Immunogen composition capable of inducing long-lasting immune response against orthopoxvirus, preparation method therefor, and use thereof

Abstract

The present invention relates to an immunogen composition capable of inducing long-lasting immune response against orthopoxvirus, a preparation method therefor, and use thereof. Specifically, provided herein is an immunogen composition, which comprises: (1) one or more first immunogens, which are derived from an RNA polymerase and / or a DNA polymerase expressed during early and / or intermediate phases after infection with orthopoxvirus and induce cellular immune response; and (2) one or more second immunogens, which are derived from antigen peptides of intracellular mature virus particles (IMV) and / or extracellular envelope particles (EEV) of orthopoxvirus and induce humoral immune response. Also provided herein is an expression vector composition encoding the immunogen composition. The immunogen composition provided herein can not only induce the production of neutralizing antibodies that can widely neutralize different viral strains, but also achieve a broad-spectrum protective effect against vaccinia virus and monkey pox virus, and provide long-lasting immune response protection.

Description

An immunogenic composition capable of inducing a long-lasting immune response against poxvirus, its preparation method and application. Technical Field

[0001] This invention belongs to the field of biomedicine, and particularly relates to the manufacture of genetically engineered drugs and vaccines. Specifically, this disclosure relates to an immunogenic composition that can induce a long-lasting immune response against orthopoxvirus, its preparation method and application, and its application in the prevention and treatment of orthopoxvirus infections such as monkeypoxvirus and vaccinia virus. Background Technology

[0002] Monkeypox is caused by the monkeypox virus (MPXV). Infection typically presents with fever, headache, swollen lymph nodes, and muscle pain, accompanied by a rash following the fever. MPXV is a double-stranded DNA virus belonging to the genus Orthopoxvirus in the family Poxviridae. It has two infectious forms: intracellular mature virus (IMV) particles and extracellular enveloped virus particles (EEV). Transmission primarily occurs through direct contact (such as sexual or skin contact) and respiratory droplets. Monkeypox mainly spreads among people with HIV / AIDS and those with weakened immune systems.

[0003] Given that routine smallpox vaccination has been suspended in various countries and regions for 40 years, the proportion of the global population susceptible to orthopoxvirus is increasing year by year. Vaccination is one of the effective means of controlling a potential future monkeypox outbreak. Currently, there is no specific treatment or drug for monkeypox. Only one small molecule drug has been approved for marketing in the United States, but the latest clinical results show that it has not shown significant relief of symptoms from monkeypox infection. The two attenuated vaccines developed for smallpox, ACAM2000 and JYNNEOS, which received emergency approval, have limited protective effects against monkeypox and may cause side effects in certain populations with compromised immune systems. Therefore, there is an urgent need to develop a safer, more effective, and long-lasting monkeypox virus vaccine to combat the potential increase in monkeypox prevalence.

[0004] Clinical studies have reported that while high levels of anti-monkeypox virus antibodies can be detected in severely ill patients with monkeypox co-infection and HIV, they have not effectively prevented the spread of monkeypox virus or alleviated infection symptoms. Humoral immunity and cellular immunity are effective barriers against viral infections. Viral clearance during monkeypox infection depends on the coordination of antibodies and T cells, with these two protective mechanisms complementing each other. Numerous clinical evidences support or reveal the effective antiviral role of T cells: even with low induced antibody levels after vaccination in individuals with monkeypox co-infection and HIV, T cell responses can effectively combat monkeypox virus infection; among naturally infected and healthy vaccinated individuals, 60% of those previously vaccinated against smallpox developed MPXV-specific T cell immune responses, providing potential protection against monkeypox virus infection; recovered individuals after monkeypox virus infection can activate more CD4+ and CD8+ T cells, and the duration and intensity of the T cell response are superior to those with neutralizing antibodies, further highlighting the importance of enhancing cellular immunity. Therefore, an ideal vaccine design needs to synergistically induce humoral and cellular immunity. On the one hand, the neutralizing antibodies induced by the humoral response can prevent the virus from infecting host cells, and on the other hand, the activated T cell response can resist viral infection and generate protective immune memory.

[0005] Based on this, in order to address the current prevalence of monkeypox virus co-infection with HIV and the possibility of its further widespread transmission in the population, there is an urgent need in the field to develop a monkeypox virus vaccine that can synergistically induce humoral and cellular immune responses, so that it can produce high-titer neutralizing antibodies and induce long-term protection, thereby providing safety guarantees for the general population and specific populations. This is also the technical problem that this invention aims to solve. Summary of the Invention

[0006] To address the aforementioned technical problems in this field, the present invention aims to provide a long-acting anti-orpoxvirus immunogen composition that can synergistically induce humoral and cellular immune responses, its preparation method, and its application. The immunogen inducing the humoral response originates from highly conserved envelope proteins of orpoxvirus; the immunogen inducing the cellular immune response originates from RNA polymerase and / or DNA polymerase expressed in the early and mid-stages of viral infection. This application provides a technical solution for loading these components separately into the same vector or different vectors, thereby achieving both broad-spectrum protection against orpoxviruses such as monkeypoxvirus and vaccinia virus, and stimulating a strong T-cell response for a long-lasting effect, thus achieving effective prevention and / or treatment of orpoxvirus.

[0007] The present invention achieves the above objectives by adopting the following technical solutions: On the one hand, the present application provides an immunogen composition comprising: (1) one or more first immunogens derived from RNA polymerase and / or DNA polymerase expressed in the early and / or mid-stages after orthopox virus infection, and inducing a cellular immune response; and (2) one or more second immunogens derived from antigenic peptides of intracellular mature viral particles (IMV) and / or extracellular envelope particles (EEV) of orthopox virus, and inducing a humoral immune response.

[0008] Secondly, this application provides an expression vector composition comprising one or more genetic expression vectors, said one or more genetic expression vectors comprising:

[0009] A genetic expression vector (a) containing a nucleic acid sequence encoding one or more first immunogens,

[0010] Genetic expression vector (b), which contains a nucleic acid sequence encoding one or more second immunogens, and / or

[0011] A genetic expression vector (c) comprising a nucleic acid sequence encoding one or more first immunogens and one or more second immunogens;

[0012] The first immunogen is derived from RNA polymerase and / or DNA polymerase expressed in the early and / or mid-stages after orthopoxvirus infection, inducing a cellular immune response; and / or the second immunogen is derived from antigenic peptides of intracellular mature viral particles (IMV) and / or extracellular envelope particles (EEV) of orthopoxvirus, inducing a humoral immune response.

[0013] Thirdly, this application provides the use of the immunogen composition or expression vector composition described herein in the preparation of a pharmaceutical composition for the prevention and / or treatment of orthopoxvirus infection in subjects.

[0014] Fourthly, this application provides a biological product comprising the immunogen composition or the expression vector composition described herein. Attached Figure Description

[0015] The present invention will be further described below with reference to the accompanying drawings. These drawings are only for illustrating embodiments of the present invention and are not intended to limit the scope of the invention.

[0016] Figure 1 illustrates the construction of the multivalent immunogen.

[0017] Figure 2 shows the expression of MHE, A35R, A35R-T-1, and A35R-T-2 mRNA transfected into HEK293T cells and detected by flow cytometry.

[0018] Figure 3 shows the long-term humoral immune response evaluation of BALB / c mice vaccinated with MHE, A35R, A35R-T-1, and A35R-T-2 mRNA.

[0019] Figure 4 shows the protection test and results of BALB / c mice vaccinated with MHE, A35R, A35R-T-1, and A35R-T-2 mRNA vaccines and challenged with vaccinia virus Tian Tan strain (TTV-752-1) one year after two injections.

[0020] Figure 5 shows the protection test and results of mice vaccinated with MHE, A35R, and A35R-T-2 mRNA, challenged with monkeypox virus 10 days after the first injection (with each mouse inoculated with 3E6 PFU of VAV vaccinia virus as a positive control).

