A recombinant herpes simplex virus type I vaccine and its application

Abstract

The present invention discloses a recombinant herpes simplex virus type I vaccine and its application, belonging to the field of vaccine technology. Specifically, it relates to a fusion protein composed of an antigen molecule gD1, a human immunoglobulin antibody Fc domain and a human IgG1 hinge region fragment, wherein the amino acid sequence of the antigen molecule gD1 is shown in SEQ ID NO.6, and a recombinant vaccine is constructed. The recombinant vaccine of the present invention fuses viral antigens, immunoglobulin heavy chain Fc domain and human IgG1 hinge region fragment, has a better ability to bind to receptors on the surface of antigen-presenting cells, and can activate specific CD4 + T and CD8 + T cells, producing high titer antibody titers and specific cellular immunity. This vaccine can prevent infection and / or reinfection with herpes simplex virus type 1.

Description

Technical Field

[0001] The present invention belongs to the technical field of vaccines, and in particular relates to a recombinant herpes simplex virus type I vaccine and an application thereof. Background Art

[0002] To date, eight herpes viruses have been confirmed to be associated with human diseases: herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus, cytomegalovirus (CMV), and herpes simplex virus types 6, 7, and 8. The human diseases caused by these eight herpes viruses vary in clinical manifestations, but they all share a common characteristic: after the initial infection of the human body, each virus may persist in the body for a long time, or even for life. This long-term infection often alternates between overt and latent infection.

[0003] Epidemiological studies indicate that an estimated 67% of the global population is infected with HSV-1. After initial infection, HSV-1 causes chronic infection in sensory ganglia and can reactivate on the mucous membranes and skin. Infection is often asymptomatic but can result in a variety of symptoms and signs. These include recurrent oral or perioral lesions ("cold sores"), skin and mucous membrane lesions (including genital lesions), eye infections (herpetic keratitis), and severe systemic disease (encephalitis and neonatal disease with multiorgan involvement).

[0004] Once infected, HSV-1 is a lifelong infection. Most often, infection only causes acute blisters on the oral and genital areas during acute episodes. HSV infection can remain latent within the trigeminal and lumbosacral ganglia. When the body experiences immunosuppression or immunodeficiency, or when exposed to triggering factors such as ultraviolet light, fever, trauma, emotional stress, or bacterial or other viral infections, the latent HSV can be activated and replicate rapidly, causing localized recurrent blisters. Furthermore, the virus lurking within the ganglia can also enter the central nervous system through retrograde neural transmission. Over the past decade, increasing evidence has suggested that the impact of HSV on humans may extend beyond herpes. Our understanding of HSV and its infection remains limited. For example, HSV-1, previously thought to primarily cause infections of the head and face, can also cause genital herpes, just as HSV-2 does. Generally speaking, HSV-1 is the primary etiologic agent of oral and labial herpes infections, while HSV-2 is the primary etiologic agent of genital herpes infections. However, epidemiological data in recent years have shown that the number of cases of HSV-1 infection of the reproductive organs has gradually increased, especially among adolescents and young men and women. The HSV-1 infection rate in the age group of 15 to 30 has exceeded that of HSV-2. From the analysis of the histopathological changes caused by viral infection, there is no obvious difference between HSV-1 and HSV-2. Both manifest as local herpes ulcers in the reproductive organs, accompanied by more severe pain.

[0005] HSV genital tract infection has become one of the most prevalent sexually transmitted infections worldwide. HSV infection increases the risk of infection with sexually transmitted viruses such as human immunodeficiency virus (HIV), increases the transmission of sexually transmitted viruses (STIs) such as human papillomavirus (HPV), and leads to abnormal immune responses, ultimately leading to the development of cancers such as cervical cancer. HSV infection in women of childbearing age can cause genital tract damage and dysfunction. In pregnant women, HSV infection can lead to miscarriage, premature birth, congenital fetal developmental abnormalities, and neonatal HSV encephalitis. Furthermore, HSV entry into the central nervous system can damage the nervous system, causing herpes simplex encephalitis, Bell's palsy, and primary trigeminal neuralgia, and is closely associated with the development of certain psychiatric disorders and Alzheimer's disease. These new epidemiological data have reshaped our understanding of HSV and prompted countries to prioritize data collection on HSV infection and the development of new vaccine strategies.

[0006] HSV-1 infection can remain latent in the nervous system for extended periods, leading to recurrent infections during periods of stress or immune deficiency. Existing antiviral drugs, such as acyclovir, can effectively control symptoms, but these drugs are highly susceptible to genetic mutations and drug resistance. Furthermore, these drugs cannot prevent primary herpes simplex virus infection or control latent and recurrent infections.

[0007] HSV-1 is also a significant etiology of herpes simplex encephalitis. Literature reports suggest that approximately 95% of cases of herpes simplex encephalitis are caused by HSV-1. Furthermore, it is the most common etiology of sporadic, fatal encephalitis. Adult patients often develop specific antibodies against HSV-1 before developing symptoms, suggesting that infection results from viral reactivation. In children, HSV-1 often causes neonatal herpes simplex encephalitis, a clinical manifestation similar to that of other herpes simplex viruses, characterized by mental disturbances, sudden loss of consciousness, and specific neurological signs, accompanied by fever. Severe cases can lead to neurological sequelae such as microcephaly, microcephaly, and polycystic encephalomalacia. Neonatal herpes first gained attention in the 1930s. Contact between maternal and fetal tissue can lead to vertical transmission from mother to child. HSV-1 is primarily transmitted in utero, during delivery, and after birth, potentially resulting in stillbirth and neonatal herpes.

[0008] From a structural biology perspective, the basic structure of herpesviruses includes the following features: a spherical core composed of a double-stranded DNA genome and DNA-binding proteins, approximately 75 nm in diameter. This core is coated with a capsid protein, which is regularly organized into a three-dimensional icosahedral structure with a diameter of 95 to 105 nm. Within these 162 capsids are 150 hexamers and 12 pentamers. The nucleocapsid is surrounded by a layer of globular protein, called the cortex. Beyond this cortex lies the envelope, which is covered with various glycoproteins. The complete virus has a diameter of 120 to 300 nm.

[0009] HSV-1 DNA is 152,260 bp long (17 strains, Genbank No. X14112), with a G+C content of 68%. The genome consists of two segments, the L segment and the S segment, with repetitive sequences at both ends. Under the strict regulatory mechanism of the genome, it operates according to a linear timeline program that can be divided into three phases: immediate-early, early, and late. The corresponding genes activated for transcription are the immediate-early gene (IE), early gene (E), and late gene (L). These genes operate according to a strict sequence: IE gene transcriptional expression -> production of IE gene products -> activation and regulation of E gene transcriptional expression -> production of E gene products -> activation and regulation of L gene products.

[0010] There are multiple membrane proteins on the herpesvirus envelope, all of which are glycosylated proteins, mainly gB, gC, gD, gE, gG, gH, gI, gL, gM, and gN. Membrane proteins bind to nonspecific or specific receptors on the host cell membrane, allowing the virus to enter the cell and perform its biological functions. Approximately five membrane proteins are directly related to the process of virus entry into the cell. The glycoproteins gB and gC act by adhering to heparin sulfate molecules bound to the cell surface. Research results on tissue culture cells suggest that gC is not absolutely necessary for viral infection, but the presence of gC can increase the efficiency of virus binding to cells by 10 times. gB, gD, gH, and gL are essential for the step of virus entry into the cell. However, in the presence of gL, they are no longer essential for the event of virus attachment to the cell surface. Therefore, based on the comprehensive experimental results, the function of gB, gD, gH, and gL is mainly to cause the fusion of the viral envelope and the cell envelope, and the main glycoprotein that can interact with the cell's fusion receptors and initiate the fusion process is gD.

