Recombinant I-type herpes simplex virus vaccine and application thereof

Abstract

The invention discloses a recombinant I-type herpes simplex virus vaccine and application thereof, and belongs to the technical field of vaccines. In particular to a fusion protein which is composed of an antigen molecule gD1, a human immunoglobulin antibody Fc structural domain and a human IgG1 hinge region fragment, the amino acid sequence of the antigen molecule gD1 is shown as SEQ ID NO.6, and a recombinant vaccine is constructed. The recombinant vaccine fuses a virus antigen, an immune globulin heavy chain Fc structural domain and a human IgG1 hinge region fragment, has better capability of being combined with a receptor on the surface of an antigen presenting cell, can activate specific CD4 + T and CD8 + T cells, generates high-titer antibody titer and specific cellular immunity, and can be used for preparing a high-titer antibody. The vaccine can prevent infection and / or reinfection of the herpes simplex type I virus.

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] So far, eight herpes viruses associated with human diseases have been confirmed, namely: herpes simplex virus type I and type II (HSV-1 and HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus, cytomegalovirus (CMV), herpes virus type 6, type 7, and type 8. The human-related diseases caused by these eight herpes viruses vary in clinical manifestations, but they all have one common feature, that is, after the initial infection of the human body, each virus may persist in the body for a long time, or even lifelong infection, and this long-term infection often alternates between different forms of overt infection and latent infection.

[0003] Epidemiological studies have shown that an estimated 67% of the world's population is infected with HSV-1. After the initial infection, HSV-1 causes chronic infection in the sensory ganglia and can reactivate on the mucous membranes and skin. The infection is usually asymptomatic, but can also cause 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 diseases (encephalitis and neonatal disease involving multiple organs).

[0004] Once infected, HSV-1 is carried for life. Most of the time, the infected person only causes herpes in the labial and genital areas during acute attacks. After HSV infection, the virus can lurk in the trigeminal ganglion or lumbosacral ganglion. When the body is immunosuppressed or immunodeficient, or encounters triggers such as ultraviolet rays, fever, trauma and emotional tension, bacteria or other viral infections, the latent HSV will be activated and replicated in large quantities, causing local recurrent herpes. At the same time, the virus lurking in the ganglion can also enter the central nervous system through reverse transmission of nerves. In the past decade, more and more evidence has shown that the impact of HSV on humans may not be as simple as herpes. Our understanding of HSV and its infection is still very limited. For example, HSV-1, which was believed to cause head and facial infections in the past, can cause genital herpes like HSV-2. Generally speaking, HSV-1 is the main pathogen of oral herpes infection, and HSV-2 is the main pathogen of genital herpes infection. However, epidemiological data in recent years show 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 people aged 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 of which manifest as local herpetic ulcers on the reproductive organs, accompanied by more severe pain.

[0005] HSV genital tract infection has become one of the most common sexually transmitted diseases in the world. HSV infection can increase the risk of infection with human immunodeficiency virus (HIV), increase the spread of sexually transmitted viruses such as human papillomavirus (HPV), lead to abnormal immune responses in the body, and then develop into cancers such as cervical cancer. HSV infection in women of childbearing age can lead to genital tract damage and dysfunction. In particular, HSV infection in pregnant women can lead to miscarriage, premature birth, congenital fetal developmental abnormalities, and neonatal HSV encephalitis. In addition, HSV that enters the central nervous system can damage the nervous system, not only causing herpes simplex virus encephalitis, Bell's palsy, and primary trigeminal neuralgia, but also is closely related to the occurrence of certain mental illnesses and Alzheimer's disease. These new epidemiological data have changed people's past understanding of HSV and prompted countries to begin to pay attention to data collection on HSV infection and develop new vaccine design strategies.

[0006] HSV-1 can remain dormant in the nervous system for a long time after infection. It can cause recurrent infection when the body is under stress or immunosuppression. Existing antiviral drugs, such as acyclovir, can effectively control the symptoms of infection, but these antiviral drugs are very likely to cause gene mutations and drug resistance. In addition, the drugs cannot prevent primary infection with herpes virus or control latent and recurrent infection.

[0007] HSV-1 is also a very important pathogen of herpes virus encephalitis. Literature reports believe that about 95% of herpes virus encephalitis pathogens are HSV-1. And it is the most common pathogen of sporadic, fatal encephalitis. Adult patients usually have specific antibodies against HSV-1 before the onset of the disease, indicating that this is an infection caused by viral reactivation. For children, HSV-1 often causes neonatal herpes encephalitis, which is similar to other herpes viruses in clinical manifestations, manifested as abnormal mental activity, sudden loss of consciousness, and specific neurological signs, accompanied by fever. In severe cases, it leads to neurological sequelae, such as microcephaly, microcephaly, polycystic encephalomalacia, etc. Neonatal herpes attracted people's attention in the 1930s. Contact between maternal and fetal and neonatal tissues can cause vertical transmission between mother and child. HSV-1 is mainly transmitted in the uterus, during delivery and after birth, which may result in stillbirth and neonatal herpes.

[0008] From the perspective of structural biology, the basic structure of herpes virus includes the following features: the core part is composed of double-stranded DNA genome and DNA binding protein, which is spherical and has a diameter of about 75nm. The core is wrapped with capsid protein, which regularly forms an icosahedral three-dimensional symmetrical structure composed of 162 shell particles, with a diameter of 95-105nm. Among the 162 shell particles, there are 150 hexamers and 12 pentamers. There is a layer of spherical protein outside the nucleocapsid, called the cortex, and outside the cortex is the capsule, on which there are various glycoproteins. The diameter of the complete virus is 120-300nm.