[0021] ns or no label: indicates no significant difference; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001. Detailed Implementation

[0022] This application provides an immunogenic composition capable of inducing a long-lasting immune protective response against orthopoxvirus, its preparation method, and its application. The immunogenic composition and vaccine prepared from it provided by this application can induce neutralizing antibodies that can broadly neutralize different strains of vaccinia virus, and also achieve broad-spectrum protection against vaccinia virus and monkeypox virus, providing long-lasting immune response protection. The immunogenic composition provided by this application can provide 100% immune protection against lethal monkeypox virus challenge after one dose; after two doses, it can still resist high-dose lethal vaccinia virus challenge for one year. Therefore, this application provides a novel technical solution for the effective prevention and / or treatment of orthopoxvirus.

[0023] This invention provides a candidate orthopoxvirus mRNA immunogen that can synergistically induce humoral and cellular immunity. Compared with existing vaccines, the immunogen of this invention can induce both high-titer neutralizing antibodies against monkeypoxvirus and / or vaccinia virus and long-lasting protection. When used in vaccine preparation, it can be prepared quickly, conveniently, and simply. It can be used for vaccination of the general population as well as for specific populations such as those with weakened immune systems. It can serve as a broad-spectrum vaccine to address the current outbreak of monkeypox Clade I b and the prevalence of monkeypox virus co-infection with HIV.

[0024] In a first aspect, this application provides an immunogenic composition comprising: (1) one or more first immunogens derived from RNA polymerases and / or DNA polymerases expressed in the early and / or mid-stages of orthopoxvirus infection, and inducing a cellular immune response; and (2) one or more second immunogens derived from antigenic peptides of intracellular mature viral particles (IMV) and / or extracellular envelope particles (EEV) of orthopoxvirus, and inducing a humoral immune response. As used herein, “antigenic peptide” means a polypeptide or antigen-derived peptide that is immunogenic and immunoreactive, including exogenous and endogenous antigenic peptides. The antigenic peptides of intracellular mature viral particles (IMV) and / or extracellular envelope particles (EEV) of orthopoxvirus described herein include their structural proteins and derivatives thereof. The phrase “derived from…” as used herein includes the substance itself or a derivative thereof. For example, “derived from antigenic peptide” includes “antigenic peptide or a derivative thereof”.

[0025] First Immunogen

[0026] In some embodiments, the first immunogen is derived from RNA polymerase and / or DNA polymerase expressed in the early and / or mid-stages after orthopoxvirus infection and induces a cellular immune response. In some embodiments, the first immunogen inducing the cellular immune response comprises one or more selected from F8L, F4L, A6R, A25R, L6R, L4R proteins, their immunogenic fragments, or homologous proteins. In a preferred embodiment, the first immunogen inducing the cellular immune response comprises one or more selected from F8L, A25R, L6R proteins, their immunogenic fragments, or homologous proteins.

[0027] In some embodiments, the first immunogen inducing a cellular immune response is a conserved T-cell antigen. The term "conserved T-cell antigen" as used herein refers to a conserved T-cell antigen obtained by analyzing the T-cell consensus sequence of polymerase protein using immunological and bioinformatics methods, excluding the allergenic and toxic characteristics of T-cell antigens, thus improving its safety. In some embodiments, the conserved T-cell antigen is provided by the method described in the fifth aspect of this application. In some embodiments, the conserved T-cell antigen provided by the method described in the fifth aspect of this application is a conserved T-cell antigen against orthopoxvirus. In some embodiments, the sequence shown in SEQ ID NO:9 is obtained by the method described in the fifth aspect of this application. The sequence shown in SEQ ID NO:9 is a recombinant sequence derived from F8L, A25R, and L6R proteins or their immunogenic fragments or homologous proteins obtained by the method described in the fifth aspect of this application. In some embodiments, the first immunogen inducing a cellular immune response comprises the full length or a portion of the amino acid sequence shown in SEQ ID NO:9. In some embodiments, the first immunogen that induces a cellular immune response comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequence shown in SEQ ID NO:9.

[0028] In the context of describing immunogens of viruses in the genus *Orthopoxvirus*, the term "homologous protein" as used herein refers to a protein that is homologous between specific viral species within the genus *Orthopoxvirus* or between different evolutionary branches. In some embodiments, "homologous proteins" within the genus *Orthopoxvirus* includes homologous proteins of monkeypoxvirus and vaccinia virus. For example, monkeypoxvirus F4L and vaccinia virus E4L are homologous proteins, monkeypoxvirus A6R and vaccinia virus A5R are homologous proteins, monkeypoxvirus A25R and vaccinia virus A24R are homologous proteins, monkeypoxvirus L6R and vaccinia virus J6R are homologous proteins, and monkeypoxvirus L4R and vaccinia virus J4R are homologous proteins.

[0029] Second Immunogen

[0030] In some embodiments, the second immunogen is derived from the antigenic peptides of intracellular mature viral particles (IMV) and / or extracellular envelope particles (EEV) of orthopoxvirus, and induces a humoral immune response. In some embodiments, the antigenic peptides of the intracellular mature viral particles (IMV) include M1R, H3L, and E8L proteins or immunogenic fragments thereof or homologous fragments thereof. In specific embodiments, the homologous fragments of the M1R, H3L, and E8L proteins include amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the sequences of the M1R, H3L, and E8L proteins and having the same or substantially the same immunogenicity. In some embodiments, the full-length sequences of the M1R, H3L, and E8L proteins are shown in SEQ ID NO:2, 3, and 4, respectively. In some embodiments, the antigenic peptide of the intracellular mature viral particle (IMV) comprises the full length or a portion thereof of the amino acid sequence shown in SEQ ID NO:2, 3, and 4, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequences shown in SEQ ID NO:2, 3, and 4. In this context, the fusion protein of the M1R, H3L, and E8L proteins is abbreviated as "MHE". In some embodiments, MHE serves as a candidate immunogen. In some embodiments, the MHE fusion protein comprises the amino acid sequence shown in SEQ ID NO:8. In some embodiments, the MHE fusion protein comprises a portion of the amino acid sequence shown in SEQ ID NO:8. In some embodiments, the MHE fusion protein includes a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequence shown in SEQ ID NO:8.

[0031] In some embodiments, the antigenic peptide of the extracellular enveloped virus (EEV) comprises the A35R protein or an immunogenic fragment thereof or a homologous protein thereof. In some embodiments, the homologous fragment of the A35R protein comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with and having the same or substantially the same immunogenicity as the A35R protein. In some embodiments, the antigenic peptide of the extracellular enveloped virus comprises the extracellular portion of the A35R protein. In some embodiments, the A35R protein or its immunogenic fragment thereof or a homologous protein thereof comprises the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the extracellular portion of the A35R protein comprises the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the antigenic peptide of the extracellular enveloped virus (EEV) comprises the full length or a portion of the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the antigenic peptide of the extracellular envelope particle (EEV) includes a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequence shown in SEQ ID NO:1.

[0032] In some embodiments, the second immunogen described herein comprises a combination of M1R-H3L-E8L and A35R. In some embodiments, the second immunogen described herein comprises M1R-H3L-E8L. In some embodiments, the second immunogen described herein M1R-H3L-E8L comprises the amino acid sequence shown in SEQ ID NO:8. In some embodiments, the second immunogen described herein M1R-H3L-E8L comprises the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, and / or SEQ ID NO:4. In some embodiments, the second immunogen described herein M1R-H3L-E8L comprises a portion of the amino acid sequence shown in SEQ ID NO:8. In some embodiments, the second immunogen M1R-H3L-E8L herein comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequence shown in SEQ ID NO:8. In some embodiments, the second immunogen M1R-H3L-E8L herein comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, and / or SEQ ID NO:4.