[0011] From the perspective of HSV-1's human host cells, there are at least three receptors involved in HSV-1 entry: the herpes simplex virus entry mediator HVEM, a member of the tumor necrosis factor receptor family; and nectin-1 and nectin-2, members of the immunoglobulin superfamily. All of these receptors are capable of binding to gD1 and inducing viral entry. Based on biological experiments, relevant research further explores the topological mechanisms of HSV-1 gD1 binding to these receptors. Some analyses of the spatial structure of gD1 and HVEM have provided a detailed interpretation of their three-dimensional conformations. In particular, some dynamic analyses have shown that the spatial conformations of gD1 when it exists alone and when it is bound to HVEM are different. The thermodynamic reason for this difference in spatial structure is determined by the sequence of approximately 32 amino acids at the amino terminus of the gD1 molecule. Amino acids 7 to 15 and 24 to 32 in this sequence are important sites for gD1 to bind to HVEM, and are also the sites that control nectin-2 recognition, but do not affect nectin-1 recognition. Downstream of this sequence is the site that binds nectin-1 and nectin-2. From a topological point of view, this sequence forms a hairpin structure.

[0012] Further epitope analysis showed that there are at least 12 regions in the structure of HSV-1gD1 molecule that may contain CD4 + T cell epitopes are approximately 27 to 34 amino acids in length, and the corresponding CD4 + T cell response, both have protective significance, CD4 + T cell response plays an important role in resisting HSV-1 virus infection and determines the intensity and scope of specific humoral immunity and specific cellular response.

[0013] Studies have shown that CD8 + The importance of T cell responses in HSV-1 infection was highlighted in an analysis of patients co-infected with HSV-1 and HSV-2, in which HSV-1-specific CD8 + The number of T cells is negatively correlated with the severity of herpes lesions in the genital area. + T cell infiltration usually occurs in CD4 + After the infiltration of T cells, and the recurrence site of such genital herpes lesions, CD8 + T cells are enriched. This CD8 + The enrichment of T cells is often associated with the possibility that the virus can be cleared from the local area in a short time. These phenomena indicate that the CD8 T cells induced during herpes virus infection +T cell response is of great significance in the body's control and clearance of viruses. Studies on corneal damage caused by HSV-1 infection have shown that CD8 + T and CD4 + T is equally important in the body's protective response to corneal damage. Further research found that when HSV-1 enters the sensory neurons of the trigeminal nerve from the periphery, it enters the latent infection stage of the virus. At this time, immunopathological tests show that CD8 + T cells are present in large numbers around the trigeminal nerve, which helps stabilize the virus in a latent state and prevents it from being activated. The corresponding tissue culture model of trigeminal ganglion cells infected with HSV-1 showed that in CD8 + In the presence of T cells, the virus can express some immediate early genes, but cannot express early genes and late genes. + The specific antibodies of T cells can reactivate the virus from the latent nerve cells after only 5 days. + T cells may be activated by IE proteins expressed by viruses in latent infection in nerve cells, and activated CD8 + T in turn forms an effective surveillance for the latent virus. Once the related virus is reactivated in the nerve cells, that is, the virus replicates and proliferates, these CD8 + T cells may destroy and kill infected nerve cells in the form of specific CTL response. Similar results were obtained with HSV-2. Therefore, a drug that can induce CD8 + Vaccines that stimulate T cell responses are the key to solving the problem.

[0014] Existing data indicate that while human herpes viruses can establish a latent infection during infection, clinically leading to chronic, persistent infections that are difficult to completely eliminate from the body, the body can nonetheless mount a comprehensive immune response to these viral infections. Based on the principles and foundations of these immune responses, some vaccine attempts have proven feasible. The HSV-1 vaccine is the most ideal method for preventing herpes simplex virus type 1, but to date, no vaccine with a robust immune response has been developed.

[0015] Inactivated virus vaccines are the most classic approach. In 1992, researchers attempted to inactivate HSV-infected rabbit brains and other animal tissues using formalin, then extract the whole virus to prepare a vaccine. Inactivated viruses retain all antigenic molecules and can elicit a broad immune response, but these vaccines are not highly immunogenic and offer limited protection. Other inactivation methods, such as heating, chemical treatment, and ultraviolet irradiation, have also been tried without success.

[0016] HSV-1 and VZV are both alpha herpes viruses. The HSV-1 live attenuated vaccine route was studied with reference to the varicella live attenuated vaccine. Researchers isolated HSV-1 virus from clinical samples and continuously passaged it on human diploid cells and guinea pig embryonic fibroblasts to obtain attenuated strains, such as KOS-63. The attenuated strains showed obvious low neurotoxicity in the rabbit model, but could still lurk in the nervous system and cause recurrent infection. To address this problem, the viral genome was edited to weaken the virulence and reduce the ability to establish latent infection and relapse, while maintaining proliferation ability and immunogenicity. However, studies have shown that repeated passage of the attenuated strain in cells may restore toxicity.

[0017] Subunit vaccines have a relatively simple antigen composition, are easy to prepare, have good tolerance, are highly safe, and have no risk of latent infection. However, the effectiveness of subunit vaccines against latent infection and recurrent infection is still unclear.

[0018] The replication-restricted vaccine approach has garnered significant attention. A gK gene-deficient strain has been constructed that induces a long-lasting T cell response in mice. Immunized mice effectively suppress disease development in genital tract challenge experiments, with a protection rate of up to 90%. Replication-restricted strains are less virulent and do not replicate in vivo, thus inducing effective immune protection. However, these strains must be grown in genetically modified cells containing a gene that is missing its expression. This preparation method directly limits their application. Genetically modified cells carry a certain risk of carcinogenesis, and passage of the strain in modified cells also carries the risk of regaining virulence.

[0019] Live vector vaccines involve inserting genes expressing HSV-1-associated antigens into replicable viral or bacterial vectors. Following immunization, the viral or bacterial vector expresses the corresponding antigenic proteins, inducing a specific immune response. Vaccinia virus, adenovirus, and Salmonella have been the most studied. Other studies have involved cloning the gD1 protein gene into a bacteriophage genome. The gD1 protein is then fused to a phage surface protein, displaying it on the phage surface. Immunizing mice with these recombinant phage particles results in the gD1 antigen being presented by cells, inducing a specific immune response. Phages offer excellent safety as vectors, are easily mass-produced, can induce an immune response, and avoid issues such as latent HSV-1 toxicity. However, many key issues regarding phage-vectored vaccines, such as dosing and administration, remain unclear.

[0020] Nucleic acid vaccines are also a research hotspot. These involve inserting antigen genes into eukaryotic expression vectors and direct inoculation to express the antigen in vivo. One study constructed the HSV-1 gD1 and human interleukin-12 genes onto DNA and encapsulated the DNA vaccine in nanoparticles. Immunization of mice with the cornea produced high levels of specific neutralizing antibodies. Secretory IgA in tears, and levels of IFN-γ and IL-4 in serum were significantly elevated. The cytotoxicity of splenocytes and natural killer (NK) cells was significantly enhanced. Furthermore, herpes simplex stromal keratitis in mice was significantly reduced after challenge with a wild-type strain.

[0021] Definition of immune complex: The minimum composition of an immune complex molecule is at least one antigen combined with two antibody molecules. The complex formed during infection can amplify the immune response by targeting antigens to Fc receptors on presenting cells, activate complement, or sequester large amounts of antigens from the blood circulation to the liver or spleen. Fc fusion proteins have been widely used as the backbone of antibody derivatives, such as antibody-drug conjugates and antibody-cytokine conjugates, and have shown good efficacy and no adverse reactions in humans. There are many advantages to using Fc fusion technology to develop vaccines: 1. Antigens can be easily purified using protein A / G affinity chromatography; 2. Fc promotes the correct folding of fusion proteins and binding to APC cells, increasing the immunogenicity of the antigen; 3. Fc fusion proteins can improve the solubility and stability of recombinant immunogens and extend their half-life.

