengthening step.

[0320] Example 42. The method as described in any one of Examples 34-41, wherein the application step is performed via topical, transdermal, subcutaneous, intradermal, oral, intranasal (e.g., intranasal spray), intratracheal, sublingual, buccal, rectal, vaginal, inhalation, intravenous (e.g., intravenous injection), intra-arterial, intramuscular (e.g., intramuscular injection), intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intra-articular, periarticular, local, or epidermal application.

[0321] Example 43. The method as described in any one of Examples 34-42, wherein the effective amount is administered as a single dose or as a series of doses at intervals of one or more time intervals.

[0322] Example 44. The method as described in any one of Examples 34-43, wherein the effective amount is administered without adjuvant.

[0323] Example 45. The method as described in any one of Examples 34-43, wherein the effective amount is administered together with one or more adjuvants.

[0324] Example 46. A method comprising administering to a subject an effective amount of a protein as described in any one of Examples 1-31 to generate neutralizing antibodies or neutralizing antiserum against HSV in the subject.

[0325] Example 47. The method as described in Example 46, wherein the subject is a mammal, optionally a human or a non-human primate.

[0326] Example 48. The method as described in Example 46 or 47, further comprising isolating the neutralizing antibody or neutralizing antiserum from the subject.

[0327] Example 49. The method of Example 48, further comprising administering an effective amount of isolated neutralizing antibody or neutralizing antiserum to a human subject via passive immunization to prevent or treat HSV infection.

[0328] Example 50. The method of any one of Examples 46-49, wherein the neutralizing antibody or neutralizing antiserum comprises a polyclonal antibody against the HSV surface antigen, optionally wherein the neutralizing antibody or neutralizing antiserum contains no or substantially no antibody against the C-terminal propeptide of collagen.

[0329] Example 51. The method of any one of Examples 46-50, wherein the neutralizing antibody comprises a monoclonal antibody against the HSV surface antigen, optionally wherein the neutralizing antibody contains no or substantially no antibody against the C-terminal propeptide of collagen.

[0330] Example 52. The protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine as described in any one of Examples 1-31, for use in inducing an immune response against HSV in a subject and / or for use in the treatment or prevention of HSV infection.

[0331] Example 53. Use of any protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine as described in any of Examples 1-31 for inducing an immune response against HSV in a subject and / or for treating or preventing HSV infection.

[0332] Example 54. Use of any protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine as described in any of Examples 1-31 for the manufacture of a medicament or prophylactic agent for inducing an immune response against HSV in a subject and / or for the treatment or prevention of HSV infection.

[0333] Example 55. A method for analyzing a sample, the method comprising: contacting the sample with a protein as described in any one of Examples 1-31, and detecting the binding between the protein and an analyte capable of specifically binding the HSV surface antigen.

[0334] Example 56. The method as described in Example 55, wherein the analyte is an antibody, receptor, or cell that recognizes the surface antigen.

[0335] Example 57. The method as described in Example 55 or 56, wherein the combination indicates the presence of the analyte in the sample and / or indicates HSV infection in the subject from whom the sample originated.

[0336] Example 58. A kit comprising a protein as described in any one of Examples 1-21 and a substrate, pad, or vial containing or immobilizing the protein, optionally wherein the kit is an ELISA or lateral chromatography assay kit.

[0337] Example The following examples are for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0338] Example 1: Method Animal Research, Facility, and Ethics Statement. Specific pathogen-free (SPF) BALB / c female mice (6–8 weeks old) and female guinea pigs (4–6 weeks old) were purchased from Charles River Co., LTD for immunogenicity studies. These animals were housed in a pathogen-free facility at the Chengdu Hi-tech Incubation Park's animal care center. The animals had unrestricted access to food and water and were housed under controlled conditions, including a 12-hour light / dark cycle, a temperature range of 16°C–26°C, and humidity levels between 40% and 70%. All mouse experiments were conducted in accordance with international standards for animal research and were approved by the Institutional Animal Care and Use Committee (IACUC) of Clover Biopharmaceuticals.

[0339] Immunization of mice. Nine-week-old female BALB / c mice obtained from Charles River Pharmaceuticals, Beijing, were administered three intramuscular injections of 50 μL each, two weeks apart. Two weeks after the last dose of HSV-2 vaccine, blood and spleen samples were collected to assess humoral immune response.

[0340] Guinea pig immunization and challenge. Female guinea pigs aged four to six weeks from Charles River Guinea Pigs, Beijing, were administered three intramuscular injections (100 μL each) at two-week intervals. On day 42, prior to HSV-2 challenge, the animals were anesthetized and the vaginal fornix was cleaned. 20 μL of a solution containing 1 x 10⁻⁶ HSV-2 was administered. 6The TCID50 HSV-2 G strain was carefully instilled into the vaginal fornix. Guinea pigs were monitored daily for signs of disease, and the severity of primary genital skin lesions was assessed using a lesion scoring scale ranging from 0 (indicating no disease) to 4 (indicating severe vesicular ulcerative dermatitis of the perineum). The scoring criteria were as follows: 0 points indicated no visible lesions; 1 point indicated 1-2 scattered small vesicles; 2 points indicated 3 or more scattered small vesicles; 3 points indicated ruptured clustered vesicles forming erosions; 4 points indicated significant ulceration; and 5 points indicated a fatal event.