[0033] In some embodiments, the second immunogen described herein comprises A35R. In the context of this application, "A35R" includes the A35R protein or an immunogenic fragment thereof or a homologous protein thereof. In some embodiments, "A35R" includes the extracellular portion of the A35R protein. In some embodiments, the second immunogen A35R described herein comprises the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the second immunogen A35R described herein comprises a portion of the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the second immunogen A35R described herein comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence shown in SEQ ID NO:1.

[0034] Immunogen Composition

[0035] In some embodiments, the immunogen composition of this application comprises M1R-H3L-E8L. In some embodiments, the immunogen composition of this application comprises a combination of M1R-H3L-E8L and A35R. In some embodiments, the immunogen composition of this application comprises a combination of A35R and a conserved T-cell antigen. In some embodiments, the immunogen composition of this application comprises a combination of M1R-H3L-E8L and a conserved T-cell antigen. In some embodiments, the immunogen composition of this application comprises a combination of M1R-H3L-E8L, A35R, and a conserved T-cell antigen.

[0036] In some embodiments, the immunogen composition of this application includes a conserved T-cell antigen as a first immunogen and a combination of M1R-H3L-E8L and A35R as a second immunogen.

[0037] The E8L, H3L, M1R, and A35R proteins mentioned in this application may be derived from the monkeypox virus MPXV-M5312_HM12_Rivers strain that emerged in 2022.

[0038] In some embodiments, the immunogen composition is provided as a multivalent vaccine or a combination vaccine. Both the multivalent and / or combination vaccine immunogen compositions can be administered via sequential or combined immunization. The immunogen compositions of the present invention, in the form of multivalent or combination vaccines, can simultaneously activate both anti-poxetine T-cell and antibody responses, achieving combined protection.

[0039] The immunogenic composition described herein can induce a long-lasting immune response against orthopoxvirus. As described herein, a "long-lasting" protective immune response means protection against high doses of lethal virus for one year or more after vaccination.

[0040] Connection sequence

[0041] In some embodiments, the immunogen composition of this application further includes a linker sequence for linking different immunogens. The linker sequence may include an IRES linker sequence and a adapter. In some embodiments, the linker sequence includes an IRES linker sequence, a rigid adapter, a shearable adapter, or a flexible adapter.

[0042] IRES connection sequence

[0043] Optionally, the "Internal Ribosome Entry Site Sequence (IRES)" described herein is derived from the non-coding region mRNA sequence of viruses, primarily ribonucleic acid viruses such as poliovirus, Coxsackievirus, enterovirus, hepatitis A virus, foot-and-mouth disease virus, and encephalomyocarditis virus. In a preferred embodiment, the IRES linker sequence includes a sequence derived from encephalomyocarditis virus, for example, as shown in SEQ ID NO:5.

[0044] connector

[0045] Optionally, the linkers described herein vary in amino acid length, including sequences of 1-28 amino acids. The linkers described herein include rigid linkers, flexible linkers, or shearable (or self-splicing) linkers. The linker sequence may contain glycine and serine to provide flexibility, glutamic acid and lysine to improve water solubility, or, when the linker is rigid, a helical structure capable of effectively separating the fusion proteins. For example, the linker sequence may include EGKSSGSGSESKST, GSAGSAAGSGEF, KESGSVSSEQLAQFRSLD, (GGGS)n, (EAAAK)n sequences, where n can be an integer of 0, 1, 2, 3, 4, or 5, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology and having the same or substantially the same function.

[0046] In some embodiments, the linker comprises a rigid linker. In some embodiments, the rigid linker comprises an EAAK linker or a (XP)n sequence rich in proline (Pro). A rigid linker having the (EAAAK)n sequence has an α-helical structure, providing rigid and stable spacers. In some embodiments, the immunogen compositions described herein employ an EAAK rigid linker. The (XP)n sequence has a proline (Pro) rich in proline (Pro), where X can specify any amino acid, preferably alanine, lysine, or glutamic acid. The (XP)n rigid linker provides rigidity and effectively separates the fusion proteins through the proline present therein. In some embodiments, the immunogen compositions described herein employ an (XP)n rigid linker.

[0047] In some embodiments, the adapter includes a shearable (or self-splicing) adapter. In some embodiments, the immunogenic compositions described herein employ adapters derived from 2A self-splicing peptide sequences, including but not limited to F2A derived from foot-and-mouth disease virus, E2A derived from Equine rhinitis A virus, P2A derived from Porcine teschovirus, T2A derived from Thosea asigna virus, or amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with and having the same or substantially the same function as those sequences.

[0048] In some embodiments, the envelope proteins M1R, H3L, and E8L derived from intracellular mature viral particles (IMV) are linked by a cleavable linker to achieve correct expression and translation of the same transcript. In some embodiments, the M1R-H3L-E8L proteins described in this application are linked by a P2A or T2A linker.

[0049] In some embodiments, the A35R protein derived from extracellular envelope particles (EEVs) is expressed as a separate transcript and / or linked to an immunogen that induces cellular immunity via a linker sequence. In some embodiments, the A35R protein is linked to a conserved T-cell antigen via a linker (e.g., a rigid linker) or a ribosome entry site sequence (IRES).

[0050] In some embodiments, the conserved T-cell antigen and A35R protein described in this application are linked via an IRES linker sequence. In some embodiments, the conserved T-cell antigen and A35R protein described in this application are linked via a rigid linker. In a preferred embodiment, the conserved T-cell antigen and A35R protein described in this application are linked via an EAAK linker.

[0051] In some embodiments, the immunogen composition described herein comprises M1R, H3L, and E8L proteins linked by P2A or T2A linkers, and a combination of A35R and a conserved T-cell antigen linked by an IRES linker sequence. In some embodiments, the immunogen composition described herein comprises M1R, H3L, and E8L proteins linked by P2A or T2A linkers, and a combination of A35R and a conserved T-cell antigen and / or a C-terminal protein degradation domain linked by an IRES linker sequence. The nucleic acid sequence of the C-terminal protein degradation domain is preferably a dihydrofolate reductase (DHFR) amino acid sequence. In some embodiments, the immunogen composition described herein comprises M1R, H3L, and E8L proteins linked by P2A or T2A linkers, and a combination of A35R and a conserved T-cell antigen linked by a rigid linker. In some embodiments, the immunogen composition described herein comprises M1R, H3L, and E8L proteins linked by P2A or T2A linkers, and a combination of A35R and a conserved T-cell antigen linked by an EAAK linker.

[0052] Secondly, this application provides an expression vector composition comprising one or more genetic expression vectors, said one or more genetic expression vectors comprising:

[0053] A genetic expression vector (a) containing a nucleic acid sequence encoding one or more first immunogens,

[0054] Genetic expression vector (b), which contains a nucleic acid sequence encoding one or more second immunogens, and / or

[0055] A genetic expression vector (c) comprising a nucleic acid sequence encoding one or more first immunogens and one or more second immunogens;

[0056] The first immunogen is derived from RNA polymerase and / or DNA polymerase expressed in the early and / or mid-stages after orthopoxvirus infection, inducing a cellular immune response; and / or the second immunogen is derived from antigenic peptides of intracellular mature viral particles (IMV) and / or extracellular envelope particles (EEV) of orthopoxvirus, inducing a humoral immune response.

[0057] In some embodiments, the first immunogen in the expression vector composition comprises a conserved T-cell antigen. The second immunogen is derived from antigenic peptides of intracellular mature viral particles (IMV) and / or extracellular envelope particles (EEV) of orthopoxvirus, inducing a humoral immune response.

[0058] In some embodiments, the coding sequence contained in the genetic expression vector has undergone codon optimization and RNA secondary structure optimization.