[0022] However, the use of antibodies alone or the Fc domain of the antibody heavy chain constant region to deliver antigens to immune cells has a major limitation. Because they are monomers containing only one Fc, they can only bind to the high-affinity FcγRI (CD64) but cannot cross-link multiple FcγRs or bind to the low-affinity FcγRII (CD32) and FcγRIII (CD16). FcγRI is primarily expressed on monocytes and neutrophils, where it binds ligands with high affinity, monomeric IgG, and immune complexes. FcγRII is expressed on every cell bearing FcγRs (except NK cells), binds ligands with low affinity, and promotes phagocytosis / endocytosis of immune complexes and B cell activation. FcγRIII is expressed on macrophages, NK cells, myeloid progenitor cells, and neutrophil lineages. FcγRIII expression on macrophages is regulated by IFN-γ, mediating antibody-dependent cellular cytotoxicity (ADCC). FcγRIIA and FcγRIII are crucial for enhancing immune responses. Classic immune complexes are formed by the binding of antigen molecules within the body to numerous antibody molecules. These complexes contain multiple Fc domains and can enhance antigen uptake by increasing binding activity to all FcγRs. This is because polyclonal antibodies bind to the same antigen to form multimers, and numerous Fc domains bind to FcRs on the surface of APCs, inducing receptor cross-linking and promoting the internalization of the complex by APCs. The antigen is then processed and presented to T cells.

[0023] Monoclonal antibodies can partially replicate this process: when antibodies bind to intact microorganisms and the surface antigen density is high enough, FcγR cross-linking is permitted. The soluble antigens in conventional subunit vaccines, however, lack this ability. Similarly, Fc fusion proteins cannot form multimeric complexes, and therefore lack the ability to enhance immune responses when used as vaccines. Despite the many advantages of Fc fusion proteins, the genetic engineering of immune complexes containing two or more Fc proteins remains a significant obstacle, hindering the development of immune complex vaccines. To date, numerous approaches have been explored internationally, and these studies have demonstrated that genetically engineering the expression of antigens and antibodies, followed by their combination to form immune complexes, is a highly attractive immunization strategy. The greatest challenges currently lie in the molecular design of immune complexes, their efficient expression and purification, and the need for large-scale production under GMP-compliant conditions. Summary of the Invention

[0024] In view of this, the present invention provides a recombinant herpes simplex virus type I vaccine and its application.

[0025] To achieve the above object, the present invention adopts the following technical solutions:

[0026] The present invention provides a fusion protein consisting of four antigen molecules gD1, two human immunoglobulin antibody Fc domains, and two human IgG1 hinge region fragments;

[0027] The C-termini of the antigen molecule gD1 peptide chains are respectively connected to the N-termini of the antibody Fc domain peptide chains, and the N-termini of one peptide chain in the antigen molecule gD1 connected to the antibody Fc domain are connected in series with a human IgG1 hinge region fragment; the two human IgG1 hinge region fragments are connected in the form of an interchain disulfide bond;

[0028] The amino acid sequence of the antigen molecule gD1 is shown in SEQ ID NO.6;

[0029] The amino acid sequence of the human IgG1 hinge region is shown in SEQ ID NO.21;

[0030] The sequence SEQ ID NO.6 and the sequence SEQ ID NO.21 are connected in series via a linker.

[0031] Furthermore, the linker sequence is 10-20 aa in length, and the linker sequence is an amino acid sequence comprising a combination of glycine and serine.

[0032] Furthermore, the linker sequence is shown as SEQ ID NO.22.

[0033] Furthermore, the human immunoglobulin antibody Fc is selected from any one of human IgG, IgM, IgD, IgA and IgE.

[0034] Furthermore, the human IgG antibody is selected from any one of IgG1, IgG2, IgG3 and IgG4.

[0035] Furthermore, when the amino acid sequences of the two IgG1 antibody Fc domain peptide chains are identical, the amino acid sequence is as shown in SEQ ID NO. 10;

[0036] When the amino acid sequences of the two IgG1 antibody Fc domain peptide chains are different, one of the Fc peptide chains contains two mutation points S354C and T366Y, and the sequence is shown in SEQ ID NO.8; the other Fc peptide chain also contains two mutation points Y349C and Y407T, and the sequence is shown in SEQ ID NO.9.

[0037] The present invention also provides the use of the fusion protein in preparing a recombinant herpes simplex virus type I vaccine.

[0038] The present invention also provides a recombinant herpes simplex virus type I vaccine comprising the above-mentioned fusion protein.

[0039] Furthermore, the vaccine also includes pharmaceutically acceptable aluminum hydroxide adjuvant, squalene, QS-21, oligonucleotide or one or more immune stimulants.

[0040] In addition, the present invention also provides the use of the vaccine in preparing a product for preventing and / or treating herpes simplex virus type I infection.

[0041] The present invention is a recombinant herpes simplex virus type I (HSV-1) vaccine. The antigen used is a viral protein, preferably the viral outer membrane glycoprotein D (hereinafter referred to as gD1). The immune complex is designed as follows: the C-termini of two gD1 polypeptide chains are respectively connected to the Fc of human immunoglobulin to form a gD1-Fc fusion protein, wherein the N-terminus of one peptide chain contains at least two cysteines to form an interchain disulfide bond. After the fusion protein is expressed in mammalian cells, two or four Fc single chains can assemble into dimers or tetramers; the tetrameric gD1-Fc fusion protein contains four gD1 polypeptide chains and two Fc domains.

[0042] In order to achieve covalent bond connection of the structural units, an amino acid sequence containing DKTHTCPPCPAPELLGGGSTSGSGKPGSGEG (SEQ ID NO. 1) is fused to the N-terminus of one of the chains. The sequence contains two cysteine ​​residues, and the 14 amino acids at the C-terminus of the sequence can be replaced by a sequence containing a combination of glycine and serine.

[0043] The constructed fusion protein containing two Fcs is similar to the immune complex formed by the natural antigen and two antibody molecules in the body (a quasi-immune complex, without the Fab part of the antibody). After immunization, it can quickly bind to the two FcγRs on the surface of antigen-presenting cells in the body, realize active antigen presentation, and initiate humoral and cellular immune responses.

[0044] The number of Fc domains is ≥2, and two or more Fc domains bind to receptors on the surface of immune cells, subsequently initiating intracellular signal transduction, and corresponding antigen presentation and immune response.

[0045] The heterodimer gD1_kFc sequence is SEQ ID No. 2, and the gD1_hFc sequence is SEQ ID No. 3. The homodimer gD1_Fc chain sequence is SEQ ID No. 4, and the SS_gD1_Fc chain sequence is SEQ ID No. 5.

[0046] The prepared high-purity antigen can activate CD4 + T cells, CD8 +T cells induce the body's immune system to produce corresponding specific antibodies and cellular immunity to prevent infection caused by HSV-1.

[0047] The present invention designs a recombinant HSV-1 herpes simplex virus vaccine, which is a unique immune complex-like antigen molecule. Each molecule contains four viral glycoprotein antigen molecules gD1 and two human IgG1Fc domains. The polypeptide chains of the preferred Fc domain are asymmetric, with a knob-in-hole design, which is easy to form heterodimers. Preferably, a short peptide sequence DKTHTCPPCPAPELLGGGSTSGSGKPGSGEG (SEQ ID NO.1) is fused and expressed in series at the N-terminus of one of the peptide chains. The sequence can form two interchain disulfide bonds, and the two immune complex molecules are coupled together through the interchain disulfide bonds to construct a complete vaccine antigen molecule containing two Fcs.

[0048] In the present invention, the two peptide chains constituting the quasi-immune complex are both derived from viral outer membrane proteins at the N-terminus, and the C-terminus is the Fc fragment of human immunoglobulin. The two peptide chains may be identical or different, with one peptide chain having the aforementioned structure capable of forming an interchain disulfide bond at its N-terminus, while the other does not. Alternatively, the peptide chain may be derived from a viral tegument protein, which is the primary antigenic epitope region of the present invention and capable of providing an effective immune stimulant. The peptide chain may be a viral structural protein or a viral non-structural protein. The present invention preferably uses the HSV-1 outer membrane glycoprotein gD1 as an example for discussion, although other effective HSV-1 antigens are also encompassed by the present invention.