[0009] The length of HSV-1 DNA is 152260bp (17 strains, Genbank No. X14112), with a G+C content of 68%. The genome consists of two segments, namely the L segment and the S segment, with repetitive sequences at both ends of the segments. Under the strict regulatory mechanism of the genome, it follows a linear time program. The linear time process can be divided into three stages, namely, immediate early, early and late. The corresponding genes that activate transcription are immediate early genes (IE), early genes (E), and late genes (L). They work according to the strict program of IE gene transcription expression -> production of IE gene products -> activation and regulation of E gene transcription expression -> production of E gene products -> activation and regulation of L gene products.

[0010] There are multiple membrane proteins on the envelope of herpes virus, all of which are glycosylated proteins, mainly including gB, gC, gD, gE, gG, gH, gI, gL, gM, gN, etc. Membrane proteins bind to each other with nonspecific or specific receptors on the host cell membrane, so that the virus enters the cell and completes its biological function. Wherein about 5 membrane proteins are directly related to the process of virus entering the cell, the effect caused by glycoprotein gB and gC is the heparin sulfate molecule attached to the cell surface, and the research results on tissue culture cells suggest that gC is not absolutely necessary for virus infection, but the existence of gC can improve the efficiency of virus binding to cells by 10 times. GB, gD, gH and gL are necessary for the step of virus entering the cell. But they are not necessary for the event of virus attachment to the cell surface when gL exists. Therefore, from the comprehensive test results, the function of gB, gD, gH and gL is mainly to cause the fusion of virus envelope and cell envelope, and the main glycoprotein that can act on the fusion receptor of the cell and start the fusion process is gD.

[0011] From the perspective of HSV-1's human host cells, there are at least three types of receptors related to HSV-1 entry, including the herpes virus entry mediator protein HVEM, a member of the tumor necrosis factor receptor family; immunoglobulin superfamily members nectin-1 and nectin-2. The above receptors can bind to the gD1 molecule and induce the virus to enter the cell. Based on biological experiments, relevant studies further explore the topological mechanism of HSV-1 gD1 binding to the corresponding receptors. Some analyses on 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 about 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 the binding of gD1 to HVEM, and are also sites that control the recognition of nectin-2, but do not affect the recognition of nectin-1. Downstream of this sequence is the site that binds nectin-1 and nectin-2. Topologically, this sequence forms a hairpin structure.

[0012] Further epitope analysis showed that there are at least 12 regions in the structure of the HSV-1gD1 molecule that may contain CD4 + T cell epitopes are approximately 27 to 34 amino acids in length. + T cell response, both have protective significance, CD4 + T cell response plays an important role in the fight against 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 period of time. These phenomena indicate that the CD8 +T cell response is important in controlling and clearing 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 without 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 not early genes and late genes. + The specific antibodies of T cells can reactivate the virus from the latent nerve cells only 5 days later. + T cells may be activated by IE proteins expressed by viruses in latent infection in nerve cells, and activated CD8 + In turn, T cells effectively monitor latent viruses. Once the relevant viruses are reactivated in 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 for 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 show that human herpes viruses can form latent infections during the process of infecting the human body, and clinically form chronic and persistent long-term infections, which are difficult to completely eliminate from the body. However, the body can form a complete immune response to these viral infections, and based on the principles and foundations of these immune responses, some vaccine attempts have also been proven to be feasible. HSV-1 vaccine is the most ideal method to prevent herpes simplex virus type I, but no vaccine with a strong immune effect has been developed so far.

[0015] Inactivated virus vaccine is the most classic technical route. In 1992, some people tried to use formalin to inactivate HSV-infected rabbit brains and other animal tissues, and then extract the whole virus to prepare vaccines. Inactivated viruses retain all antigen molecules and can cause a wide range of immune responses, but this type of vaccine has low immunogenicity and poor protection. Other inactivation methods, such as heating, chemical treatment, and ultraviolet irradiation, have been tried, but no good results have been achieved.

[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. The researchers isolated the 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 significantly low neurotoxicity in the rabbit model, but could still lurk in the nervous system and cause recurrent infections. To address this issue, the viral genome was edited to weaken the virulence, 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 route has attracted much attention. The constructed gK gene-deficient strain induced a long-lasting T cell response in mice. The immunized mice effectively suppressed the occurrence of the disease in the reproductive tract attack experiment, with a protection rate of up to 90%. Replication-restricted strains are less virulent and will not replicate in the body. They can induce effective immune protection, but such strains must be grown in genetically modified cells that contain genes that are missing in expression. The preparation method directly limits the application of such strains. Genetically modified cells have a certain risk of carcinogenicity, and there is also a risk of restoring virulence when the strain is propagated in modified cells.

[0019] Live vector vaccines are vaccines that insert genes expressing HSV-1 related antigens into replicable viral or bacterial vectors. After immunization, the viral or bacterial vectors can express the corresponding antigenic proteins and induce the body to produce corresponding immune responses. The most studied are vaccinia virus, adenovirus, and Salmonella. There are also studies that clone the gD1 protein gene into the phage genome. The gD1 protein is expressed by fusion with the phage surface protein and displayed on the phage surface. The recombinant phage particles are used to immunize mice, and the gD1 antigen is presented by the cells, inducing a specific immune response. Phages are safe as vectors, easy to mass produce, can induce immune responses, and avoid problems such as HSV-1 latent toxicity. However, many issues such as the dosage and procedure of phage vector vaccines have not been studied clearly.