[0059] For the genetic expression vector (a), it comprises a nucleic acid sequence encoding one or more first immunogens. The first immunogen is derived from RNA polymerase and / or DNA polymerase expressed in the early and / or mid-stages after orthopoxvirus infection, inducing a cellular immune response. In some embodiments, the first immunogen inducing the cellular immune response comprises one or more proteins selected from F8L, F4L, A6R, A25R, L6R, L4R, or immunogenic fragments thereof, or homologous proteins thereof. In some embodiments, the genetic expression vector (a) comprises a genetic expression vector (a') comprising a nucleic acid sequence encoding one or more proteins selected from F8L, F4L, A6R, A25R, L6R, L4R, or immunogenic fragments thereof, or homologous proteins thereof. In a preferred embodiment, the first immunogen inducing the cellular immune response comprises one or more proteins selected from F8L, A25R, L6R, or immunogenic fragments thereof, or homologous proteins thereof. In some embodiments, the genetic expression vector (a) comprises a genetic expression vector (a”) containing a nucleic acid sequence encoding one or more of the F8L, A25R, L6R proteins or their immunogenic fragments or homologous proteins. In some embodiments, the first immunogen inducing the cellular immune response is a conserved T-cell antigen. As used herein, “conserved T-cell antigen” refers to a conserved T-cell antigen obtained by analyzing the T-cell consensus sequence of polymerase protein using immunological and bioinformatics methods, excluding the allergenic and toxic characteristics of T-cell antigens, thus improving its safety. In some embodiments, the genetic expression vector (a) comprises a genetic expression vector (a”') containing a nucleic acid sequence encoding a conserved T-cell antigen. In some embodiments, the conserved T-cell antigen is provided by the method described in the fifth aspect of this application. In some embodiments, the conserved T-cell antigen provided by the method described in the fifth aspect of this application is a conserved T-cell antigen against orthopoxvirus. In some embodiments, the conserved T-cell antigen encoded in the genetic expression vector (a”') that induces a cellular immune response comprises the full length or a portion thereof of the amino acid sequence shown in SEQ ID NO:9. In some embodiments, the conserved T-cell antigen encoded by the genetic expression vector (a”') comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence shown in SEQ ID NO:9. In some embodiments, the nucleic acid sequence encoding one or more first immunogens that induce a cellular immune response is codon- and RNA secondary structure optimized, including the nucleic acid sequence shown in SEQ ID NO:18.In some embodiments, the nucleic acid sequence encoding one or more first immunogens includes a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the nucleic acid sequence shown in SEQ ID NO:18.

[0060] For the genetic expression vector (b), it comprises a nucleic acid sequence encoding one or more second immunogens. In some embodiments, the genetic expression vector (b) comprises a genetic expression vector (b') comprising a nucleic acid sequence encoding at least one antigenic peptide of an intracellular mature viral particle (IMV) derived from orthopoxvirus. In some embodiments, the antigenic peptide of the intracellular mature viral particle (IMV) comprises M1R, H3L, and E8L proteins, or immunogenic fragments thereof, or homologous fragments thereof. In some embodiments, the genetic expression vector (b') comprises a nucleic acid sequence encoding M1R protein, H3L protein, and E8L protein, or immunogenic fragments thereof, or homologous fragments thereof. In some embodiments, after codon optimization and RNA secondary structure optimization, the nucleic acid sequences encoding M1R, H3L, and E8L proteins, or immunogenic fragments thereof, or homologous fragments thereof, respectively comprise the nucleic acid sequences shown in SEQ ID NO:14, 15, and 16. In some embodiments, the genetic expression vector (b') comprises the nucleic acid sequences shown in SEQ ID NO:14, 15, and 16. In some embodiments, the genetic expression vector (b') comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the nucleic acid sequences shown in SEQ ID NO:14, 15, and 16. In some embodiments, the genetic expression vector (b') comprises a nucleic acid sequence as shown in SEQ ID NO:19. In some embodiments, the genetic expression vector (b') comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the nucleic acid sequence shown in SEQ ID NO:19. The linkers between different proteins of the IMV (e.g., M1R, H3L, and E8L proteins) are preferably sequences derived from Porcine teschovirus P2A or from Thosea asigna virus T2A, in any order. P2A comprises the amino acid sequence shown in SEQ ID NO:6. T2A comprises the amino acid sequence shown in SEQ ID NO:7.

[0061] In some embodiments, the genetic expression vector (b) further includes a genetic expression vector (b”) comprising a nucleic acid sequence encoding an antigenic peptide derived from at least one extracellular envelope particle (EEV) of orthopoxvirus. In some embodiments, the antigenic peptide of the extracellular envelope particle (EEV) includes the A35R protein or an immunogenic fragment thereof or a homologous protein thereof. In some embodiments, the genetic expression vector (b”) comprises a nucleic acid sequence encoding the A35R protein or an immunogenic fragment thereof or a homologous fragment thereof. In some embodiments, the genetic expression vector (b”) comprises a nucleic acid sequence encoding the extracellular portion of the A35R protein. In some embodiments, the genetic expression vector (b”) comprises a nucleic acid sequence as shown in SEQ ID NO:17. In some embodiments, the genetic expression vector (b”) includes a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the nucleic acid sequence shown in SEQ ID NO:17.

[0062] For the genetic expression vector (c), it comprises nucleic acid sequences encoding one or more first immunogens and one or more second immunogens. In some embodiments, the genetic expression vector (c) includes a genetic expression vector (c') comprising nucleic acid sequences encoding at least one first immunogen and at least one antigenic peptide derived from extracellular envelope particles (EEVs). In some embodiments, the genetic expression vector (c) comprises, from its 5' end to its 3' end, nucleic acid sequences encoding a second immunogen, a linker sequence, and a first immunogen, and / or a nucleic acid sequence of a C-terminal protein degradation domain. In some embodiments, the genetic expression vector (c') comprises, from its 5' end to its 3' end, nucleic acid sequences of A35R protein or its immunogenic fragment or its homologous fragment, a linker sequence, and a conserved T-cell antigen. In some embodiments, the genetic expression vector (c') comprises, from its 5' end to its 3' end, nucleic acid sequences of A35R protein or its immunogenic fragment or its homologous fragment, a linker sequence, and a conserved T-cell antigen, and / or a nucleic acid sequence of a C-terminal protein degradation domain. In some embodiments, the linker sequence includes a rigid linker, an IRES linker sequence, a shearable linker, or a flexible linker. In a preferred embodiment, the linker sequence is a rigid linker. In a specific embodiment, the rigid linker is an EAAK linker. In some embodiments, the rigid linker encodes a rigid linker peptide that is helical and effectively separates the fusion protein segments, and its amino acid sequence is shown in SEQ ID NO:10. In some embodiments, the genetic expression vector (c') contains, from its 5' end to its 3' end, a nucleic acid sequence encoding the A35R protein or its immunogenic fragment or its homologous fragment, an IRES linker sequence, and a conserved T-cell antigen. In some embodiments, the genetic expression vector (c') contains, from its 5' end to its 3' end, a nucleic acid sequence encoding the A35R protein or its immunogenic fragment or its homologous fragment, a rigid linker, and a conserved T-cell antigen.

[0063] In some embodiments, or those referred to as "A35R-T-1", the genetic expression vector (c") comprises, from its 5' end to its 3' end, a nucleic acid sequence encoding the A35R protein or an immunogenic fragment or homologous fragment thereof, an IRES linker sequence, a conserved T-cell antigen, and / or a C-terminal protein degradation domain. The amino acid sequence of the C-terminal protein degradation domain is preferably a dihydrofolate reductase (DHFR) amino acid sequence, for example, as shown in SEQ ID NO:12. In the A35R-T-1 embodiment, the genetic expression vector (c") includes a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:13. In the A35R-T-1 embodiment, the genetic expression vector (c”) comprises the nucleic acid sequence shown in SEQ ID NO:20. In the A35R-T-1 embodiment, the genetic expression vector (c”) comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the nucleic acid sequence shown in SEQ ID NO:20.