[0049] In the design of the present invention, the constructed immune complex molecule is a human antibody constant region domain Fc connected to the C-terminus of the viral antigen peptide chain. In terms of the choice of Fc type, it can be derived from any of human IgG, IgM, IgD, IgA and IgE, preferably IgG. According to the requirements of antigen presentation, IgG antibody subtypes are also within the range of optimization selection, including IgG1, IgG2, IgG3 and IgG4. Different subtypes have different Fc receptor binding preferences. The present invention preferably uses IgG1 Fc as an example for technical description. The function of the Fc domain is to bind to immune cell receptors and complete antigen presentation of a specific pathway. Therefore, a portion of the IgG1 Fc constant region is intercepted for use in the present invention to meet the design requirements. Preferably, the amino acid sequence of the hinge region, CH2, and CH3 regions of the human IgG1 antibody is selected, and the two cysteines in the hinge region form an interchain disulfide bond, that is, a dimer is formed. Such constant regions appear in pairs, which are referred to as Fc domains in the present invention. The Fc domain preferably has a natural sequence that retains complete FcRs receptor binding activity. Of course, non-natural Fc sequences can also be selected, but it is crucial to retain FcRs binding activity while also retaining the amino acid sequence corresponding to complement system activation activity.

[0050] In the design of the present invention, the Fc domain can also adopt a symmetrical design, that is, not using the Kih design, and the molecular structure designed by the present invention can also be achieved. Specifically, the peptide chain SEQ ID No. 5 containing the sequence SEQ ID No. 1 and the peptide chain SEQ ID No. 4 not containing SEQ ID No. 1 are constructed into the same expression vector and transferred into CHO cells. The two peptide chains are co-expressed under the action of their respective promoters to obtain the antigen gD1 dimer. At the same time, a dual immune complex molecule containing dual Fc domains can also be obtained. According to test results, it also meets the design criteria of the present invention. However, in theory, the Kih design has a lower mismatch rate and a higher yield.

[0051] In summary, the molecules designed by the present invention have dual Fc domains, which bind to the surface receptors of APC presenting cells, promote the phagocytosis of gD1 antigen molecules by presenting cells, and activate CD4 + T, CD8 + T and produce neutralizing antibodies. Fc number ≥ 2, rapid presentation of antigens, activation of corresponding signaling pathways, and production of protective immunity. Experiments have shown that more manifestations are MHC I cross-presentation, activation of CD8 + T cells, according to the previous discussion, we know that activating CD8 + T is a crucial element for the herpes vaccine to work, and of course it also activates CD4 + T and B cell immune responses.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The pathogenic microorganism antigen fusion protein containing two Fcs of the present invention can present antigens in a targeted manner, activate the body's humoral immunity and cellular immunity, prevent HSV-1 infection and / or prevent reinfection and morbidity; the fusion protein can be expressed in mammalian cells, and the antigen molecules self-assemble into an immune complex-like fusion protein, which can be purified to prepare a vaccine for human or animal use. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Schematic diagram of the interchain disulfide bond formed by the short peptide sequence at the N-terminus of the antigen;

[0055] Figure 2 Schematic diagram of the molecular structure of LZ920 immune complex;

[0056] Figure 3 Schematic diagram of the molecular structure of LZ921 immune complex;

[0057] Figure 4 This is the SDS-PAGE electrophoresis diagram of the LZ920 vaccine antigen in Example 3;

[0058] Figure 5 This is the SDS-PAGE electrophoresis diagram of the LZ921 vaccine antigen in Example 3;

[0059] Figure 6 This is the SEC-HPLC purity test chart of the LZ920 vaccine stock solution in Example 4;

[0060] Figure 7 This is the SEC-HPLC purity test chart of LZ921 vaccine stock solution in Example 4;

[0061] Figure 8 This is a graph showing the antibody titer test results of serum from mice after immunization in Example 5;

[0062] Figure 9 CD4 in Example 8 + T-specific immune response results diagram;

[0063] Figure 10 CD8 in Example 8 + T-specific immune response results diagram; DETAILED DESCRIPTION

[0064] To better illustrate the present invention, the following embodiments are listed. Obviously, the embodiments described are only part of the present invention, not all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without making any creative efforts are also within the scope of protection of the present invention.

[0065] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that the numbers used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0066] The implementation of the present invention will be described below using HSV-1 gD1 as an example.

[0067] The amino acid sequences involved in the present invention are shown in Table 1 below:

[0068] Table 1 Amino acid sequences and primer sequences involved in the present invention

[0069]

[0070]

[0071]

[0072]

[0073] Example 1: Gene sequence design

[0074] The SS sequence SEQ ID No. 1: DKTHTCPPCPAPELLGGGSTSGSGKPGSGEG (31aa) contains two cysteine ​​residues. This sequence is extracted from the 17 amino acids DKTHTCPPCPAPELLGG (SEQ ID NO. 21) of the hinge region of human IgG1, and a 14 amino acid linker: GSTSGSGKPGSGEG (SEQ ID NO. 22) is added to the C-terminus. After expression, it can form an interchain disulfide bond, such as Figure 1 shown.

[0075] The gD1_kFc sequence SEQ ID No. 2 is a Knob chain, which is composed of the glycoprotein sequence gD1 SEQ ID No. 6 and the sequence kFc SEQ ID No. 8 chain. The wild-type gD1 (KOS standard strain) is 394 amino acids in length, forming a gB-gH / gL-gD complex, binding to the cell receptor HVEM, NECTIN-1 or heparan sulfate, and triggering cell membrane fusion. Amino acids 1 to 25 of the wild-type gD1 amino acid sequence are signal peptides, amino acids 26 to 340 are located on the surface of the virus particle, amino acids 341 to 361 are α-helical structures that constitute the transmembrane region, and amino acids 362 to 394 are inside the virus particle. The functional region of gD1 that interacts with glycoproteins gB and gH / gL to trigger membrane fusion is located within 26 to 340 amino acids. This region contains the key antigenic determinant sequence for viral infection. Referring to the technical solutions of other R&D teams in this field, starting from the 26th amino acid residue, 307 amino acids were intercepted as the candidate antigen sequence of the vaccine of the present invention. The sequence contains three intrachain disulfide bonds, 91<->214, 131<->227, and 143<->152, and three N-glycosylation sites, at amino acid residues 119, 146, and 287.

[0076] The kFc peptide chain attached to the C-terminus of the gD1 glycoprotein is a knob chain. This sequence is derived from the constant region of the human IgG1 heavy chain and comprises the hinge, CH2, and CH3 regions. The cysteine ​​residue connecting the wild-type CH1 region to the light chain has been removed to reduce the likelihood of disulfide bond mispairing. Additionally, two amino acids in the CH3 region, S354C and T366Y, have been mutated. The T366Y mutation has a larger side chain, forming a "knob"-like protrusion that facilitates proper kFc pairing.

[0077] The SS_gD1_hFc sequence (SEQ ID No. 3) is a hole chain, consisting of three parts: SS SEQ ID No. 1, gD1 SEQ ID No. 6, and hFc SEQ ID No. 9, starting from the N-terminus. The hFc is a hole chain containing two mutations: Y349C and Y407T. The Y407T mutation changes the large amino acid side chain of tyrosine (Y) to the smaller side chain of threonine (T), forming a "hole"-like concave structure that cooperates with the knob chain to facilitate proper hFc pairing.

[0078] KiH design is a classic technical approach, often used in bispecific antibody design to reduce the probability of mispairing. The KiH mutation scheme used in this invention is provided to better describe the technical method of this invention. This method can obtain the desired vaccine antigen structure. Other Fc mutation schemes can also achieve the goals of improving yield and reducing mispairing of the present invention and are also within the scope of protection of this invention.

[0079] Interestingly, when we do not use KiH design, but use the sequence gD1_Fc SEQ ID No.4 and the sequence SS_gD1_Fc SEQ ID No.5, the N-terminal short peptide SS SEQ ID No.1 of SEQ ID No.5 can also realize the interchain disulfide bond, and the correctly paired antigen can also be obtained. Both strategies are within the scope of protection of the present invention. Different Fc mutations have different effects on the binding activity of antigen molecules to cell receptors. The two strategies need to balance the yield and efficacy. See the schematic diagram of the molecular design structure for details. Figure 2 and Figure 3 .

[0080] Example 2: Construction of cloning plasmids

[0081] The amino acid sequence was converted into a nucleotide sequence, the gene codon was optimized, and Nanjing GenScript Biotech Co., Ltd. was commissioned to synthesize the sequence gD1 SEQ ID No.6, the sequence SS_gD1 SEQ ID No.7, the sequence kFc SEQ ID No.8, the sequence hFc SEQ ID No.9, and the sequence Fc SEQ ID No.10, and loaded into a cloning plasmid (the plasmid was selected from the commonly used plasmids in the GS Xceed system). BGI was commissioned to synthesize primers SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, and SEQ ID No.20. The specific sequences are shown in Table 1 above.