[0020] Nucleic acid vaccines are also one of the research hotspots. The antigen gene is inserted into a eukaryotic expression vector and directly inoculated to express the antigen in vivo. A study constructed the gD1 of HSV-1 and the human interleukin 12 gene together on DNA, and encapsulated the DNA vaccine in nanoparticles. The corneal immunization of mice produced high levels of specific neutralizing antibodies; the secretory IgA in tears, IFN-γ and IL-4 levels in serum were significantly increased; the cytotoxic effects of spleen cells and NK cells were significantly enhanced; after the wild strain attack, the herpes stromal keratitis in mice was also significantly weakened.

[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 of 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. It is easy to purify antigens using protein A / G affinity chromatography; 2. Fc promotes the correct folding of fusion proteins and APC cell binding, increasing the immunogenicity of antigens; 3. Fc fusion proteins can improve the solubility and stability of recombinant immunogens and extend their half-life.

[0022] However, there is a major limitation in using a single antibody or the Fc domain of the constant region of the heavy chain of an antibody to deliver antigens to immune cells. Since it is a monomer containing only one Fc, it 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 mainly expressed on monocytes and neutrophils, binds to ligands with high affinity, and binds to monomeric IgG and immune complexes. FcγRII can be expressed on every cell with FcγR (except NK cells), and FcγRII binds to ligands with low affinity. FcγRII can promote phagocytosis / endocytosis of immune complexes and B cell activation. FcγRIII is expressed on macrophages, NK cells, bone marrow precursor cells, and neutrophil cell lines. FcγRIII expression on macrophages is regulated by IFN-γ and can mediate antibody-dependent cellular cytotoxicity (ADCC). FcγRIIA and FcγRIII are very important for enhancing immune response. Classic immune complexes are formed by the combination of antigen molecules in the body and numerous antibody molecules. They contain multiple Fc domains and can enhance antigen uptake by increasing the binding activity with all FcγRs. This is because polyclonal antibodies bind to the same antigen to form polymers, and numerous Fcs bind to FcRs on the surface of APC cells, inducing receptor cross-linking, promoting the internalization of the complex by APC cells, and the antigen is 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 is allowed to form cross-links. The soluble antigens in conventional subunit vaccines do not have this ability. Similarly, Fc fusion proteins cannot form multimeric complexes, so vaccines do not have the effect of enhancing immune effects. Although Fc fusion proteins have many advantages, there are still considerable obstacles in the use of genetic engineering methods to prepare immune complexes containing two or more Fcs, which is a difficulty in the development of immune complex vaccines. So far, many schemes have been tried internationally. These studies have shown that the use of genetic engineering methods to express antigens and antibodies and then combine them to form immune complexes is also a very attractive immune strategy. The biggest challenge at present is the molecular design of immune complexes, effective expression and purification, and the need for large-scale production under GMP conditions. Summary of the invention

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

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

[0026] The present invention provides a fusion protein, which is composed of 4 antigen molecules gD1, 2 human immunoglobulin antibody Fc domains, and 2 human IgG1 hinge region fragments;

[0027] The C-terminal of the peptide chain of the antigen molecule gD1 is respectively connected to the N-terminal of the peptide chain of the antibody Fc domain, and the N-terminal of one peptide chain in the antigen molecule gD1 connected to the antibody Fc domain is 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 hinge region of human IgG1 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 has a length of 10-20 aa, 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] Further, 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 one or more immunostimulants including pharmaceutically acceptable aluminum hydroxide adjuvant, squalene, QS-21, oligonucleotides.

[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 a viral outer membrane glycoprotein D (hereinafter referred to as gD1), the quasi-immune complex design is: 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, and after the fusion protein is expressed in mammalian cells, two or four Fc single chains can be assembled into dimers or tetramers; the gD1-Fc fusion protein in the form of a tetramer contains four gD1 polypeptide chains and two Fc domains.

[0042] In order to realize the connection of structural units through covalent bonds, 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 (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 sequence of the heterodimer gD1_kFc is SEQ ID No. 2, and the sequence of gD1_hFc is SEQ ID No. 3. The sequence of the homodimer gD1_Fc chain is SEQ ID No. 4, and the sequence of the SS_gD1_Fc chain is SEQ ID No. 5.

[0046] The prepared high-purity antigen can activate CD4 after intramuscular or subcutaneous injection in the range of 5 to 200 micrograms per dose. + 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, preferably the polypeptide chains of the Fc domain are asymmetrically combined, which is a knob into hole design and is easy to form heterodimers, wherein 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, and 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 N-terminal antigenic peptide chains of the two peptide chains constituting the quasi-immune complex are both derived from the outer membrane protein of the virus, and the C-terminal is the Fc fragment of human immunoglobulin. The two peptide chains can be the same or different, and the N-terminal of one of the peptide chains contains the above-mentioned structure that can form an interchain disulfide bond, while the other does not. It can also be derived from the tegument protein of the virus, which is the main antigenic epitope region of the present invention and can provide an effective immune stimulant. It can be a viral structural protein or a viral non-structural protein. The present invention preferably uses the outer membrane glycoprotein gD1 of HSV-1 as an example for discussion, and of course other effective HSV-1 antigens are also included in the present invention.