[0064] In some embodiments, the genetic expression vector (c”') comprises, from its 5’ end to its 3’ end, a nucleic acid sequence encoding the A35R protein or an immunogenic fragment or a homologous fragment thereof, a rigid linker, and a conserved T-cell antigen. In some embodiments, or embodiments referred to as “A35R-T-2”, the genetic expression vector (c”') comprises, from its 5’ end to its 3’ end, a nucleic acid sequence encoding the A35R protein or an immunogenic fragment or a homologous fragment thereof, an EAAK linker, and a conserved T-cell antigen. In the A35R-T-2 embodiment, the genetic expression vector (c”') includes a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:11. In the A35R-T-2 embodiment, the genetic expression vector (c”') includes the nucleic acid sequence shown in SEQ ID NO:21. In the A35R-T-2 embodiment, the genetic expression vector (c”) includes a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the nucleic acid sequence shown in SEQ ID NO:21.

[0065] In some embodiments, the expression vector composition comprises a genetic expression vector (b') and a genetic expression vector (b”).

[0066] In some embodiments, the expression vector composition comprises a genetic expression vector (b') and a genetic expression vector (c").

[0067] In some embodiments, the expression vector composition comprises a genetic expression vector (b') and a genetic expression vector (c”').

[0068] In some embodiments, the expression vector composition comprises a genetic expression vector (a'), a genetic expression vector (b'), and a genetic expression vector (b''). In some embodiments, the expression vector composition comprises a genetic expression vector (a''), a genetic expression vector (b'), and a genetic expression vector (b''). In some embodiments, the expression vector composition comprises a genetic expression vector (a'''), a genetic expression vector (b'), and a genetic expression vector (b'').

[0069] In some embodiments, the genetic expression vector includes mRNA vectors, DNA plasmid vectors, recombinant viral vectors, and recombinant bacterial vectors. The mRNA vectors include, but are not limited to, linear, circular, and self-replicating vectors. The recombinant viral vectors include, but are not limited to, vectors containing poxvirus, adenovirus, adeno-associated virus, herpes simplex virus, measles virus, reovirus, rhabdovirus, tick-borne encephalitis virus, influenza virus, respiratory syncytial virus, and poliovirus. In some embodiments, the mRNA vector includes the basic elements required for in vitro transcription from the 5' end to the 3' end, including the T7 promoter, the 5' untranslated region (UTR), the coding sequence (CDS), the 3' untranslated region (UTR), and the polyadenylated tail (poly A) sequence. The 5'-UTR sequence, the 3'-UTR sequence, and the polyadenylated tail (poly A) sequence are as shown in the optimal combination described in Chinese Patent Application No. 202211129466.2. The coding sequences include, but are not limited to, sequences encoding immunogens that encode orthopoxvirus multiprotective epitopes that can induce humoral immune responses as described herein, and sequences encoding conserved T-cell antigens that can induce cellular immune responses.

[0070] In some embodiments, the expression vectors in the expression vector composition can be inserted into vectors of the same type or different types. Insertion of the expression vectors into multiple vectors of the same type constitutes a multivalent vaccine, or insertion into different vectors constitutes a combination vaccine. Both the multivalent and / or combination vaccines can be administered via sequential or combined immunization. The expression vector compositions of the present invention, inserted into vectors of the same type or different types in various ways, can simultaneously activate anti-poxetine T-cell and antibody responses, achieving combined protection.

[0071] The term "orthopoxvirus" as used in this article refers to the genus Orthopoxviridae within the subfamily Poxvirinae of chordates. Orthopoxviruses are double-stranded DNA viruses, including variola virus, vaccinia virus (VACV), cowpox virus, rabbitpox virus, monkeypox virus (MPXV), Akhmeta virus, and Alaska pox virus, along with their corresponding evolutionary branches. The evolutionary branches described in this article include, but are not limited to, the Tian Tan strain (TTV-752-1), the North American vaccine strain, the Wyeth derivative strain, the Listeria strain, the Ankara derivative strain, the Copenhagen strain, and the New York strain.

[0072] Monkeypox virus belongs to the genus Orthopoxviridae. Similar to many other members of the orthopoxviridae family, monkeypox virus produces two different forms of viral particles during its replication cycle: extracellular enveloped viral particles (EEV) and intracellular mature viral particles (IMV). The latter is released later in the infection cycle through lysis of the infected host.

[0073] Due to the conservation of protein-coding genes among members of the orthopoxvirus genus, particularly the conservation of viral surface proteins, infection with orthopoxvirus or vaccination with orthopoxvirus can provide cross-protection against other members of the genus. In response to the global health emergency, the FDA approved two smallpox vaccines, JYNNEOS and ACAM2000, for conditional use to prevent the spread of monkeypox virus. Previously, this cross-protection was also utilized by administering vaccinia virus (VACV) as a live virus vaccine to control smallpox transmission. Therefore, those skilled in the art can anticipate that although the vaccine provided in this application is developed against monkeypox virus and / or vaccinia virus, it may also provide partial cross-protection against other orthopoxviruses. Therefore, based on the experimental results demonstrated in this application, those skilled in the art can anticipate that the technical solution provided in this application can be used to prepare therapeutic and / or prophylactic pharmaceutical compositions for the treatment and / or prevention of orthopoxvirus.

[0074] Technical solutions for codon optimization

[0075] In a preferred embodiment, the expression vector composition is an mRNA vaccine, the mRNA vaccine comprising:

[0076] i) The codon-optimized M1R antigen sequence as described above, and the codon-optimized nucleic acid sequence is shown in SEQ ID NO:14;

[0077] ii) The H3L antigen sequence as described above is encoded with codon optimization, and the codon-optimized nucleic acid sequence is shown in SEQ ID NO:15;

[0078] iii) The codon-optimized E8L antigen sequence as described above, and the codon-optimized nucleic acid sequence is shown in SEQ ID NO:16;

[0079] iv) The codon-optimized A35R antigen sequence as described above, the codon-optimized nucleic acid sequence is shown in SEQ ID NO:17;

[0080] v) Encoding a codon-optimized conserved T-cell antigen sequence as described above, the codon-optimized nucleic acid sequence being shown in SEQ ID NO:18;

[0081] vi) The candidate immunogen (MHE) is a fusion protein encoding the mature intracellular viral particles of monkeypox virus as described above. The codon-optimized nucleic acid sequence is shown in SEQ ID NO:19.

[0082] vii) encodes the fusion protein (abbreviated as A35R-T-1, A35R-T-2) constructed from the monkeypox virus extracellular envelope particles and conserved T-cell antigens as described above, and the codon-optimized nucleic acid sequences are shown in SEQ ID NO:20 and SEQ ID NO:21, respectively.

[0083] Technical solutions involving NTP analogs

[0084] In a preferred embodiment, for the expression vector composition capable of simultaneously inducing humoral and cellular immune responses, when the vector is an mRNA vector, some or all of the uracil and / or cytosine nucleosides may be replaced during in vitro transcription to prepare mRNA. The replacement includes replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the uracil nucleosides in the mRNA with at least one substance selected from the group consisting of: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2'-thiouridine, 4'-thiouridine, 5'-methylcytosine, 5'-methyluridine, 2-thio-1-methyl-1-deaza- Pseudoruriidine, 2-thio-T-methyl-pseudoruriidine, 2-thio-5-aza-uridine, 2-thio-dihydropseudoruriidine, 2-thio-dihydrouridine, 2-thio-pseudoruriidine, 4-methoxy-2-thio-pseudoruriidine, 4-methoxy-pseudoruriidine, 4-thio-1-methyl-pseudoruriidine, 4-thio-pseudoruriidine, 5-aza-uridine, dihydropseudoruriidine, or 5-methoxyuridine and 2'-O-methyluridine. In a preferred embodiment, uracil nucleoside is replaced with pseudouridine or N1-methylpseudoruriidine or N1-ethylpseudoruriidine. In a more preferred embodiment, uracil nucleoside is replaced with N1-methylpseudoruriidine.