[0082] Construction of SEQ ID No. 2 expression vector Ek2: Using the plasmid containing SEQ ID No. 6 as a template, primers SEQ ID No. 12 and SEQ ID No. 18 were used to PCR amplify the target gene fragment kP1. Using the plasmid containing SEQ ID No. 8 as a template, primers SEQ ID No. 17 and SEQ ID No. 16 were used to PCR amplify the target gene fragment kP2. The amplification conditions were 98°C for 10 s, 55°C for 5 s, and 72°C for 7 s for 35 cycles, and extension at 72°C for 10 min.

[0083] The two products were linked using overlap extension PCR. Using primers with complementary ends, the PCR products formed overlapping strands. This allowed the amplified fragments from different sources to be spliced ​​together by extension in the subsequent amplification reaction. 15 μl of each PCR product, kP1, and kP2, were mixed, and 15 μl of PrimeSTAR Polymerase and 15 μl of water for injection were added. PCR was repeated using the following reaction conditions: 10 cycles of 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 15 seconds, followed by extension at 72°C for 10 minutes. The resulting spliced ​​product was kP3.

[0084] The expression vector plasmid was transformed into DH5α and positive clones were screened. Single colonies were randomly selected and expanded for culture. The plasmid was extracted using a commercial kit and double-digested with restriction endonucleases (HindIII / EcoRI), and the vector fragment was recovered. The KP3 target gene fragment prepared by PCR was mixed with the double-digested vector fragment (HindIII / EcoRI). Exnase II homologous recombinase and buffer were added. The reaction was carried out in a gene amplification instrument at 37°C for 30 minutes. The reaction was immediately ice-bathed and then mixed with DH5α competent cells that had also been thawed in an ice-bath. The cells were heat-shocked at 42°C for 90 seconds. An appropriate amount of LB medium was added and incubated at 37°C for 45 minutes. The cells were then plated on resistance plates and positive clones were screened.

[0085] Construction of SEQ ID No. 3 expression vector Eh3: Using the plasmid containing SEQ ID No. 7 as a template, primers SEQ ID No. 11 and SEQ ID No. 20 were used to PCR amplify the target gene fragment hP1. Using the plasmid containing SEQ ID No. 9 as a template, primers SEQ ID No. 19 and SEQ ID No. 16 were used to PCR amplify the target gene fragment hP2. The amplification conditions were 98°C for 10 s, 55°C for 5 s, and 72°C for 7 s for 35 cycles, and extension at 72°C for 10 min.

[0086] The two products were joined using overlap extension PCR. 15 μl of each PCR product, hP1, and hP2, were mixed, and 15 μl of PrimeSTAR Polymerase and 15 μl of water for injection were added. PCR was repeated using 10 cycles of 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 15 seconds, followed by extension at 72°C for 10 minutes. The resulting spliced ​​product was hP3.

[0087] The expression vector plasmid was transformed into DH5α and positive clones were screened. Single colonies were randomly selected and expanded. Plasmids were extracted using a commercial kit and double-digested with restriction endonucleases (HindIII / EcoRI) to recover the vector fragment. The hP3 target gene fragment prepared by PCR was mixed with the double-digested vector fragment (HindIII / EcoRI). Exnase II homologous recombinase and buffer were added and the reaction was carried out in a gene amplification instrument at 37°C for 30 minutes. The reaction was immediately ice-bathed and then mixed with DH5α competent cells that had also been thawed in an ice-bath. The cells were heat-shocked at 42°C for 90 seconds. An appropriate amount of LB medium was added and incubated at 37°C for 45 minutes. The cells were then plated on resistance plates and positive clones were screened.

[0088] Construction of SEQ ID No. 4 expression vector EL4: Using the plasmid containing SEQ ID No. 6 as a template, primers SEQ ID No. 11 and SEQ ID No. 13 were used to PCR amplify the target gene fragment L1. Using the plasmid containing SEQ ID No. 10 as a template, primers SEQ ID No. 14 and SEQ ID No. 15 were used to PCR amplify the target gene fragment L2. The amplification conditions were 98°C for 10 s, 55°C for 5 s, and 72°C for 7 s for 35 cycles, and extension at 72°C for 10 min.

[0089] The two products were joined using overlap extension PCR. 15 μl of each PCR product, L1, and L2, were mixed, and 15 μl of PrimeSTAR Polymerase and 15 μl of water for injection were added. PCR was repeated using the following reaction conditions: 10 cycles of 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 15 seconds, followed by extension at 72°C for 10 minutes. The resulting joined product was L3.

[0090] The expression vector plasmid was transformed into DH5α and positive clones were screened. Single colonies were randomly selected and expanded for culture. The plasmid was extracted using a commercial kit and double-digested with restriction endonucleases (HindIII / EcoRI) to recover the vector fragment. The L3 target gene fragment prepared by PCR was mixed with the double-digested vector fragment (HindIII / EcoRI). Exnase II homologous recombinase and buffer were added and the reaction was carried out in a gene amplification instrument at 37°C for 30 minutes. The reaction was immediately ice-bathed and then mixed with DH5α competent cells that had also been thawed in an ice-bath. The cells were heat-shocked at 42°C for 90 seconds. An appropriate amount of LB medium was added and incubated at 37°C for 45 minutes. The cells were then plated on resistance plates and positive clones were screened.

[0091] Construction of SEQ ID No. 5 expression vector ES5: Using the plasmid containing SEQ ID No. 7 as a template, primers SEQ ID No. 12 and SEQ ID No. 13 were used to PCR amplify the target gene fragment H1. Using the plasmid containing SEQ ID No. 10 as a template, primers SEQ ID No. 14 and SEQ ID No. 15 were used to PCR amplify the target gene fragment H2. The amplification conditions were 98°C for 10 s, 55°C for 5 s, and 72°C for 7 s for 35 cycles, and extension at 72°C for 10 min.

[0092] The two products were joined using overlap extension PCR. 15 μl of each PCR product, H1, and H2, were mixed, and 15 μl of PrimeSTAR Polymerase and 15 μl of water for injection were added. PCR was repeated using the following reaction conditions: 10 cycles of 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 15 seconds, followed by extension at 72°C for 10 minutes. The resulting joined product was H3.

[0093] The expression vector plasmid was transformed into DH5α and positive clones were screened. Single colonies were randomly selected and expanded for culture. The plasmid was extracted using a commercial kit and double-digested with restriction endonucleases (HindIII / EcoRI) to recover the vector fragment. The H3 target gene fragment prepared by PCR was mixed with the double-digested (HindIII / EcoRI) vector fragment, and Exnase II homologous recombinase and buffer were added. The reaction was carried out in a gene amplification instrument at 37°C for 30 minutes. The reaction was immediately ice-bathed and then mixed with DH5α competent cells that had also been thawed in an ice-bath. The cells were heat-shocked at 42°C for 90 seconds. An appropriate amount of LB medium was added and incubated at 37°C for 45 minutes. The cells were then plated on resistance plates and positive clones were screened.

[0094] The above positive clones were amplified and plasmids Ek2, Eh3, EL4 and ES5 were prepared in large quantities. Double enzyme digestion (PvuI / NotI) was performed and the linearized expression vectors Ek2_P / N, Eh3_P / N, EL4_P / N and ES5_P / N were recovered by gel excision.

[0095] Ek2_P / N and Eh3_P / N were mixed together, DNA ligase and reaction buffer were added, and the reaction was allowed to proceed overnight at 2-8°C. The ligation product was then transformed into DH5α competent cells to prepare a dual-expression plasmid. Plasmids were extracted in large quantities using a commercial kit and digested with the restriction endonuclease PvuI to linearize the dual-expression vector, thus completing the construction of the heterodimer expression gene cassette, designated 921.