[0049] In the design of the present invention, the constructed immune complex molecule is connected to the human antibody constant region domain Fc at the C-terminus of the viral antigen peptide chain. In the selection of Fc types, it can be derived from any one of human IgG, IgM, IgD, IgA and IgE, preferably IgG. According to the requirements of antigen presentation, IgG antibody subtypes are also within the scope of optimization selection, including IgG1, IgG2, IgG3 and IgG4. Different subtypes have different Fc receptor binding tendencies. The present invention preferably uses IgG1 Fc as an example for technical description. The function of the Fc domain is to bind to the immune cell receptor and complete the antigen presentation of a specific path. Therefore, a part of the Fc constant region of IgG1 is intercepted for the present invention, which can meet the design requirements. It is preferred to select the amino acid sequence of the hinge region, CH2, and CH3 region of the human IgG1 antibody, and form an interchain disulfide bond through two cysteines in the hinge region, that is, to form a dimer. 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 the FcRs binding activity while also retaining the amino acid sequence corresponding to the complement system activation activity.

[0050] In the design of the present invention, the Fc domain can also adopt a symmetrical design, that is, without using the KiH design, the molecular structure designed by the present invention can also be realized. 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, transferred into CHO cells, and the two peptide chains are co-expressed under the action of their respective promoters, and the antigen gD1 dimer can be obtained, and the double immune complex molecule containing the double Fc domain can also be obtained. According to the test results, it also meets the design standards of the present invention. However, in theory, the KiH scheme has a lower mismatch rate and a higher harvest rate.

[0051] In summary, the molecules designed by the present invention have double Fc domains, 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 antigen presentation, activation of corresponding signaling pathways, and protective immunity. Experiments show that more performance is MHC I cross-presentation, activation of CD8 + T cells, according to the previous discussion, we know that activation of 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 by mammalian cells, and the antigen molecules self-assemble into an immune complex-like fusion protein, which is purified and made into 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 It is a schematic diagram of the molecular structure of LZ920 immune complex;

[0056] Figure 3 It is a schematic diagram of the molecular structure of LZ921 immune complex;

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

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

[0059] Figure 6 This is the SEC-HPLC purity test chart of 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 test results of serum antibody titer after mouse immunization in Example 5;

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

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

[0064] In order to better illustrate the present invention, the following embodiments are listed. Obviously, the described embodiments are only a part of the present invention, not all embodiments. Based on the embodiments in the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0065] It should be noted that the terms "first", "second", etc. in the specification 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 data 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 that are 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 is described below by taking gD1 of HSV-1 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] SS sequence SEQ ID No. 1: DKTHTCPPCPAPELLGGGSTSGSGKPGSGEG (31aa) contains two cysteine ​​residues. The 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 connected by the glycoprotein sequence gD1 SEQ ID No.6 and the sequence kFc SEQ ID No.8 chain. The wild-type gD1 (KOS standard strain) has a total length of 394 amino acids, forming a gB-gH / gL-gD complex, which binds to the cell receptor HVEM, NECTIN-1 or heparan sulfate to trigger cell membrane fusion. The 1-25 amino acids of the wild-type gD1 amino acid sequence are signal peptides, the 26-340 amino acids are located on the surface of the virus particles, the 341-361 amino acids are α-helical structures that constitute the membrane-penetrating region, and the 362-394 amino acids are inside the virus particles. The functional region of gD1 that interacts with glycoproteins gB and gH / gL to trigger membrane fusion is located within 26-340 amino acids. This region contains the key antigenic determinant sequence of viral infection. Referring to the technical solutions of other research and development teams in this field, starting from the 26th amino acid residue, 307 amino acids are intercepted as the candidate antigen sequence of the vaccine of the present invention. The sequence contains three pairs of intrachain disulfide bonds, 91<->214, 131<->227 and 143<->152. Three N-glycosylation sites, at amino acid residues 119, 146 and 287.

[0076] The kFc peptide chain connected to the amino acid C-terminus of gD1 glycoprotein is a Knob chain. The sequence is extracted from the constant region of the heavy chain of human IgG1, including the hinge region, CH2 and CH3 regions, in which the cysteine ​​connecting the wild-type CH1 region to the light chain is removed to reduce the probability of disulfide bond mismatch. In addition, two amino acids S354C and T366Y in the CH3 region are mutated. The amino acid mutated by T366Y has a larger side chain, forming a "Knob"-like protruding structure to assist the correct pairing of kFc.

[0077] SS_gD1_hFc sequence SEQ ID No.3 is a Hole chain, which consists of three parts, starting from the N-terminus, including sequence SSSEQ ID No.1, sequence gD1 SEQ ID No.6 and sequence hFc SEQ ID No.9. Among them, hFc is a Hole chain, which contains two mutation points: Y349C and Y407T. The Y407T mutation changes the large amino acid side chain tyrosine (Y) into a small side chain of threonine (T), forming a "Hole"-like concave structure, which cooperates with the Knob chain to assist hFc in correct pairing.