[0085] Thirdly, this application provides the use of the immunogen composition or expression vector composition described herein in the preparation of a pharmaceutical composition for the prevention and / or treatment of orthopoxvirus infection in a subject. In some embodiments, the subject has immunodeficiency or immunodeficiency. In some embodiments, the subject has HIV. In some embodiments, the subject is a patient with monkeypox co-infection with HIV. In some embodiments, the subject is a severely ill patient with monkeypox co-infection with HIV. In some embodiments, the subject has not been vaccinated against smallpox. In some embodiments, the subject is a human. In some embodiments, the pharmaceutical composition further includes one or more pharmaceutically acceptable carriers or excipients, preferably suitable for oral, intradermal, subcutaneous, intramuscular, or intranasal administration. In some embodiments, the preparation includes loading nucleic acids encoding the immunogen composition described herein, which can simultaneously induce humoral and cellular immune responses, into an mRNA vector and / or DNA plasmid vector to prepare a nucleic acid vaccine, or loading them into an adenovirus vector to prepare a viral vector vaccine, or displaying them onto a cell membrane to prepare an inactivated cell vaccine. The mRNA vectors include, but are not limited to, linear, circular, and self-replicating vectors. In some embodiments, the preparation includes preparing an mRNA vaccine from the expression vector composition described herein. In some embodiments, the preparation includes separately mixing the mRNA vaccine with cationic lipid nanoparticles to prepare a pharmaceutical composition. In some embodiments, the preparation includes mixing two different mRNA vaccines in a certain molar ratio and then mixing them with cationic lipid nanoparticles to prepare a pharmaceutical composition. In some embodiments, the mRNA vaccine prepared from the fusion protein candidate immunogen of mature intracellular monkeypox virus particles (MHE), and the mRNA vaccine prepared from the fusion protein constructed from monkeypox virus extracellular envelope particles and / or conserved T-cell antigens (A35R, A35R-T-1, A35R-T-2), can be separately mixed with cationic lipid nanoparticles to prepare a formulation, or the two different mRNA vaccines can be first mixed in a certain molar ratio and then mixed with cationic lipid nanoparticles to prepare a formulation. In some embodiments, the "certain molar ratio" includes 1:10 to 10:1, 1:9 to 9:1, 1:8 to 8:1, 1:7 to 7:1, 1:6 to 6:1, 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, or 1:1, or any molar ratio within the above ranges. In a preferred embodiment, the preparation involves mixing two different mRNA vaccines in a 1:1 molar ratio, and then mixing them with cationic lipid nanoparticles to prepare a pharmaceutical composition. In some embodiments, the preparation involves separately mixing the two mRNA vaccines with cationic lipid nanoparticles before preparing the pharmaceutical composition. In some embodiments, the pharmaceutical composition can be used in combination with other anti-vaccinia virus antibodies or antiviral drugs.

[0086] Fourthly, this application provides a biological product comprising the immunogenic composition or the expression vector composition described herein. In some embodiments, the biological product is a prophylactic and / or therapeutic biological product for the prevention and / or treatment of orthomyxovirus. In some embodiments, the biological product simultaneously induces humoral and cellular immune responses against orthomyxovirus. In some embodiments, the biological product induces a long-lasting protective immune response against orthomyxovirus.

[0087] In some embodiments, the bioproduct further includes one or more pharmaceutically acceptable carriers or excipients, preferably suitable for oral, intradermal, subcutaneous, intramuscular, or intranasal administration. In some embodiments, the bioproduct is delivered via cationic lipid nanoparticles.

[0088] In some embodiments, the biological product comprises mRNA and cationic lipid nanoparticles. In some embodiments, the biological product comprises a formulation formed by separately mixing an mRNA vaccine and cationic lipid nanoparticles. In some embodiments, the biological product comprises a formulation formed by first mixing two different mRNA vaccines in a specific molar ratio and then mixing them with cationic lipid nanoparticles. In some embodiments, the "specific molar ratio" includes 1:10 to 10:1, 1:9 to 9:1, 1:8 to 8:1, 1:7 to 7:1, 1:6 to 6:1, 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, or 1:1, or any molar ratio within the range of the above values. In some embodiments, the biological product comprises a formulation formed by first mixing two different mRNA vaccines in a 1:1 molar ratio and then mixing them with cationic lipid nanoparticles. In some embodiments, the biological product comprises a formulation formed by separately mixing two mRNA vaccines and cationic lipid nanoparticles.

[0089] In some embodiments, the biological products are provided in the form of multivalent or combination vaccines. Both the multivalent and / or combination vaccine biological products can be administered via sequential or combined immunization. The biological products of the present invention, in the form of multivalent or combination vaccines, can simultaneously activate both anti-orchiopeptidosis T-cell and antibody responses, achieving combined protection.

[0090] In some embodiments, the biological product may be used in combination with other anti-poxvirus antibodies or antiviral drugs. In some embodiments, the administration methods of the multivalent fusion immunogen monkeypox nucleic acid vaccine biological agent that induces humoral and cellular immune responses include, but are not limited to, intramuscular injection, nasal drop administration, nebulization administration, microneedle injection, subcutaneous injection, intradermal injection, and intramuscular injection. In preferred embodiments, the formulation or pharmaceutical composition is administered via intramuscular and subcutaneous injection. Further, considering the ease of administration, in a preferred embodiment, the administration is by intramuscular injection.

[0091] Biological agent technology solutions involving mRNA vaccines

[0092] In a preferred embodiment, the biological agent comprises:

[0093] a) An mRNA vaccine encoding a fusion protein candidate immunogen (MHE) of monkeypox virus intracellular mature viral particles (IMV);

[0094] b) mRNA vaccines encoding monkeypox virus extracellular envelope particles (EEV);

[0095] c) mRNA vaccines encoding monkeypox virus extracellular envelope particles (EEV) and conserved T-cell antigens;

[0096] d) mRNA vaccines containing multivalent fusion immunogens that simultaneously induce humoral and cellular immune responses; and / or

[0097] e) The delivery vector for the above-mentioned mRNA vaccine.

[0098] The biological agent can be used to prevent and / or treat monkeypox virus or its highly homologous vaccinia virus infection.

[0099] carrier / delivery tool

[0100] Furthermore, the transport carrier includes cationic liposomes, polymers, proteins, or lipid nanoparticles. In a preferred embodiment, the transport carrier includes cationic liposomes and cationic lipid nanoparticles. In a more preferred embodiment, the transport carrier includes cationic lipid nanoparticles. The formulation can be prepared by repeatedly mixing the cationic lipid nanoparticles with the mRNA vaccine.

[0101] In some embodiments, the mRNA vaccine prepared from the fusion protein candidate immunogen (MHE) of the monkeypox virus intracellular mature viral particles, and the mRNA vaccine prepared from the fusion protein (A35R, A35R-T-1, A35R-T-2) constructed from monkeypox virus extracellular envelope particles and / or conserved T-cell antigens, can be prepared by mixing with cationic lipid nanoparticles separately to form a formulation. Alternatively, the two different mRNA vaccines can be mixed in a certain molar ratio before being mixed with the cationic lipid nanoparticles to form a formulation. In some embodiments, the "certain molar ratio" includes 1:10 to 10:1, 1:9 to 9:1, 1:8 to 8:1, 1:7 to 7:1, 1:6 to 6:1, 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, or 1:1, or any molar ratio within the above ranges. In some embodiments, the two different mRNA vaccines are first mixed in a 1:1 molar ratio before being mixed with the cationic lipid nanoparticles to form a formulation. In some embodiments, the prepared mRNA vaccine is mixed with cationic lipid nanoparticles separately to prepare a formulation.