[0096] EL4_P / N and ES5_P / N were mixed together, DNA ligase and reaction buffer were added, and the reaction was allowed to proceed overnight at 2-8°C. The ligation product was then transformed into DH5α competent cells to prepare a dual-expression plasmid. Plasmids were extracted in large quantities using a commercial kit and digested with the restriction endonuclease PvuI to linearize the dual-expression vector. A homodimer expression gene cassette, designated 920, was constructed.

[0097] Example 3 Construction of high expression engineered cell lines

[0098] Establishment and screening of stable clones: Prepare 0.8 ml of CHO K1 cell suspension (1.5×10 6 Cells / ml) were added, 20 μg of linearized plasmid and transfection reagent were added in sequence, and the perforation voltage of the Gene Pulser Xcell (Bio-Rad) was set to 300 V, 900 μF single pulse, infinite resistance, and a disposable shock cup (Bio-Rad) with a gap of 4 mm. The shock time was 12-20 ms. The cells in the shock cup were transferred to a culture flask, 30 ml of CHO culture medium was added, and the cells were cultured on a shaker at 37°C and 5% CO2 at 136 rpm. After culturing for 24 hours, the cells were collected by low-speed centrifugation at 800 rpm and replaced with 50 μM MSX CHO culture medium (without glutamine) was used for pressure screening. The cells were transferred into 96-well flat-bottom culture plates by limiting dilution. The culture plates were cultured in a 37°C, 5% CO2 incubator and observed under an inverted microscope. The monoclonal cell wells were marked. Then, positive cells in 24-well and 6-well cell culture plates were screened in sequence by ELISA. Monoclonal strains with higher expression levels were screened, and continuous subculture and testing were performed to finally obtain cell clones with high expression of the target gene.

[0099] The positive cell line obtained by transfecting CHO K1 with linearized plasmid 920 was collected, the culture supernatant was collected, and the antigen was captured and purified by affinity chromatography, and identified by SDS-PAGE electrophoresis. The results are shown in Figure 4 The cell line that met the standards was named LZ920; the positive cell line obtained by transfecting CHO K1 with linearized plasmid 921 was collected, the culture supernatant was collected, and the antigen was captured and purified by affinity chromatography, and identified and tested by SDS-PAGE electrophoresis. The results are shown in Figure 5 The cell line that met the standards was named LZ921.

[0100] Example 4: Fermentation of engineered cells and purification of vaccine antigens

[0101] After the cell line is expanded and cultured, it is inoculated into a 2L Erlenmeyer flask containing 500ml of CHO cell culture medium and cultured on a shaker at 37°C and 5% CO2 at 130-140 rpm. After 4-5 days of culture, the cell line is transferred to a bioreactor for culture. Reactor culture conditions: initial inoculation density of the reactor: 500,000-1,000,000 cells / ml, culture temperature 37°C, pH 6.5-7.5, dissolved oxygen 50%±20%, stirring speed 50-120 rpm, and intermittent feeding (such as sugar, culture medium or alkali solution) is used during the culture period. Samples are taken every day to check the cell count and cell activity. Generally, the culture is carried out for 12-14 days. When the proportion of viable cells drops below 85%, the fermentation culture is stopped and the fermentation broth is harvested.

[0102] Centrifuge the harvested fermentation broth at 10,000 rpm for 30 minutes to remove large insoluble particles such as cell debris, or use a depth filter to remove cells and cell debris, and collect the cell culture supernatant. Filter the culture supernatant through a 0.45 μm clarification filter. The filtered intermediate can be stored at 2-8°C or directly transferred to the next purification step.

[0103] The filtered harvested fluid can be directly loaded onto a protein A affinity chromatography gel column (ProteinAt Bead LX, Mabselect Sure LX, etc.) for purification. The affinity chromatography column is equilibrated with 20mM PBS (pH 7.4, containing 135mM NaCl) buffer. After loading, the column is washed with 20mM PBS buffer until the UV detector display returns to near the baseline; then, it is dissociated with 0.1mol / L citric acid (pH 3.5), and the elution peak is collected based on the A280 absorbance value. The protein purity after affinity chromatography reaches more than 90%. The affinity chromatography fluid is incubated at room temperature for 60 minutes to inactivate the virus, and then an appropriate amount of 2MTris solution is added to neutralize it to a pH of 7.2-7.6.

[0104] Load the neutralized affinity chromatography solution directly onto a pre-packed Sephacryl S-400HR (or Sepharose 4 Fast Flow, or Sephadex 200PG) gel chromatography column, with the sample volume not exceeding 2% of the column bed volume. Use 40 mM phosphate buffer (pH 7.4, containing 150 mM NaCl) as the mobile phase. Collect the column effluent in sections as the absorbance on the UV detector A280 increases. Stop collecting samples when the absorbance decreases close to the baseline level. Combine the collected fractions with a purity exceeding 95%.

[0105] The purified liquid was diluted with purified water to 4 times of its original volume and loaded onto the equilibrated DEAE Sepharose 4 Fast Flow column. After loading, the column was washed with equilibration buffer for 3 column volumes, then eluted with a liquid containing an appropriate sodium chloride concentration and the eluate was collected.

[0106] The eluate was filtered through a 20 nm pore size virus removal filter connected to a depth filter, and then filtered through a 0.2 μm sterilization filter membrane to obtain the vaccine stock solutions, which were named LZ920 antigen protein stock solution and LZ921 antigen protein stock solution, respectively. Samples were taken for SEC-HPLC purity test. The test profile is detailed in Figure 6 and Figure 7 The results showed that the HPLC purity reached more than 94%, which met the design standards and satisfied the needs of subsequent efficacy evaluation.

[0107] Example 5: Mouse macrophage phagocytosis assay

[0108] Macrophage Isolation and Culture: Randomly select BALB / c mice and intraperitoneally inject 5 ml of 1640 culture medium without fetal bovine serum. Gently massage the abdomen for 2-3 minutes, let it rest for 5 minutes, and then extract the peritoneal fluid with a sterile syringe. Centrifuge at 1000 rpm for 5 minutes, wash the cells twice with 1640 culture medium, and resuspend the macrophages in 1640 culture medium supplemented with 10% fetal bovine serum. Seed the cells in a 96-well plate and culture in a 37°C, 5% CO2 incubator for 12 hours, after which the culture medium is replaced.

[0109] FITC-labeled LZ920 antigen protein, LZ921 antigen protein, LZ901 protein (VZV gE-Fc fusion protein containing two Fc residues, positive control), and IgG (negative control) were diluted in 1640 culture medium to a concentration of 10 μg / 100 μl. The diluted LZ920-FITC, LZ921-FITC, LZ901-FITC, and IgG-FITC were added to a 96-well cell culture plate seeded with macrophages, with 100 μl per well. Place the 96-well cell culture plate in a 37°C, 5% CO2 incubator and incubate for 2 hours. Wash twice with 20mM PBS and add 100μl of RBITC-labeled CD68 polyclonal antibody (100-fold dilution). This antibody can specifically bind to macrophages and show red fluorescence. After incubation for 1 hour in a 37°C, 5% CO2 incubator, wash twice with 20mM PBS and place the 96-well cell culture plate under a fluorescence microscope to photograph and record. The results of the macrophage phagocytosis of FITC-labeled LZ920 and LZ921 antigen protein stock solution are shown in the figure. Figure 8 .

[0110] The results showed that after adding LZ920-FITC and LZ921-FITC to macrophages, the LZ920 and LZ921 antigen proteins were phagocytosed by the macrophages, producing obvious green fluorescence. However, the control group with IgG-FITC did not produce obvious fluorescence, indicating that they were not phagocytosed by macrophages.

[0111] Experimental results show that the two Fc fragments of LZ920 and LZ921 protein concentrates can bind to Fc receptors on the surface of macrophages, activate macrophages to initiate phagocytosis, further play the role of macrophage antigen presentation, and initiate specific immune responses.

[0112] Example 6: Mouse Immunization Experiment

[0113] The vaccine stock solution was diluted with aluminum hydroxide adjuvant to prepare vaccines containing 20 μg / 0.5 ml, 10 μg / 0.5 ml, 5 μg / 0.5 ml, 2 μg / 0.5 ml, 1 μg / 0.5 ml, and 0.5 μg / 0.5 ml of LZ920 and LZ921 antigens, respectively. Female BLAB / c mice aged 4 to 7 weeks were randomly divided into 14 groups of 5 mice each. Each mouse in the control group received an intraperitoneal injection of 0.5 ml of aluminum adjuvant.