[0341] Adjuvants. CAS-1 and MF-59 were manufactured by Clover Biopharmaceuticals. AS01B and AS01E were purchased from GSK Vaccines. CpG 1018 was purchased from Dynavax Technologies. CpG 1018 (CPG) is a TLR-9 agonist, a synthetic CpG-B oligonucleotide with a phosphate thioester backbone, the sequence of which is 5′-TGACTGTGAACGTTCGAGATGA-3′ (SEQ ID NO: 32). Aluminum hydroxide was manufactured by Croda under GMP conditions. Prior to each immunization, the candidate vaccine antigen and adjuvant were mixed at a 1:1 volume ratio by gentle inversion.

[0342] Protein expression and purification. To generate secretory gD-His, gD-trimer, gB-His, gB-trimer, and gD-gB-trimer fusion proteins, cDNAs encoding the signal peptide + extracellular domain of HSV-2 gD protein (amino acids 1-331, GenBank: QBH77835.1) and gB protein (amino acids 1-727, GenBank: YP_009137179.1) were synthesized by GenScript using codons optimized for the Chinese hamster (Cricetulus griseus). The extracellular domain of the HSV-2 gD protein is amino acids 26-331, and the extracellular domain of the HSV-2 gB protein is amino acids 23-727. A His6 tag was added to the 3' end of the gD or gB extracellular domain, separated by a 10-amino acid linker sequence (Gly-Ser)5 (SEQ ID NO: 33) to construct gD-His and gB-His. The cDNA of the gD and gB extracellular domains was subcloned into the pTRIMER expression vector (GenHunter Corporation). Hind III and Bgl IIThe site allows for in-frame fusion of the soluble gD or gB protein with the trimer tag (amino acids 1156-1464 from human type I (α) collagen with a mutant BMP-1 site), as previously described. For the gD-gB-trimer fusion protein, the gB sequence (amino acids 23-727) is linked to the 3' end of gD, followed by the trimer tag coding sequence, to produce an in-frame fusion of the soluble gD-gB protein with the trimer tag.

[0343] Expression vectors for gD-His, gD-trimer, gB-His, gB-trimer, and gD-gB-trimer were stably transfected into GH-CHO (dhfr) using electroporation (Celetrix LE+). - / - Cell lines (KingHunter Biosciences) were used. Cells were cultured in SFM-4 CHO serum-free medium (Cytiva BioSciences) and selected with 10 nM methotrexate (MTX, Sigma). Fusion proteins were generated in shake flasks supplemented with Cell Boost 7A / 7B (Cytiva) using a fed-batch process according to the manufacturer's guidelines. The sequences of the signal peptide-free gD-trimer, gB-trimer, and gD-gB-trimer are shown in SEQ ID Nos: 1, 2, and 4, respectively.

[0344] After centrifugation at 4000 g for 30 min to remove cells, the clarified culture medium was collected, and the trimer fusion protein was purified to homogeneity by chromatography. The trimer fusion protein was captured using a protein A affinity column (MabSelect PrismA, GE Healthcare) preloaded with D6-Fc (Clover Biopharmaceuticals). Unbound impurities were removed, and the trimer fusion protein was eluted with 0.6–0.8 M NaCl in phosphate-buffered saline.

[0345] gD-His and gB-His proteins were purified using a Ni-affinity column (General Health Medical). gD-His was eluted with 60 mM imidazole, and gB-His was eluted with 80 mM imidazole, both prepared in phosphate-buffered saline.

[0346] For the connexin 1-Fc and HVEM-Fc expression vectors, genes encoding the extracellular domains of human connexin 1 and HVEM were synthesized and subcloned into the pGH-hFc expression vector (KingHunter) at Hind III and Bgl II restriction sites. This allows in-frame fusion with the human IgG Fc domain. The vector was transfected into GH-CHO (dhfr- / - The cells were cultured in cell lines under the conditions described previously. Connector protein 1-Fc and HVEM-Fc were generated in shake flasks and purified using protein A affinity chromatography with MabSelect PrismA (General Health Medical).

[0347] Similarly, ligand 1-trimer and HVEM-trimer expression vectors were generated by subcloning synthetic cDNA into pTRIMER vectors (KingHunter) and transfecting them into the GH-CHO cell line. These proteins were produced and purified using the same methods as other trimer fusion proteins.