[0078] KiH design is a classic technical route, which is often used in bispecific antibody design to reduce the probability of mismatching. The KiH mutation scheme adopted in the present invention is a technical method provided to better describe the present invention, through which the desired vaccine antigen structure can be obtained. Other Fc mutation schemes can also achieve the goals of improving the yield and reducing mismatching of the present invention, and are also within the scope of protection of the present 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 and 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 onto the cloning plasmid (the plasmid selected the commonly used plasmid in the GS Xceed system). Huada Gene 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 SEQID No.20, and 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, and using the plasmid containing SEQ ID No.8 as a template, primers SEQID No.17 and SEQ ID No.16 were used to PCR amplify the target gene fragment kP2. The amplification conditions were 98°C for 10s, 55°C for 5s, 72°C for 7s for 35 cycles, and extension at 72°C for 10min.

[0083] The two products were connected by overlapping extension PCR technology. By using primers with complementary ends, the PCR products formed overlapping chains, so that the amplified fragments from different sources were overlapped and spliced ​​together by extending the overlapping chains in the subsequent amplification reaction. 15μl of PCR products kP1 and kP2 were mixed, 15μl of PrimeSTAR Polymerase and 15μl of injection water were added, and PCR was performed again. The reaction conditions were: 98℃10s, 55℃5s, 72℃15s 10 cycles, and 72℃ extension for 10min. The spliced ​​product was kP3.

[0084] The expression vector plasmid was transformed into DH5α and positive clones were screened. Single colonies were randomly selected for expansion and culture. The plasmid was extracted using a commercial kit, and the plasmid was double-digested with restriction endonucleases (HindIII / EcoRI) to recover the vector fragment. The KP3 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 at 37°C for 30 minutes in a gene amplifier, and then ice-bathed immediately. Then, the competent DH5α that had also been melted in an ice-bath was mixed, heat-shocked at 42°C for 90 seconds, and an appropriate amount of LB medium was added to incubate at 37°C for 45 minutes, and the resistant plate was coated to screen positive clones.

[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, and using the plasmid containing SEQ ID No.9 as a template, primers SEQID No.19 and SEQ ID No.16 were used to PCR amplify the target gene fragment hP2. The amplification conditions were 98°C for 10s, 55°C for 5s, and 72°C for 7s for 35 cycles, and extension at 72°C for 10min.

[0086] The two products were connected by overlapping extension PCR. 15 μl of PCR products hP1 and hP2 were mixed, 15 μl of PrimeSTAR Polymerase and 15 μl of injection water were added, and PCR was performed again. The reaction conditions were: 98℃10s, 55℃5s, 72℃15s 10 cycles, and 72℃ extension for 10min. The spliced ​​product was hP3.

[0087] The expression vector plasmid was transformed into DH5α and positive clones were screened. Single colonies were randomly selected for expansion and culture. The plasmid was extracted using a commercial kit, and the plasmid was 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 (HindIII / EcoRI) vector fragment, and Exnase II homologous recombinase and buffer were added. The reaction was carried out at 37°C for 30 minutes in a gene amplifier, and then ice-bathed immediately. Then, the competent DH5α that had also been melted in an ice-bath was mixed, heat-shocked at 42°C for 90 seconds, and an appropriate amount of LB medium was added to incubate at 37°C for 45 minutes, and the resistant plate was coated to screen positive clones.

[0088] Construct 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, and 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 10s, 55°C for 5s, and 72°C for 7s for 35 cycles, and extension at 72°C for 10min.

[0089] The two products were connected by overlapping extension PCR. 15 μl of PCR products L1 and L2 were mixed, 15 μl of PrimeSTAR Polymerase and 15 μl of injection water were added, and PCR was performed again. The reaction conditions were: 98℃10s, 55℃5s, 72℃15s 10 cycles, and 72℃ extension for 10min. The spliced ​​product was L3.

[0090] The expression vector plasmid was transformed into DH5α and positive clones were screened. Single colonies were randomly selected for expansion and culture. The plasmid was extracted using a commercial kit, and the plasmid was 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 (HindIII / EcoRI) vector fragment, and Exnase II homologous recombinase and buffer were added. The reaction was carried out at 37°C for 30 minutes in a gene amplification instrument, and then ice-bathed immediately. Then, the competent DH5α that had also been melted in an ice-bath was mixed, heat-shocked at 42°C for 90 seconds, and an appropriate amount of LB medium was added to incubate at 37°C for 45 minutes, and the resistant plate was coated to screen positive clones.

[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, and using the plasmid containing SEQ ID No.10 as a template, primers SEQID No.14 and SEQ ID No.15 were used to PCR amplify the target gene fragment H2. The amplification conditions were 98°C for 10s, 55°C for 5s, and 72°C for 7s for 35 cycles, and extension at 72°C for 10min.

[0092] The two products were connected by overlapping extension PCR. 15 μl of PCR products H1 and H2 were mixed, 15 μl of PrimeSTAR Polymerase and 15 μl of injection water were added, and PCR was performed again. The reaction conditions were: 98℃10s, 55℃5s, 72℃15s 10 cycles, and 72℃ extension for 10min. The spliced ​​product was H3.

[0093] The expression vector plasmid was transformed into DH5α and positive clones were screened. Single colonies were randomly selected for expansion and culture. The plasmid was extracted using a commercial kit, and the plasmid was 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 at 37°C for 30 minutes in a gene amplification instrument, and then ice-bathed immediately. Then, the competent DH5α that had also been melted in an ice-bath was mixed, heat-shocked at 42°C for 90 seconds, and an appropriate amount of LB medium was added to incubate at 37°C for 45 minutes, and the resistant plate was coated to screen positive clones.