[0114] The six experimental groups of LZ920 antigen were intraperitoneally injected with 0.5 ml of antigen containing 20 μg, 10 μg, 5 μg, 2 μg, 1 μg, and 0.5 μg respectively. After the first immunization, they were immunized again at an interval of 3 weeks, for a total of 2 immunizations. Blood was collected from the tail vein before the second immunization, and the serum was separated and frozen below -20°C. Blood was collected from the retro-orbital vein 14 days after the second immunization, and the serum was separated and frozen below -20°C.

[0115] The six experimental groups for the LZ921 antigen were administered in the same manner as the LZ920 group. A third immunization was performed three weeks after the second immunization, for a total of three immunizations. Blood was collected from the tail vein before the second and third immunizations, and the serum was separated and frozen below -20°C. Fourteen days after the third immunization, blood was collected from the retro-orbital vein, and the serum was separated and frozen below -20°C. Splenectomy was also performed to isolate lymphocytes. Detailed groupings are shown in Table 2.

[0116] Table 2 Immunization groups and immunization procedures of LZ920 and LZ921 mice

[0117]

[0118]

[0119] Example 7: Serum Antibody Titer Determination (ELISA)

[0120] The recombinant HSV gD1-His protein was diluted to 0.2 μg / ml with carbonate buffer and coated on a 96-well ELISA plate with 100 μl per well. After incubation at 37°C for 2 hours, the plate was incubated at 2-8°C overnight. The liquid in the 96-well plate was discarded and the plate was washed three times with 20 mM PBS-T. After that, 200 μl of blocking solution (2% bovine serum albumin solution) was added to each well and the plate was blocked at 37°C for 60 minutes. The blocking solution in the well was aspirated and the plate was washed three times with 20 mM PBS-T solution. Mouse serum pre-diluted at 1:100 (or 1:1000) was added to the first column of the 96-well plate and then serially diluted by 2 times. The negative control was the serum of mice immunized intraperitoneally with aluminum adjuvant only (1:100). After 60 minutes of reaction at 37°C, the blocking solution in the well was aspirated and the plate was incubated with 20 mM Wash three times with PBS-T solution, add 1:100 diluted goat anti-mouse IgG-HRP conjugate, 100 μl per well, and react at 37°C for 60 minutes; aspirate the liquid in the wells, wash three times with 20mM PBS-T solution, add 100 μl of TMB colorimetric solution to each well, and after 10 minutes, add 50 μl of stop solution to terminate the reaction. Then, measure the A450 absorbance of each well using a TECAN Infinit200 microplate reader. The cutoff value is 3 times the mean absorbance of the negative control (aluminum hydroxide adjuvanted mouse serum) (if the absorbance value is less than 0.1, it is calculated as 0.1). The maximum dilution corresponding to the mean absorbance of the mouse immune serum is higher than the cutoff value, which is the mouse immune serum antibody titer.

[0121] The geometric mean and standard deviation of anti-HSV gD antibody titers in the serum of mice treated with LZ920 are shown in Table 3. With the exception of one mouse in the 20 μg / 0.5 ml / mouse group that failed to convert to positive status after the first immunization, the seroconversion rate of BALB / c mice in all other dose groups was 100%. After the second immunization, all mice converted to positive status. Statistical analysis of antibody titers measured in animals across experimental groups showed that after the first immunization, antibody titers were essentially consistent across all dose groups, ranging from 1131 to 2111. After the second immunization, with the exception of the 10 μg / 0.5 ml / mouse group, which showed individual differences, serum antibody titers in all other mice ranged from 128,000 to 512,000. This indicates that serum antibody titers in BALB / c mice significantly increased three weeks after the second immunization with LZ920 and reached a stable level, with no significant differences in serum antibody titers among the groups. There was no significant difference in the serum antibody titer of BALB / c mice immunized with LZ920 at a dose of 0.5 μg / 0.5 ml / mouse compared with other immunization doses, indicating that the 0.5 μg / 0.5 ml / mouse dose group had a strong immunogenicity.

[0122] Table 3 Results of serum antibody titer test of BALB / c mice in the LZ920 experimental group (ELISA)

[0123]

[0124] The geometric mean and standard deviation of anti-HSV gD antibody titers in the serum of mice in the LZ921 experimental group are shown in Table 4. With the exception of the 2μg / 0.5ml / mouse and 0.5μg / 0.5ml / mouse groups, which had seroconversion rates of 80% and 60% after the first immunization, the seroconversion rates of BALB / c mice in all other dose groups were 100%. After the second and third immunizations, the seroconversion rates of BALB / c mice in all dose groups were 100%. Statistical analysis of the antibody titers measured in the animals across the experimental groups showed that antibody titers were essentially the same across all dose groups after the first immunization, ranging from 528 to 2425. After the second immunization, antibody titers in all dose groups further increased and reached a relatively stable level. There was essentially no significant difference in serum antibody titers among the groups, ranging from 97,006 to 256,000. After the third immunization, antibody titers in all dose groups increased significantly compared to those after the second immunization. Serum antibody titers did not differ significantly between groups, ranging from 844485 to 1688970. In summary, serum antibody titers in BALB / c mice immunized with LZ921 at a dose of 0.5 μg / 0.5 ml / mouse were essentially unchanged compared to those in the other doses, indicating that the 0.5 μg / 0.5 ml / mouse dose exhibited strong immunogenicity.

[0125] Table 4 Results of serum antibody titer detection (ELISA) of BALB / c mice after immunization in the LZ921 experimental group

[0126]

[0127]

[0128] Example 8: CD4 + T and CD8 + T cell response detection

[0129] Extraction of spleen lymphocytes from mice in the LZ921 experimental group: The mice were killed by cervical dislocation and immersed in 75% ethanol. The spleen of the mouse was removed in a clean bench. 4ml-5ml of mouse lymphocyte separation solution was added to the grinder (return to room temperature and shake well before use). After grinding, the separation solution containing the spleen cells was immediately transferred to a 15ml centrifuge tube and covered with 1000μl of RPMI1640 culture medium (keeping the liquid surface boundary clear). Centrifuge at room temperature and 2500rpm for 30min. Set a slower acceleration and deceleration. After the centrifugation, the cells were separated and the lymphocyte layer was aspirated. 10ml of RPMI1640 culture medium was added and washed upside down. Centrifuge at room temperature and 1500rpm for 10min to collect the cells, resuspend them and count them.

[0130] Cytokine detection: Blood lymphocytes (4.0×10 6 cells / ml) and added to a 24-well cell plate at 500 μl / well. Each lymphocyte was added to two wells: one well was added with cell culture medium at 500 μl / well as an unstimulated control; the other well was added with diluted gD-His protein solution at 500 μl / well as a sample after specific antigen stimulation. The final cell count in each well was 2.0 × 10 6 . Take 500μl of a certain amount of lymphocytes and add them to the corresponding position in a 24-well cell culture plate, then add 500μl of cell culture fluid to serve as a blank control for the experiment. Take 500μl of a certain amount of human peripheral blood lymphocytes and add them to the corresponding position in a 24-well cell culture plate, then add 500μl of cell culture fluid to serve as a single positive control and a fluorescence minus one control for the experiment. Place the 24-well cell culture plate in a 37.0℃, 5.0% CO2 incubator and culture for 24 hours.

[0131] Sampling: Remove the cell suspension from the 24-well plate and place it in a centrifuge tube. Place the tube in a benchtop centrifuge and centrifuge at 3500 rpm for 3 minutes. Discard the supernatant and gently tap the bottom of the tube until the cells are loose.

[0132] Block Fc receptors: Use an Fc receptor-specific antibody (BD Fc Block) to block nonspecific staining caused by fluorescent antibodies against Fc receptors. Dilute BD Fc Block 50-fold, add 100 μl / well, and incubate at 4°C for 15 minutes. Wash cells twice with Staining Buffer.