[0348] Finally, the His6 tag was attached to the 3' end of the human LIGHT extracellular domain, separated by a (Gly-Ser)5 (SEQ ID NO:33) linker to construct the LIGH-His protein. The expression vector was transfected into the GH-CHO cell line, and the protein was produced and purified using a nickel (Ni) column, eluted in 60 mM imidazole in phosphate-buffered saline.

[0349] SEC-HPLC. Purity of gD-His, gD-trimer, gB-trimer, and gD-gB-trimer proteins was evaluated using size exclusion chromatography (SEC-HPLC) on an Agilent 1260 Infinity HPLC system equipped with a TSK gel G3000SWxL column (Tosoh Corporation). The mobile phase used for separation was phosphate-buffered saline (PBS). Proteins were monitored at a flow rate of 1 mL / min at a wavelength of 280 nm throughout the 20-minute run. This method enables the analysis of protein homogeneity and allows purity determination by evaluating the chromatograms, ensuring the presence of a single peak corresponding to the respective protein.

[0350] The binding of gD to human connexin 1 and HVEM receptors was studied. Proteins containing connexin 1-Fc, connexin 1-trimer, HVEM-Fc, and HVEM-trimer (1 μg / mL) were immobilized on enzyme-linked immunosorbent assay (ELISA) plates (Thermo) and incubated overnight at 4°C. The plates were then blocked at 37°C with 2% skim milk powder for 2 hours to prevent nonspecific binding. After washing three times with PBS (PBST) containing 0.05% Tween 20 to remove unbound proteins, the plates were incubated with a series of concentrations of biotin-labeled gD-His, gD-trimer, and gD-gB-trimer at 37°C for 1 hour. After washing another set three times with PBST, the plates were treated with a 1:5000 dilution of streptavidin-horseradish peroxidase (SA-HRP) conjugate (Jackson, 1:5000 dilution) at 37°C for 1 hour to facilitate the detection of bound biotin-tagged proteins. Following three further washes with PBST to remove excess SA-HRP, a 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Thermo Scientific) was added to generate a signal, allowing colorimetric detection of protein interactions.

[0351] HVEM activity assay. This was achieved by subcloning a synthetic cDNA template (GenScript) encoding the full-length human HVEM sequence into the pCLV-puro expression vector (Clover Biotech). Hind III and XbaI Restriction sites were used to construct the HVEM expression vector. This vector was then stably introduced into the HEK293-NFKB-Luc cell line (Clover Biopharmaceuticals) using electroporation (Celetrix LE+). Transfected cells were cultured in DMEM supplemented with 10% FBS and selected for puromycin resistance using 2 µg / mL puromycin (Solarbio).

[0352] Subsequently, freshly trypsinized HEK293-NFKB-Luc cells stably expressing HVEM were seeded at a density of 40,000 cells per well. After incubation at 37°C in a 5% CO2 incubator with serially diluted (3-fold) concentrations of gD or LIGHT protein for 8 hours, the cells were lysed, and luciferase activity was measured using a luciferase assay system (Vazyme, Novizan) according to the manufacturer's instructions. This assay quantifies the activation of the NF-κB pathway as an indirect measure of the binding and functional interaction between HVEM and gD / LIGHT.

[0353] Negative staining electron microscopy. Sample preparation before negative staining: Dilute the sample to the appropriate concentration for negative staining and mix thoroughly by repeated pipetting to ensure uniform dilution.

[0354] Negative staining grid preparation: The protein sample was applied to a glow discharge hydrophilic negative staining grid (Beijing XinXingBaiRui Technology Co., Ltd., catalog number: T11032). After allowing the grid to stand, excess sample was removed with filter paper, and the grid was rinsed with deionized water. Subsequently, the grid was stained with the staining solution at room temperature, and any remaining liquid at the grid edges was absorbed with filter paper. The grid was then allowed to air dry naturally.

[0355] Data Collection and Processing: Sample observation and data collection were performed on a Thermo Fisher Talos L120 TEM equipped with a C120 kV TEM. The electron detector used was a CETA direct electron counting detector. Data collection was performed in super-resolution mode using SerialEM software. The contrast transfer function (CTF) was corrected using Cistem software to mitigate the effects of varying defocusing and radiation damage. Particles were automatically extracted from the photomicrographs using boxes 2–3 times the longest diameter of the particles. Poor-quality particles (e.g., understained particles, particles too close together, or particles near the edges of the photomicrograph) were discarded. The remaining particles were classified in two dimensions (2D) using Cistem software.

[0356] Neutralizing antibody assay. Two serial dilutions (50 µL per well) of heat-inactivated serum samples were prepared in 96-well microtiter plates. Then, 60 µL of a solution containing 100 TCID50 of HSV-2 strain G was added to each well. For complement-dependent neutralization assays, 10% guinea pig serum was incorporated into the culture medium. The plates were incubated at 37°C for two days under a 5% CO2 atmosphere for virus infection. After incubation, the culture medium was discarded, and the plates were stained with crystal violet. The plates were then washed and examined for viral plaques. The neutralizing titer was calculated as the reciprocal of the serum dilution that resulted in a 50% reduction in cell monolayer lysis.