[0102] In a fifth aspect, this application provides a method for obtaining the conserved T-cell antigen sequence for inducing cellular immune responses as described herein, the method comprising:

[0103] a) Search and download the DNA polymerase F8L, RNA polymerase A25R, and L6R protein sequences of various viruses in the Orthopodovirus genus from the Genebank database for comparison;

[0104] b) Using the predictive tools NetMHCIpan and NetMHCIIpan, 12 MHC-I and 54 MHC-II supertypes covering more than 90% of the population were selected as pre-bound HLA types;

[0105] c) Analyze the three protein sequences one by one, screen out the MHC-I and MHC-II epitopes with high binding capacity, and map the potential dominant epitopes to the original sequence;

[0106] d) Improve vaccine safety by eliminating the allergenic and toxic characteristics of T antigens using tools ALLERGENFP V1.0, ALLERGENFP V2.0, and AllergenFP. Obtain conserved T-cell antigens.

[0107] The "conserved T-cell antigen" described herein refers to a conserved T-cell antigen obtained by analyzing the T-cell consensus sequence of polymerase protein using immunological and bioinformatics methods, excluding the allergenic and toxic characteristics of T-cell antigens, thus improving its safety. In some embodiments, the conserved T-cell antigen provided by the method described in the fifth aspect is a conserved T-cell antigen against orthopoxvirus. In some embodiments, the amino acid sequence of the conserved T-cell antigen obtained by this method is shown in SEQ ID NO:9. The sequence shown in SEQ ID NO:9 is derived from F8L, A25R, and L6R proteins or their immunogenic fragments or homologous proteins. In some embodiments, the codon-optimized nucleic acid sequence encoding the conserved T-cell antigen sequence described herein is shown in SEQ ID NO:18.

[0108] All numerical ranges provided herein are intended to clearly include all values ​​falling between the endpoints of the range and the range of values ​​between them. Features mentioned in the invention or embodiments may be combined. All features disclosed in this specification may be used in any combination form, and each feature disclosed in the specification may be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0109] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0110] Example

[0111] To further illustrate the technical means and effects of this invention, specific embodiments are described below. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art can make appropriate modifications and variations to this invention, all of which are within the scope of this invention.

[0112] Unless otherwise specified, all techniques or conditions used in the examples were conventional methods, performed according to the techniques or conditions described in the literature in this field, or generally according to conventional conditions such as those described in Sambrook et al., *Molecular Cloning: A Laboratory Guide* (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations in the product instructions. All reagents and instruments used were commercially available products through legitimate channels. Unless otherwise stated, percentages and parts are by weight.

[0113] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to one skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this application. The preferred embodiments and materials described herein are for illustrative purposes only.

[0114] Materials, Methods and Animals

[0115] The mRNA preparation, mRNA vaccine preparation, animal immunization protocols, and detection methods involved in the experiments described in this example are as follows:

[0116] Biosafety and Ethics Statement

[0117] The mouse rearing and related experiments involved in this study were conducted at Fudan University. Routine immunization was performed in a specific pathogen-free (SPF) laboratory, vaccinia virus challenge experiments were conducted in a biosafety level 2 laboratory, and monkeypox virus challenge experiments were conducted in a biosafety level 3 laboratory. All experimental procedures were approved by the Ethics Committee of Shanghai Public Health Clinical Center before implementation, and the experimental process was strictly carried out in accordance with the established procedures.

[0118] laboratory animals

[0119] All experimental mice were specific pathogen-free (SPF) grade. Among them, BALB / c mice (female, 6-8 weeks old) were purchased from Suzhou Huachang Biotechnology Co., Ltd. and housed in the SPF experimental area of ​​Shanghai Public Health Clinical Center. Vaccine virus challenge experiments were conducted in the biosafety level 2 laboratory of Shanghai Public Health Clinical Center. Monkeypox virus challenge experiments were conducted in the animal biosafety level 3 (ABSL3) facility of Shenzhen Third People's Hospital.

[0120] Cells and viruses

[0121] HEK-293T and Vero E6 cell lines (human embryonic kidney cells 293) were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. Monkeypox virus CladeIIb was obtained from a clinical isolate from the Third People's Hospital of Shenzhen, and vaccinia virus Tiantan strain TTV-752-1 was obtained from the Shanghai Public Health Clinical Center. Both monkeypox virus and vaccinia virus were propagated in Vero E6 cells.

[0122] Experimental methods

[0123] I. Design and synthesis of immunogens

[0124] The E8L, H3L, M1R, and A35R proteins involved in this invention are derived from the monkeypox virus MPXV-M5312_HM12_Rivers strain that emerged in 2022.

[0125] The various fusion immunogens involved in this invention are constructed as follows:

[0126] (1) M1R-H3L-E8L construct (MHE): The full-length M1R protein, H3L protein, and E8L protein of monkeypox virus (amino acid sequences are shown in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively) were used to construct the fusion immunogen.

[0127] (2) The extracellular envelope particle A35R immunogen construct uses the extracellular terminus encoding the monkeypox virus A35R protein, as shown in SEQ ID NO:1.

[0128] (3) The fusion protein construct of extracellular envelope particle A35R and conserved T cell antigen was constructed as follows:

[0129] Conserved T-cell antigens were obtained through the following methods:

[0130] a) Search and download the DNA polymerase F8L, RNA polymerase A25R, and L6R protein sequences of various viruses in the Orthopodovirus genus from the Genebank database for comparison;

[0131] b) Using the predictive tools NetMHCIpan and NetMHCIIpan, 12 MHC-I and 54 MHC-II supertypes covering more than 90% of the population were selected as pre-bound HLA types;

[0132] c) Analyze the three protein sequences one by one, screen out the MHC-I and MHC-II epitopes with high binding capacity, and map the potential dominant epitopes to the original sequence;

[0133] d) Improve vaccine safety by eliminating the allergenic and toxic characteristics of T antigens using tools ALLERGENFP V1.0, ALLERGENFP V2.0, and AllergenFP. Obtain conserved T-cell antigens.

[0134] The conserved T-cell antigen eliminates the allergenic and toxic characteristics of T-cell antigens, thus improving safety. The conserved T-cell antigen obtained by the method is a conserved T-cell antigen against orthopoxvirus. The amino acid sequence of the conserved T-cell antigen obtained by the method is shown in SEQ ID NO:9.

[0135] The specific constructs of the fusion protein between the extracellular envelope particle A35R and the conserved T-cell antigen (abbreviated as A35R-T-1 and A35R-T-2, whose encoded amino acid sequences are shown in SEQ ID NO:13 and SEQ ID NO:11, respectively) of different immunogens MHE, A35R, A35R-T-1, and A35R-T-2 were optimized using eukaryotic cell codons. The codon-optimized nucleic acid sequences are shown in SEQ ID NO:19, SEQ ID NO:17, SEQ ID NO:20, and SEQ ID NO:21, respectively. These were synthesized by Shanghai Sangon Biotech Co., Ltd., and the four nucleic acid sequences were loaded into mRNA expression vectors to prepare the corresponding DNA template sequences for mRNA, as shown in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively.

[0136] II. Preparation of mRNA vaccines containing various immunogens

[0137] The different immunogenic expression vectors MHE, A35R, A35R-T-1, and A35R-T-2 constructed in step I were linearized using a single enzyme digestion method. mRNA molecules were then prepared in vitro via co-transcriptional capping. Following the nearshore T7 in vitro co-transcription kit (E331), N1-methylpseudouridine triphosphate was used to completely replace uridine triphosphate during in vitro transcription, resulting in a one-step co-transcriptional capping method to produce the native Cap1 structure. The corresponding modified mRNAs were synthesized according to the manufacturer's instructions, purified by lithium chloride precipitation, washed three times with 75% ethanol, air-dried, dissolved in RNase-free water, and the concentration was determined using Nanodrop. The prepared mRNA molecules were sent to Nohhai Life Sciences Instruments (Shanghai) Co., Ltd. According to the specific grouping scheme described below, two different mRNA vaccines were first mixed in a 1:1 molar ratio, and then mixed with cationic lipid nanoparticles to prepare an mRNA cationic lipid nanoparticle vaccine for subsequent mouse experiments.