[0133] Cell surface staining: Dispense 4μl, 1μl, and 2μl of CD3, CD4, and CD8, respectively, into 39μl Stain Buffer and add to the sample group at 50μl / well. For the single positive control group, add 50μl of Staining Buffer, followed by the corresponding single fluorescent antibody. For the fluorescence minus one control group, add 50μl of Stain Buffer, followed by the corresponding mixed fluorescent antibody after removing the single antibody (4μl, 4μl, 1μl, and 2μl for CD3, CD4, and CD8, respectively). For the blank control group, resuspend the cells in 50μl of Staining Buffer and incubate at 4°C in the dark for 30 minutes. Wash the cells twice with Staining Buffer.

[0134] Cell fixation and permeabilization: Add 250 μl of Fixation Permeabilization solution to the centrifuge tube and incubate at 4°C in the dark for 20 min. Then wash the cells twice with 1× BD Perm / Wash Buffer.

[0135] Intracellular factor staining: 2μl, 2μl, 2μl, and 2μl of INF-γ, IL-2, IL-4, and TNF-α, respectively, were added to 42μl of BD Perm / wash buffer and added to the sample group, 50μl / well. For the single positive control group and the fluorescence minus one control group, 50μl of BD Perm / wash buffer was added, followed by the addition of a single fluorescent antibody and a single fluorescent antibody mixture (2μl, 2μl, 2μl, and 2μl for INF-γ, IL-2, IL-4, and TNF-α, respectively). For the blank control group, cells were resuspended in 50μl of Stain Buffer and incubated at 4°C in the dark for 30 minutes. Cells were then washed twice with BD Perm / wash buffer, resuspended in Staining Buffer, and analyzed by flow cytometry.

[0136] Sample data acquisition: BD FACS Canto PLUS flow cytometer parameter settings, start the BD FACS CantoPLUS flow cytometer, after the instrument preheating and liquid flow start program is completed, select the channel parameters as FSC, SSC, FITC, PE, PE-Cy7, APC, APC-Cy7, BV450, BV650 test area (A), the sample flow rate is medium speed; mix the sample before loading, stop the acquisition event number to P3 gate (CD3 + )100,000 events. After the sample is resuspended in Staining Buffer, it is transferred to the flow tube. First, a blank control tube is loaded to collect data and set the gate, circling the P2 gate for debonding. Then, the voltage is adjusted using the single positive control (adjust during the reading process). After the voltage adjustment is completed, the data of the single positive control and the fluorescence minus one control group can be read. The P2 gate collects 20,000 events. The fluorescence compensation is adjusted using the fluorescence minus one control data. After all parameters are set, the P3 gate (CD3 + ), collect test samples, P3 gate (CD3 + )100000events.

[0137] Result analysis: The sample data read by flow cytometry were marked with CD4 + and CD8 + , each circle, in CD4 + and CD8 + The number of IL-2, IL-4, INF-γ, and TNF-α positive cells was counted within the gate, and the changes in cell number were compared between unstimulated and stimulated cells.

[0138] Cellular immunity CD4 + T cell results: Results as Figure 9As shown: Comparison of the mice in each dose group before and after stimulation showed that two or more cytokines were positive for IL-2, IL-4, INF-γ, and TNF-α cytokines. In the 0.5ug / 0.5ml dose group, 20% of the mice were positive for 2 factors, 20% for 3 factors, and 60% for 4 factors. In the 1ug / 0.5ml dose group, 20% of the mice were positive for 2 factors, 20% for 3 factors, and 60% for 4 factors. In the 2ug / 0.5ml dose group, The positive ratio of 2 factors was 20%, the positive ratio of 3 factors was 60%, and the positive ratio of 4 factors was 20%. In the 5ug / 0.5ml dose group, the positive ratio of 3 factors was 60%, and the positive ratio of 4 factors was 40%. In the 10ug / 0.5ml dose group, the positive ratio of 2 factors was 60%, and the positive ratio of 3 factors was 40%. In the 20ug / 0.5ml dose group, the positive ratio of 2 factors was 60%, and the positive ratio of 3 factors was 20%, and the positive ratio of 4 factors was 20%. In the adjuvant group, 60% of the mice did not convert positively, and 40% were single-factor positively converted. In summary, the CD4 + The positive rates of two or more cytokines in T cells were all 100%, indicating that there were no significant differences among the groups. In addition, the positive conversion rate and types of positively converted cytokines were significantly better than those in the adjuvant group.

[0139] Cellular immunity CD8 + T cell results: Results as Figure 10 As shown: in the 0.5ug / 0.5ml dose group, the proportion of no cytokine positive conversion was 40%, the proportion of single factor positive was 40%, and the proportion of 4 factor positive was 20%; in the 1ug / 0.5ml dose group, the proportion of no cytokine positive conversion was 40%, the proportion of single factor positive was 40%, and the proportion of 2 factor positive was 20%; in the 2ug / 0.5ml dose group, the proportion of no cytokine positive conversion was 40%, the proportion of single factor positive was 20%, and the proportion of 2 factor positive was 40%; in the 5ug / 0.5ml dose group, The proportion of mice without cytokine positive conversion was 20%, single factor positive accounted for 20%, 2 factor positive accounted for 20%, 3 factor positive accounted for 20%, 4 factor positive accounted for 20%, 10ug / 0.5ml dose group, but factor positive accounted for 40%, 3 factor positive accounted for 60%, 20ug / 0.5ml dose group, the proportion of mice without cytokine positive conversion was 60%, single factor positive accounted for 20%, 2 factor positive accounted for 20%, 60% of mice in the adjuvant group were not positively converted, and 40% were single factor positively converted. In summary, the CD8 + The positive rates of T cells, one or more cytokines were between 40% and 100%, indicating that there were no significant differences among the groups. In addition, the positive conversion rate and types of positively converted cytokines were significantly better than those in the adjuvant group.

[0140] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A fusion protein, characterized in that It consists of 4 antigen molecules gD1, 2 human immunoglobulin antibody Fc domains, and 2 human IgG1 hinge region fragments; The C-termini of the antigen molecule gD1 peptide chains are respectively connected to the N-termini of the antibody Fc domain peptide chains, and the N-termini of one peptide chain in the antigen molecule gD1 connected to the antibody Fc domain are connected in series with a human IgG1 hinge region fragment; the two human IgG1 hinge region fragments are connected in the form of an interchain disulfide bond; The amino acid sequence of the antigen molecule gD1 is shown in SEQ ID NO.6; The amino acid sequence of the human IgG1 hinge region fragment is shown in SEQ ID NO.21; The sequence SEQ ID NO.6 and the sequence SEQ ID NO.21 are connected in series via a linker.

2. The fusion protein according to claim 1, characterized in that The linker sequence has a length of 10-20 aa, and is an amino acid sequence comprising a combination of glycine and serine.

3. The fusion protein according to claim 2, characterized in that The linker sequence is shown as SEQ ID NO.

22.

4. The fusion protein according to claim 1, characterized in that The human immunoglobulin antibody Fc is selected from any one of human IgG, IgM, IgD, IgA and IgE.

5. The fusion protein according to claim 4, characterized in that The human IgG antibody is selected from any one of IgG1, IgG2, IgG3 and IgG4.

6. The fusion protein according to claim 5, characterized in that When the amino acid sequences of the two IgG1 antibody Fc domain peptide chains are identical, the amino acid sequence is as shown in SEQ ID NO. 10; When the amino acid sequences of the two IgG1 antibody Fc domain peptide chains are different, one of the Fc peptide chains contains two mutation points S354C and T366Y, and the sequence is shown in SEQ ID NO.8; the other Fc peptide chain also contains two mutation points Y349C and Y407T, and the sequence is shown in SEQ ID NO.

9.

7. Use of the fusion protein according to any one of claims 1 to 6 in the preparation of a recombinant herpes simplex virus type 1 vaccine.

8. A recombinant herpes simplex virus type I vaccine, characterized in that: Comprising the fusion protein according to any one of claims 1 to 6.

9. The vaccine according to claim 8, characterized in that The vaccine further comprises a pharmaceutically acceptable adjuvant, which is selected from any one or more of aluminum hydroxide, squalene, QS-21, and oligonucleotides.

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