[0357] ADCC assay. Screening of mouse ADCC effector cell lines: The pNFAT-TA-Luc vector (Beyotime) was stably integrated into the Jurkat cell line using electroporation (Celetrix LE+), and the cells were cultured in RPMI-1640 medium supplemented with 10% FBS. After transfection, cells were selected with 1 mg / mL G418 (Sigma). After establishing a stable NFAT-Luc cell line, the mouse Fcγ3A gene (Genscript) was introduced via electroporation, and the cells were then selected with 2 µg / mL puromycin (Solepro).

[0358] Screening of target cells expressing HSV gD and gB proteins: The full-length sequences of gD and gB were cloned into the pLV vector and used to generate lentiviral particles. 293 cell lines were infected with lentivirus and then selected using 2 µg / mL puromycin (Solepro).

[0359] For the assay, fresh trypsinized target cells were added to each well at a density of 10,000 cells per well and incubated overnight. Heat-inactivated immune serum (collected on day 42) or control pre-immune serum (day 0) was diluted 1:10 in DMEM and added to the target cells. These were then co-cultured with mouse ADCC effector cells at a target-to-effector ratio of 1:15 and incubated for another 6 hours at 37°C and 5% CO2. After incubation, the cells were lysed, and luciferase activity was measured using a luciferase assay system (Novozymes) according to the manufacturer's instructions.

[0360] Cell-mediated immunization. Two weeks after the last HSV-2 vaccination, mice were humanely euthanized, and their spleens were harvested. Spleen cells were prepared into single-cell suspensions and plated into 96-well ELISPOT plates pre-coated with antibodies against IFN-γ, IL-2, IL-4, and IL-5 (MABTECH). Cells were then plated at different cell densities: 1 × 10⁻⁶ cells for IFN-γ, IL-2, IL-4, and IL-5. 5 5 × 10 4 5 × 10 5 and 1 × 10 6Cells / well. Cells were stimulated with 100 nM gD / gB protein or a positive control (PMA / ionomycin) to measure background response without sample addition. ELISPOT plates were incubated overnight at 37°C with 5% CO2. Plate development was performed using substrate solution BCIP / NBT (Sigma-Aldrich) according to the manufacturer's protocol. Spots were quantified using an ImmunoSpot Analyzer S6 Entry (CTL Ltd.) and the results are expressed as the number of spot-forming units (SFU) per well.

[0361] Statistical analysis. Prism 10.1.2 software (GraphPad software) was used for data organization and statistical evaluation. For comparisons between two experimental groups, the two-tailed Mann-Whitney U test was applied. When evaluating multiple groups, one-way ANOVA was used for multiple comparisons. A p-value less than 0.05 was considered statistically significant. Results that were not statistically significant were expressed as “ns”.

[0362] Example 2: Results Expression, purification, and purity analysis of HSV-2 trimer gD-trimer and gD-gB-trimer antigens We engineered DNA constructs to generate recombinant HSV-2 gD-His, gD-trimer, gB-His, gB-trimer, and gD-gB-trimer proteins in CHO cells. Figure 1 , Figure 2 Adding the His6 tag to the C-terminus of the extracellular domain of HSV-2 gD or gB (separated by a 10-amino acid linker consisting of (Gly-Ser)5 (SEQ ID NO: 33)) to generate the gD-His or gB-His fusion protein ( Figure 1 C Figure 1 E, Figure 2 A, Figure 2 C). The cDNA encoding the extracellular domains of HSV-2 gD and gB was subcloned into the pTRIMER expression vector to enable in-frame fusion of the soluble gD or gB protein with the trimer tag. Figure 1 D、 Figure 1 F, Figure 2 B Figure 2 D、 Figure 2 E). For the gD-gB-trimer construct, the gB sequence encoding amino acids 23-727 is fused to the 3' end of gD, followed by the trimer tag coding sequence, thereby allowing in-frame fusion of the soluble gD-gB protein with the trimer tag. Figure 1 G, Figure 2 F, Figure 2G). The furin protease site (RRAR (SEQ ID NO: 31)) has also been incorporated into the gD-gB-trimeric fusion protein located at the junction between the gD and gB proteins. Figure 1 H, Figure 2 H, Figure 2 I). After stable transfection into CHO cells, we performed a screening process to identify high-titer production clones and employed fed-batch, serum-free cell culture techniques in shake flasks.