[0094] The above positive clones were amplified and plasmids Ek2, Eh3, EL4 and ES5 were prepared in large quantities. Double restriction 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 as a group, DNA ligase and reaction buffer were added, and the reaction was carried out at 2-8°C overnight. The ligation product was then transformed into DH5α competent cells to prepare a dual expression plasmid. The plasmid was extracted in large quantities using a commercial kit and digested with restriction endonuclease PvuI to linearize the dual expression vector, thus completing the construction of the heterodimer expression gene cassette, numbered 921.

[0096] EL4_P / N and ES5_P / N were mixed as a group, DNA ligase and reaction buffer were added, and the reaction was carried out at 2-8°C overnight. The ligation product was then transformed into DH5α competent cells to prepare a dual expression plasmid. The plasmid was extracted in large quantities using a commercial kit, and the restriction endonuclease PvuI was used for enzyme digestion to linearize the dual expression vector and construct a homodimer expression gene cassette, numbered 920.

[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), 20 μg of linearized plasmid and transfection reagent were added in sequence, the perforation voltage of the gene pulse generator Gene Pulser Xcell (Bio-Rad) was set to 300 V voltage, 900 μF single pulse, infinite resistance, a disposable shock cup (Bio-Rad) with a gap of 4 mm, and the shock time was 12-20 ms. The cells in the shock cup were transferred to a culture bottle, 30 ml of CHO culture medium was added, and the culture was carried out 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 The CHO culture medium of MSX (without glutamine) was used for pressure screening, and the cells were transferred into 96-well flat-bottom culture plates by limiting dilution method. The culture plates were cultured in a 37°C, 5% CO2 incubator and observed under an inverted microscope. The monoclonal cell wells were marked, and then the positive cells in 24-well and 6-well cell culture plates were screened in turn by ELISA method to screen out the monoclonal strains with higher expression levels, which were continuously subcultured and tested 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 purified by affinity chromatography, and the identification was performed by SDS-PAGE electrophoresis. The results are shown in Figure 4 The cell line that met the standard was named LZ920; the positive cell line obtained by transfecting CHO K1 with linearized plasmid 921 was collected from the culture supernatant, purified by affinity chromatography, and identified 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 strain 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, it 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 (feeding such as sugar, culture medium or alkali solution) is used during the culture period. Samples are taken every day to check the number of cells and cell activity. Generally, the culture is carried out for 12-14 days. When the proportion of live cells drops below 85%, the fermentation culture is stopped and the fermentation liquid is harvested.

[0102] The harvested fermentation liquid is centrifuged at 10,000 r / min for 30 minutes to remove large insoluble particles such as cell debris or use a deep filter to remove cells and cell debris, and the cell culture supernatant is collected. The culture supernatant is then filtered with a 0.45 μm clarification filter. The filtered intermediate can be stored at 2 to 8 ° C or directly enter the next purification process.

[0103] The above-mentioned harvested liquid after filtration 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 balanced with a 20mM PBS (pH7.4, containing 135mM NaCl) buffer, and after loading, it is washed with a 20mM PBS buffer until the UV detector display value returns to near the baseline; then it is dissociated with 0.1mol / L citric acid (pH3.5), and the elution peak is collected according to the A280 absorbance value. The protein purity after affinity chromatography reaches more than 90%. The affinity chromatography solution is incubated at room temperature for 60min, the virus is inactivated, and then an appropriate amount of 2MTris solution is added to neutralize to a pH value of 7.2 to 7.6.

[0104] The neutralized affinity chromatography solution is directly loaded onto a loaded Sephacryl S-400HR (or Sepharose 4Fast Flow, or Sephadex 200PG) gel chromatography column, with the loading volume not exceeding 2% of the column bed volume. A 40 mM phosphate buffer (pH 7.4, containing 150 mM NaCl) is used as the mobile phase, and when the absorbance value of the UV detector A280 rises, the effluent from the chromatography column is collected in sections until the absorbance value drops to a level close to the baseline, and the collection of samples is stopped, and the collected solutions with a purity of more than 95% are combined.

[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, and 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 solution, which was named LZ920 antigen protein stock solution and LZ921 antigen protein stock solution, respectively. Samples were taken for SEC-HPLC purity test. The test spectrum is shown in Figure 6 and Figure 7 The results showed that the HPLC purity reached more than 94%, which met the design standards and met the needs of subsequent efficacy evaluation.

[0107] Example 5: Mouse macrophage phagocytosis assay

[0108] Isolation and culture of macrophages: Randomly select BALB / c mice, inject 5 ml of 1640 culture medium without fetal bovine serum into the peritoneum, gently massage the abdomen for 2-3 minutes, let it stand for 5 minutes, extract the peritoneal fluid with a sterile syringe, centrifuge at 1000r / min for 5 minutes, wash the cells twice with 1640 culture medium, and resuspend the macrophages with 1640 culture medium containing 10% fetal bovine serum. Inoculate in 96-well plates, place in a 37°C, 5% CO2 incubator for 12 hours, and then replace the culture medium.