[0363] To isolate the high-purity trimer fusion protein, we utilized chromatography, leveraging the high-affinity interaction between the trimer and its receptor Endo180 within the critical binding domain 6 (D6). The D6-Fc fusion protein was immobilized onto a Protein A column, where it was captured by the resin through high-affinity binding to the human IgG1 Fc domain of D6-Fc. Subsequently, serum-free cell culture medium rich in CHO cell-secreted trimer fusion protein was applied to the Protein A column pre-immobilized with D6-Fc. After washing away any unbound host cell protein (HCP) and other impurities, the bound trimer fusion protein was purified to near homogeneity in a single step using moderate salt elution, which preserved the interaction between D6-Fc and the Protein A column. SDS-PAGE analysis under both non-reducing and reducing conditions confirmed that the purified gD-trimer consisted of disulfide-linked trimers, while gD-His remained a monomer. Figure 3 A, Figure 3 B), such as those produced by CHO cells. Similarly, gB-trimer and gD-gB-trimer were also found to be disulfide-linked trimers (B). Figure 3 C Figure 3 D).

[0364] Protein purity was assessed using size exclusion SEC-HPLC. gD-His exhibited approximately 90% purity in the main peak. Figure 3 E). In contrast, the purities of gD-trimer, gB-trimer, and gD-gB-trimer exceeded 95%, as confirmed by size exclusion SEC-HPLC (respectively). Figure 3 F, Figure 3 G, Figure 3 H).

[0365] Characterization of HSV-2 trimer gD-trimer and gD-gB-trimer antigens Negative staining electron microscopy and two-dimensional (2D) classification of gD-His and gD-trimer showed that gD-His particles were significantly smaller than gD-trimer particles, indicating that a trimer structure was formed in gD-trimer. Figure 4 A, Figure 4B). Similarly, negative staining electron microscopy and two-dimensional (2D) classification of gB-trimer and gD-gB-trimer also showed that both gB and gD adopt a trimer configuration ( Figure 4 C Figure 4 D). We used cryo-electron microscopy to elucidate the structure of the gD-gB-trimer ( Figure 5 A- Figure 5 J), in which gB was observed to exhibit a post-fusion conformation ( Figure 6 A- Figure 6 F). Despite limited structural orientation, precise determination of the gD structure is not feasible. However, the results reveal the trimer structure of gD, showing top and side views of the gD trimer (F). Figure 7 A, Figure 7 B).

[0366] ELISA was used to determine the EC50 values ​​of monomeric and trimer gD binding to their receptors, linker protein 1 and HVEM. The results showed that the binding affinity of trimer gD was significantly higher than that of monomeric gD. Figure 8 A, Figure 8 B). The binding affinity of gD-trimer and gD-gB-trimer to the gD receptor is essentially equivalent. Figure 8 A, Figure 8 B). Monomeric gD largely fails to activate the HVEM pathway, while trimer gD activates the HVEM pathway more strongly than the natural HVEM ligand LIGHT. Figure 8 C Figure 8 D). Activation of the HVEM pathway contributes to ADCC-mediated antibody production, and gD-trimers can promote a higher proportion of ADCC-active antibodies, which are primarily responsible for clearing and preventing HSV-2 infection.

[0367] Comparison of the immunogenicity of gD-gB-trimer in the presence of different adjuvants.

[0368] The choice of adjuvant has a significant impact on the immunogenicity of subunit vaccines, as it can not only enhance the strength of the immune response but also alter the type of immunization. To determine the optimal adjuvant, we conducted a comparative screening of several adjuvants. Mice were immunized with a gD-gB-trimeric complex in combination with several adjuvants: CpG / CAS-1 (a proprietary adjuvant with properties similar to AS03), CpG / alum, AS01B, and AS01E. The immunization regimen was administered at three different time points (specifically on day 0, day 14, and day 28) to assess the immunogenicity of the vaccine formulation. Figure 9 A), and on day 42, serum binding antibody, HSV-2 neutralizing antibody, and ADCC activity were assessed. Results showed that CpG / CAS-1 induced the highest level of binding antibody (A). Figure 9B). There were no significant differences in neutralizing antibody titers and ratios among CpG / CAS-1, CpG / alum, AS01B, and AS01E. Figure 9 C Figure 9 D). However, in terms of ADCC activity, CpG / CAS-1, AS01B, and AS01E showed significantly higher levels than CpG / alum ( Figure 9 E, Figure 9 F). Considering the immunogenicity and availability of adjuvants, we identified CpG / CAS-1 as the most promising adjuvant candidate.

[0369] Immunogenicity of gD-trimer in mice The immunogenicity of gD-trimer was evaluated in BALB / c mice. gD-trimer and gD-His (…) were used. Figure 10 Both (A) and (CpG / CAS-1) were used as adjuvants, and mice were intramuscularly immunized with a three-dose primate-boost regimen (on days 0, 14, and 28). On day 42, serum samples were tested for gD-binding antibody, HSV-2 neutralizing antibody, and ADCC activity. No significant difference in gD-binding antibody titers was observed between mice immunized with monomeric and trimeric gD. Figure 10 B). However, the neutralizing antibody response in the gD-trimer group was significantly higher than that in the monomeric gD group ( Figure 10 C). ADCC activity in mouse serum on day 42 showed that the gD-trimer-induced response was significantly higher than that of the monomer gD ( Figure 10 D、 Figure 10 E), which is consistent with the role of gD-trimer in activating the HVEM pathway, thereby promoting the production of ADCC active antibodies.

[0370] Immunogenicity of gD-gB-trimer in mice Glycoprotein D (gD) serves as a ligand for HSV entry into cells and plays a crucial role in HSV infection. In addition to receptor binding of gD, the trimeric glycoprotein B (gB) is essential for fusion function during HSV infection, and gB is key for complement-dependent neutralizing antibodies and cellular immunity. To enhance immunogenicity, we constructed a gD-gB-trimeric fusion protein.

[0371] Following the immunization schedule on days 0, 14, and 28 ( Figure 11(A) We compared the immune responses of gD-His / gB-His+MF-59 (Qilong Pharmaceutical Co., Ltd.), gD-His+CpG / CAS-1, gD-trimer+CpG / CAS-1, gB-trimer+CpG / CAS-1, gD-trimer / gB-trimer+CpG / CAS-1, and gD-gB-trimer+CpG / CAS-1. Blood samples were collected on day 42 for cellular immune assessment. The complement-dependent and complement-independent HSV-2 neutralizing antibody and ADCC activities in serum on day 42 were tested. Results showed that the complement-independent HSV-2 neutralizing antibody titers were highest in the gD-trimer+CpG / CAS-1 group, followed by the gD-gB-trimer+CpG / CAS-1 group, with no significant difference between the two groups. Figure 11 B). In contrast, the gD-His / gB-His+MF-59 (Qilong Company) and gB-trimer+CpG / CAS-1 groups showed almost undetectable levels of complement-independent neutralizing antibodies (B). Figure 11 B). Complement-dependent HSV-2 neutralizing antibodies are highly dependent on gB, with the group immunized using gB as the antigen showing significantly higher titers ( Figure 11 C). Neither monomeric gD nor trimeric gD, as antigens, induces higher complement-dependent neutralizing antibodies; in fact, trimeric gD formulations induce lower titers. Figure 11 C).

[0372] Regarding ADCC activity in immune serum, the gD-His / gB-His+MF-59 (Qilong Company) group induced almost no ADCC activity. Consistent with previous findings, gD-trimer+CpG / CAS-1 induced higher ADCC activity than gD-His+CpG / CAS-1. Figure 11 D、 Figure 11 E). gB-trimer + CpG / CAS-1 induced the strongest ADCC activity, followed by gD-trimer / gB-trimer + CpG / CAS-1 and gD-gB-trimer + CpG / CAS-1, with no significant difference among the three groups. Figure 11 D、 Figure 11 E). The gD-His / gB-His+MF-59 (Qilong Company) group induced a balanced Th1 and Th2 cell-mediated immune response without significant bias. Figure 12 A, Figure 12 B). In contrast, all CpG / CAS-1 adjuvant immunizations biased the cellular immune response towards the Th1 phenotype ( Figure 12 A, Figure 12 B). Although gB induces a primary cellular immune response, gD also contributes to some degree of cellular immunity (B). Figure 12 A, Figure 12 B). Considering the levels of complement-dependent and complement-independent HSV-2 neutralizing antibodies induced in mice, ADCC activity, and cellular immunity, the gD-gB-trimer + CpG / CAS-1 combination is the optimal HSV-2 vaccine formulation.

[0373] Immunogenicity of gD-gB-trimer in guinea pigs The immunogenicity of the HSV-2 gD-gB-trimer + CpG / CAS-1 vaccine was further evaluated in guinea pigs. On days 0, 14, and 28, guinea pigs (n = 6 per group) were administered gD-gB-trimer + CpG / CAS-1, gD-His / gB-His + MF-59 (Qilon Company), or a saline control via intramuscular injection. Figure 13 As described in A. Serum samples were obtained on day 42, 14 days after the last immunization, followed by vaginal challenge with the HSV-2 G strain, as... Figure 13 As shown in Figure A. We assessed the levels of complement-dependent and complement-independent HSV-2 neutralizing antibodies in serum on day 42. Data showed that the gD-gB-trimer + CpG / CAS-1 group exhibited significantly higher titers of both complement-dependent and complement-independent HSV-2 neutralizing antibodies compared to the gD-His / gB-His + MF-59 (Qilong Pharmaceutical Co., Ltd.) group. Figure 13 B Figure 13 C). Following HSV-2 challenge, the saline control group experienced a significant decrease in body weight, while the gD-gB-trimer + CpG / CAS-1 and gD-His / gB-His + MF-59 (Qilong Company) groups showed normal weight gain. Figure 13 D). The saline control group also showed a significant increase in genital skin lesions, while the gD-gB-trimer + CpG / CAS-1 and gD-His / gB-His + MF-59 (Qilong Company) groups showed very mild to no lesions. Figure 13 E). Therefore, both the gD-gB-trimer + CpG / CAS-1 and gD-His / gB-His + MF-59 (Qilong Pharmaceutical) groups showed robust protection against HSV-2 infection, although the gD-gB-trimer + CpG / CAS-1 group was superior to the gD-His / gB-His + MF-59 (Qilong Pharmaceutical) group in terms of complement-independent HSV-2 neutralizing antibody titers.