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

[0110] The results showed that after adding LZ920-FITC and LZ921-FITC to macrophages, LZ920 and LZ921 antigen proteins could be engulfed by macrophages and produced obvious green fluorescence. However, the control group with IgG-FITC did not produce obvious fluorescence, indicating that it was not engulfed 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 20μg / 0.5ml, 10μg / 0.5ml, 5μg / 0.5ml, 2μg / 0.5ml, 1μg / 0.5ml, and 0.5μg / 0.5ml of LZ920 and LZ921 antigens, respectively. Female BLAB / c mice aged 4 to 7 weeks were randomly divided into 14 groups, with 5 mice in each group. Each mouse in the control group was intraperitoneally injected with 0.5ml of aluminum adjuvant.

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

[0115] The six experimental groups of LZ921 antigen were operated in the same way as the experimental group of LZ920. The third immunization was performed 3 weeks after the second immunization, for a total of 3 immunizations. Blood was collected from the tail vein before the second and third immunizations, and the serum was separated and frozen below -20°C. Blood was collected from the retro-orbital vein 14 days after the third immunization, and the serum was separated and frozen below -20°C. The spleen was taken to separate lymphocytes. Detailed grouping is 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, coated on a 96-well ELISA plate, 100 μl per well, placed at 37°C for 2 hours, and then placed 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, and then 200 μl of blocking solution (2% bovine serum albumin washing solution) was added to each well, and the plate was blocked at 37°C for 60 min; the blocking solution in the well was aspirated, and the plate was washed three times with 20 mM PBS-T washing solution, and mouse serum pre-diluted at 1:100 (or 1:1000) was added to the first column of wells of the 96-well plate, and then a 2-fold serial dilution was performed, and the negative control was the mouse serum (1:100) immunized intraperitoneally with aluminum adjuvant only. After reacting at 37°C for 60 min, the blocking solution in the well was aspirated, and the plate was washed with 20 mM Wash 3 times with PBS-T solution, add 1:100 times diluted goat anti-mouse IgG-HRP conjugate, 100μl per well, react at 37℃ for 60min; aspirate the liquid in the well, wash 3 times with 20mM PBS-T solution, add 100μl TMB colorimetric solution to each well, add 50μl stop solution after 10min to terminate the reaction, and then use TECAN Infinit200 microplate reader to measure the A450 absorbance of each well. Take 3 times of the mean absorbance of the negative control (aluminum hydroxide adjuvant mouse serum) (if the absorbance is less than 0.1, calculate it as 0.1) as the Cutoff value, and the maximum dilution corresponding to the mean absorbance of mouse immune serum higher than the Cutoff value is the mouse immune serum antibody titer.

[0121] The geometric mean and standard deviation of the anti-HSV gD antibody titer in the serum of the mice in the LZ920 experimental group are shown in Table 3. Except for one mouse in the 20μg / 0.5ml / mouse group that did not convert positively after the first immunization, the serum positive conversion rate of BALB / c mice in other dose groups was 100%, and all mice converted positively after the second immunization. Statistical analysis of the antibody titers measured in the animals in each experimental group showed that the antibody titers of each dose group after the first immunization were basically the same: ranging from 1131 to 2111. After the second immunization, except for the 10μg / 0.5ml / mouse group that showed individual differences in mice, the serum antibody titers of all other mice were between 128000 and 512000, indicating that the serum antibody titer of BALB / c mice obtained 3 weeks after the second immunization with LZ920 increased significantly and reached a stable level, and there was no significant difference in the serum antibody titer of mice in each group. 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 the anti-HSV gD antibody titer in the serum of the mice in the LZ921 experimental group are shown in Table 4. Except for the 2μg / 0.5ml / mouse and 0.5μg / 0.5ml / mouse dose groups, which had positive conversion rates of 80% and 60% respectively after the first immunization, the serum positive conversion rates of BALB / c mice in other dose groups were all 100%. After the second and third immunizations, the serum positive conversion rates of BALB / c mice in each dose group were all 100%. Statistical analysis of the antibody titers measured in the animals in each experimental group showed that the antibody titers of each dose group after the first immunization were basically the same, ranging from 528 to 2425. After the second immunization, the antibody titers of each dose group further increased and reached a relatively stable level. There was basically no significant difference in the serum antibody titers of mice in each group, ranging from 97006 to 256000. After the third immunization, the antibody titers of each dose group were significantly increased compared with the second immunization, and there was no significant difference in the serum antibody titers of mice in each group, ranging from 844485 to 1688970. In summary, there was basically no significant difference in the serum antibody titers of mice in the 0.5μg / 0.5ml / mouse dose group of LZ921 immunized BALB / c mice compared with other immunization doses, indicating that the 0.5μg / 0.5ml / mouse dose group has strong immunogenicity.

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

[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 soaked 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 (restored to room temperature and shaken before use). After grinding, the separation solution containing 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 stratified, the lymphocyte layer was aspirated, and 10ml of RPMI1640 culture medium was added, and the cells were washed upside down. Centrifuge at room temperature and 1500rpm for 10min to collect the cells, and count them after resuspending.

[0130] Cytokine detection: Take the quantitative resuspended blood lymphocytes of each mouse (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 number of cells in each well was 2.0×10 6 . Take 500μl of the quantitative lymphocytes and add them to the corresponding position in the 24-well cell culture plate, and then add 500μl of cell culture fluid as the blank control of the experiment. Take 500μl of the quantitative human peripheral blood lymphocytes and add them to the corresponding position in the 24-well cell culture plate, and then add 500μl of cell culture fluid as the single positive control and fluorescence minus one control of the experiment. Place the 24-well cell culture plate in a 37.0℃, 5.0% CO2 incubator and culture for 24 hours.

[0131] Sampling: Take out the cell suspension in the 24-well plate and put it into a centrifuge tube. Place the centrifuge tube in a desktop centrifuge and centrifuge at 3500 rpm for 3 minutes. Discard the supernatant and flick the bottom of the tube until the cells become loose.

[0132] Block Fc receptors: Use an antibody specific for Fc receptors (BD Fc Block) to block nonspecific staining caused by fluorescent antibody Fc receptors. Dilute BD Fc Block 50 times, 100 μl / well, incubate at 4°C for 15 minutes. Then wash the cells twice with Staining Buffer.

[0133] Cell surface staining: Take 4μl, 1μl and 2μl of CD3, CD4 and CD8 respectively to 39μl Stain Buffer solution, add to the sample group, 50μl / well. Add 50μl Staining Buffer to the single positive control group, and then add the corresponding single fluorescent antibody. Add 50μl Stain Buffer to the fluorescence minus one control group, and then add the corresponding mixed fluorescent antibody without single antibodies (the amount of CD3, CD4 and CD8 added is 4μl, 4μl, 1μl and 2μl respectively). Add 50μl Staining Buffer to the blank control group to resuspend the cells and incubate at 4℃ in the dark for 30min. Then wash the cells twice with Staining Buffer.

[0134] Cell fixation and membrane 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 were added to 42μl BD Perm / wash buffer solution, and added to the sample group, 50μl / well. 50μl BD Perm / wash buffer was added to the single positive control group and the fluorescence minus one control group, and then a single fluorescent antibody and a mixed fluorescent antibody without a single fluorescent antibody were added (the amount of INF-γ, IL-2, IL-4 and TNF-α added was 2μl, 2μl, 2μl and 2μl, respectively). 50μl StainBuffer was added to the blank control group to resuspend the cells and incubate at 4℃ in the dark for 30min. Then the cells were washed twice with BD Perm / wash buffer, resuspended in Staining Buffer, and detected by flow cytometry.

[0136] Sample data acquisition: BD FACS Canto PLUS flow cytometer parameter setting, start the BD FACS CantoPLUS flow cytometer, after the instrument preheating and liquid flow startup 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 as P3 gate (CD3 + )100000 events. After the sample is resuspended in Staining Buffer, it is transferred to the flow tube. First, a blank control tube is used to load the sample to collect data and set the gate, and the P2 gate for debonding is circled. Then the voltage is adjusted using the single positive control (adjusted 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 20000 events. The fluorescence compensation is adjusted using the fluorescence minus one control group data. After all parameters are set, the P3 gate (CD3 + ), collect test samples, P3 gate (CD3 + )100000 events.

[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 the number of cells were compared between the unstimulated and stimulated cells.

[0138] Cellular ImmunityCD4 + T cell results: Results as Fig. 9As shown: the mice in each dose group were compared before and after stimulation, and two or more cytokines were positive for IL-2, IL-4, INF-γ, and TNF-α. 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, 20% of mice were positive for 2 factors, 60% for 3 factors, and 20% for 4 factors. In the 5ug / 0.5ml dose group, 60% were positive for 3 factors, and 40% were positive for 4 factors. In the 10ug / 0.5ml dose group, 60% were positive for 2 factors, and 40% were positive for 3 factors. In the 20ug / 0.5ml dose group, 60% were positive for 2 factors, 20% were positive for 3 factors, and 20% were positive for 4 factors. In the adjuvant group, 60% of mice were not positively converted, and 40% were positively converted for a single factor. 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, and 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 Fig.10 As shown: In the 0.5ug / 0.5ml dose group, 40% of the cases had no cytokine positive conversion, 40% had a single factor positive, and 20% had a four-factor positive. In the 1ug / 0.5ml dose group, 40% of the cases had no cytokine positive conversion, 40% had a single factor positive, and 20% had a two-factor positive. In the 2ug / 0.5ml dose group, 40% of the cases had no cytokine positive conversion, 20% had a single factor positive, and 40% had a two-factor positive. In the 5ug / 0.5ml dose group, The percentage 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, no cytokine positive conversion accounted for 60%, single factor positive accounted for 20%, 2 factor positive accounted for 20%, 60% of mice in the adjuvant group did not positively convert, 40% were single factor positively convert. In summary, the CD8 + For T cells, the positive rate of one or more cytokines was between 40% and 100%, indicating that there was no significant difference among the groups, and the positive conversion rate and the types of positively converted cytokines were significantly better than those in the adjuvant group.

[0140] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A fusion protein, characterized in that It is composed of 4 antigen molecules gD1, 2 human immunoglobulin antibody Fc domains, and 2 human IgG1 hinge region fragments; The C-terminal of the peptide chain of the antigen molecule gD1 is respectively connected to the N-terminal of the peptide chain of the antibody Fc domain, and the N-terminal of one peptide chain in the antigen molecule gD1 connected to the antibody Fc domain is 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 hinge region of human IgG1 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 I 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 also includes pharmaceutically acceptable aluminum hydroxide adjuvant, squalene, QS-21, oligonucleotide or one or more immune stimulants.

10. Use of the vaccine according to claim 9 in the preparation of a product for preventing and / or treating herpes simplex virus type I infection.

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