[0374] in conclusion The trimeric gD-gB-trimer + CpG / CAS-1 formulation is a promising vaccine candidate against HSV-2. It exhibited strong immunogenicity in mice and guinea pigs, inducing a balanced mix of complement-dependent and complement-independent neutralizing antibodies, strong ADCC activity, and Th1-biased cellular immune responses. These findings highlight its potential as an effective and balanced approach to preventing HSV-2 infection.

[0375] Example 3: Detection of HSV binding and neutralizing antibodies using colloidal gold assay.

[0376] For HSV-binding antibodies: 1) A purplish-red binding pad containing recombinant HSV-2 gD-gB-trimer and rabbit IgG conjugated to colloidal gold ( Figure 14 A). 2). Nitrocellulose membrane strips containing test lines (T lines) and control lines (C lines). The T line is coated with mouse anti-human IgG, and the C line is coated with goat anti-rabbit IgG polyclonal antibody (…). Figure 14 A).

[0377] When a sufficient volume of test sample is dispensed into the well, the sample migrates through the cartridge via capillary action. If the sample contains an appropriate concentration of HSV IgG antibody, it will bind to the colloidal gold-conjugated HSV-2 gD-gB-trimer on the test strip and, under the action of lateral chromatography, move along the nitrocellulose membrane to the T-line, where it binds to mouse anti-human IgG antibody to form a colloidal gold complex, thus forming a deep purple T-line. Figure 14 B Figure 14 C Figure 14 D、 Figure 14 E). If the sample contains no HSV IgG antibody, or the amount is too small, the colloidal gold-conjugated HSV-2 gD-gB-trimer migrates on the nitrocellulose membrane using lateral chromatography and does not bind to mouse anti-human IgG at the T-line. Figure 14 B Figure 14 C Figure 14 D、 Figure 14 E). The result is determined by the presence or absence of a deep purple T-line. Colloidal gold-conjugated rabbit IgG continues to move forward to the control C-line. The combination of colloidal gold-conjugated rabbit IgG and goat anti-rabbit IgG polyclonal antibody forms a neat and uniform deep purple band, indicating that the detection reaction system is effective. Figure 14 B Figure 14 C Figure 14 D、 Figure 14 E).

[0378] For HSV neutralizing antibodies: 1) A purplish-red binding pad containing recombinant HSV-2 gD-gB-trimer and rabbit IgG conjugated with colloidal gold. Figure 15 A).

[0379] 2) Nitrocellulose membrane strips containing a test line (T line) and a control line (C line). The T line is coated with recombinant connexin-1 or HVEM receptor protein, and the C line is coated with goat anti-rabbit IgG polyclonal antibody (…). Figure 15 A).

[0380] When a sufficient volume of test sample is dispensed into the wells, the sample migrates through the cassette via capillary action. If the sample does not contain HSV neutralizing antibodies, or the amount is too small, the colloidal gold-conjugated HSV-2 gD-gB-trimer will migrate along the nitrocellulose membrane to the T-line using lateral chromatography. It combines with recombinant connexin-1 or HVEM to form a colloidal gold complex, thus forming the purplish-purple T-line. Figure 15 B Figure 15 C Figure 15 D、 Figure 15 E). If a neutralizing antibody is present in the sample, it will bind to the colloidal gold-conjugated HSV-2 gD-gB-trimer and competitively prevent the colloidal gold-conjugated HSV-2 gD-gB-trimer from binding to recombinant connexin-1 or HVEM, thereby reducing the color development of the T line, or even causing the T line to disappear. Figure 15 B Figure 15 C Figure 15 D、 Figure 15 E). The colloidal gold-conjugated rabbit IgG continued to move forward to the control line C. The colloidal gold-conjugated rabbit IgG combined with the goat anti-rabbit IgG polyclonal antibody formed a neat and uniform deep purple band, indicating that the detection reaction system was effective. Figure 15 B Figure 15 C Figure 15 D、 Figure 15 E).

[0381] sequence 。

Claims

1. A fusion polypeptide comprising a soluble HSV viral surface antigen linked by in-frame fusion to a protein trimerizing tag capable of self-trimerization.

2. The fusion polypeptide of claim 1, wherein the protein trimer tag is selected from the group consisting of: the C-terminal portion of procollagen capable of forming disulfide-linked homotrimers, the T4 foldon of fibrin from bacterial bacteriophage T4, and the leucine zipper from yeast GCN4.

3. The fusion polypeptide of claim 2, wherein the procollagen is selected from the group consisting of: proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI), or proα3(XI).

4. The fusion polypeptide of claim 2 or 3, wherein the C-terminal portion of the procollagen is a C-propeptide and does not contain any glycine repeat triple helix region in the procollagen that is linked to the C-propeptide.

5. The fusion polypeptide of claim 2 or 3, wherein the C-terminal portion of the procollagen comprises a triple helix region of a glycine repeat sequence of collagen linked to a C-propeptide.

6. The fusion polypeptide of any one of claims 2-5, wherein the C-terminal portion of the procollagen has a mutated or missing BMP-1 protease recognition sequence.

7. The fusion polypeptide of any one of claims 1 or 2, wherein the soluble HSV surface antigen comprises an extracellular domain of the HSV surface antigen or a fragment or variant thereof.

8. The fusion polypeptide according to any one of claims 1-7, wherein the soluble HSV viral surface antigen comprises an extracellular domain of gD or a fragment or variant thereof.

9. The fusion polypeptide according to any one of claims 1-7, wherein the soluble HSV viral surface antigen comprises an extracellular domain of gB or a fragment or variant thereof.

10. The fusion polypeptide of any one of claims 1-7, wherein the extracellular domain of the HSV viral surface antigen comprises a fusion of the extracellular domain of a gD or a fragment or variant thereof and the extracellular domain of a gB fusion or a fragment or variant thereof.

11. The fusion polypeptide according to any one of claims 1-10, wherein the fusion polypeptide comprises the sequence shown in any one of SEQ ID No: 1-28 or a sequence having at least 80% sequence identity with any one of SEQ ID NO: 1-28.

12. The fusion polypeptide according to any one of claims 1-11, wherein the fusion polypeptide further comprises a signal peptide.

13. A polynucleotide encoding a fusion polypeptide as described in any one of claims 1-12.

14. A vector comprising the polynucleotide as described in claim 13.

15. A cell expressing a fusion polypeptide as described in any one of claims 1-12 or a vector comprising the polynucleotide as described in claim 12 or the vector as described in claim 13.

16. A vaccine comprising the fusion polypeptide as described in claims 1-12.

17. The vaccine of claim 16, wherein the vaccine comprises two or more of the fusion polypeptides of claims 1-12.

18. The vaccine of claim 17, wherein the vaccine comprises a fusion polypeptide containing the extracellular domain of gD or a fragment or variant thereof, and a fusion polypeptide containing the extracellular domain of gB or a fragment or variant thereof.

19. A method for preventing or treating infection caused by a herpes simplex virus (HSV) selected from human HSV-1 and HSV-2 by immunizing a subject with the vaccine as described in any one of claims 16-18.

20. The method of claim 19, wherein the vaccine is administered without adjuvant.

21. The method of claim 19, wherein the vaccine is administered together with one or more adjuvants.

22. The method of claim 21, wherein the one or more adjuvants is CpG or alum, or both.

23. The method of claim 21, wherein the one or more adjuvants is CpG or an oil-in-water adjuvant, or both.

24. The method of claim 23, wherein the oil-in-water adjuvant is an oil-in-water emulsion comprising squalene, α-tocopherol and polysorbate 80.

25. The method of claim 23, wherein the oil-in-water adjuvant is an oil-in-water emulsion comprising squalene, Span 85 and polysorbate 80.

26. The method of any one of claims 19-25, wherein the vaccine is administered via intramuscular or subcutaneous injection.

27. The method of any one of claims 19-26, wherein the vaccine is administered in a single dose or in a series of doses spaced several weeks or months apart.

28. A method for detecting HSV antibodies in mammalian samples, the method comprising the following steps: (1) Contact the sample with the fusion polypeptide as described in any one of claims 1-12, (2) Detect the antibodies that bind to the HSV virus surface antigen in the fusion polypeptide.

29. The method of claim 28, wherein the antibody is a neutralizing antibody.

30. The method of claim 28 or 29, wherein the method comprises detecting the binding of the antibody to the HSV viral surface antigen in the fusion polypeptide using a secondary antibody.

31. The method of claim 28 or 29, wherein the method comprises detecting a neutralizing antibody, the resulting reading reflecting the inhibition of binding of the HSV viral surface antigen in the fusion polypeptide to the soluble HSV receptor.

32. The method of claim 31, wherein the soluble HSV receptor is fused with an Fc domain or a trimerized domain.

33. The method of claim 31 or 32, wherein the soluble HSV receptor is connexin-1 or HVEM.

34. The method of any one of claims 31-33, wherein the soluble HSV receptor has the sequence shown in any one of SEQ ID No:29-30.

35. The method of any one of claims 28-34, wherein the fusion polypeptide is labeled with a detection reagent.

36. The method of any one of claims 28-35, wherein the fusion polypeptide binds to an antibody that recognizes the C-terminal portion of collagen, wherein the antibody that recognizes the C-terminal portion of collagen is labeled with a detection reagent.

37. The method of claim 35 or 36, wherein the detection agent is colloidal gold.

38. The method of any one of claims 28-37, wherein the HSV antibody is detected by lateral chromatography.

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