[0138] III. Animal Immunization Program

[0139] The first protocol immunization experiment was designed to evaluate the differences in immunogenicity and induced long-term immune responses of the MHE, A35R, A35R-T-1, and A35R-T-2 mRNA vaccines in BALB / c mice.

[0140] (1) Mice were immunized with immunogen.

[0141] Using 6-8 week old BALB / c mice as a model, 5-6 mice in each group were administered the vaccine via intramuscular injection. In each group, 5 μg of different immunogens were injected (MHE and A35R or A35R-T-1 or A35R-T-1 were prepared as mRNA vaccines and then mixed in equal weights before injection). The control group received an equal volume of Empty-LNP (or "airborne vector"), 100 μL. Two injections were administered at 3-week intervals according to the "Prime-Boost" immunization strategy. The mouse groups are shown in the table below.

[0142] Table 1. Grouping of mouse immunization experiments

[0143] (2) To evaluate the differences in immunogenicity of MHE, A35R, A35R-T-1, and A35R-T-2 mRNA vaccines in BALB / c mice and the long-term immune responses they induce.

[0144] Blood samples were collected at different time points / days after vaccination and monitored for one year. The levels of neutralizing antibodies against monkeypox virus in the serum of different groups of mice were assessed by the FRNT (Focus Reduction Neutralization Test) and used for subsequent immunoassays.

[0145] The second immunization protocol was to evaluate the long-term protective efficacy of MHE, A35R, A35R-T-1, and A35R-T-2 mRNA vaccines against vaccinia virus challenge in BALB / c mice. BALB / c mice aged 6-8 weeks were used as a challenge model. One year after two injections, mice were challenged via intranasal drip, with a vaccinia virus dose of 5E6 PFU. Each virus was inoculated at a volume of 30 μL. Body weight changes and survival were continuously observed in different groups for 14 days.

[0146] The third immunization experiment evaluated the protective efficacy of the mRNA vaccines MHE, A35R, and A35R-T-2 against monkeypox virus challenge in BALB / c mice. Six- to eight-week-old BALB / c mice were used as a challenge model and were challenged intranasally on day 11 after inoculation with vaccinia virus strain TTV-752-1 (dose: 3E6 PFU / mouse). The monkeypox virus challenge dose was 5E5 FFU, and the inoculation volume for each virus was 30 μL. Body weight changes and survival were continuously observed in different groups for 14 days.

[0147] IV. Detection Methods

[0148] (1) Flow cytometry detection of target protein expression

[0149] a) 24 hours after transfection, cells were collected by flow cytometry and centrifuged at 500g for 5 minutes;

[0150] b) Wash once with 0.5 mL of staining buffer, centrifuge at 500 g for 5 minutes; carefully aspirate the supernatant with a pipette, being careful not to aspirate cells from the bottom of the tube;

[0151] c) Prepare the fluorescent antibody mixture (all four antibodies were purchased from Yiqiao Shenzhou: E8L antibody catalog number 40890-MM14, H3L antibody catalog number 40893-T62, A35R antibody catalog number 40886-T62, and M1R antibody catalog number 40889-T62, diluted 1:100). Mix the antibodies thoroughly, centrifuge briefly for 10 seconds to remove any remaining antibody from the tube wall. Add 50 μL / sample to the sample and mix well. Incubate at 4°C for 30 min. The antibodies should be handled on ice to prolong their shelf life. Staining system: 1*10 6 50 μL reaction system.

[0152] d) After the reaction, wash once with 0.8 mL of staining buffer, incubate at 500 g for 5 minutes, and discard the supernatant. Then add mouse anti-rabbit IgG-FITC and goat anti-mouse IgG-FITC antibodies at a ratio of 1:100, incubate at 4°C for 30 minutes, wash the cells, resuspend in staining buffer, and perform detection using a Fortessa instrument. Resuspend in 250 μL and perform detection using flow cytometry.

[0153] (2) Virus neutralization test of monkeypox virus and vaccinia virus (Focus reduction neutralization test, FRNT)

[0154] a) Seed Vero-E6 cells into 96-well plates one day in advance, with 1.5 × 10⁶ cells seeded per well. 4 Cells were cultured overnight at 37°C with 5% CO2. The next day, monkeypox virus neutralization experiments were conducted in the BSL-3 laboratory, and vaccinia virus neutralization experiments were conducted in the BSL-2 laboratory.

[0155] b) On the day of the experiment, in 96-well U-bottom dilution plates, plasma was serially diluted 4-fold with maintenance medium or neutralizing antibody was serially diluted 3-fold with maintenance medium, creating 6 or 9 concentration gradients. Virus control (VC) and cell control (CC) were added with maintenance medium. 10% complement was added to each 50 μL system.

[0156] c) Remove the virus solution from the -80°C freezer and thaw it at room temperature inside the biosafety cabinet.

[0157] d) Dilute the melted virus stock solution (5 μL / 100 μL) with an appropriate amount of maintenance medium, and add an equal volume to the 96-well dilution plate containing the sample. Add culture medium to the cell control.

[0158] e) Place the dilution plate in a 37°C incubator and incubate for 1 hour.

[0159] f) Take Vero-E6 cells from a 96-well plate, discard the cell culture medium, add 50 μL of sample-virus mixture to each well, and add 50 μL of maintenance culture medium to each cell control well.

[0160] g) Place the 96-well plate of cells in a 37-degree incubator and incubate for 18 hours.

[0161] h) After 18 hours, Vero-E6 cells infected for 18 hours were removed from the incubator, and the sample-virus mixture in the 96-well cell culture plate was discarded.

[0162] i) Add 200 μl of 4% paraformaldehyde to each well of the culture plate in the biosafety cabinet, then immerse it in 4% paraformaldehyde, fix it at room temperature for 30 minutes, and then take it out of the laboratory.

[0163] j) Discard the paraformaldehyde, add 100 μl of 0.2% Triton X-100 to each well, and break the membrane for 10 minutes.

[0164] k) Discard 0.2% Triton X-100, add 200 μl PBS to each well, and let stand for 5 minutes.

[0165] 1) Discard the PBS, add 200 μl of PBS to each well, and let stand in the biosafety cabinet for 5 minutes.

[0166] m) Discard PBS and add 50 μl of HRP-conjugated anti-VACV (Invitrogen) polyclonal antibody to each well.

[0167] n) After incubating at room temperature for 2 hours, wash 3 times with PBS, add 50 μl of substrate per well for color development for 10 minutes.

[0168] o) Wash once with PBS, and detect the number of spots using an ELISpot scanner. Calculate the neutralization inhibition rate: Inhibition rate % = [1 - (mean number of spots in sample wells - mean number of spots in cell controls) / (mean number of spots in virus control - mean number of spots in cell controls)] × 100%. Calculate the ID using Graphpad Prism 9.0 software. 50 .

[0169] The present patent will be further described in detail below with reference to the accompanying drawings and specific experiments. Unless otherwise specified, the reagents, instruments, equipment and methods used in this patent are all commercially available reagents, instruments, equipment and methods conventional in this technical field.

[0170] Example 1. Construction of fusion protein candidate immunogen

[0171] The fusion immunogens MHE, A35R, A35R-T-1, and A35R-T-2 were constructed according to Experimental Method I described above, and the schematic diagrams of the construction are shown in Figure 1.

[0172] Example 2. Flow cytometry detection of MHE, A35R, A35R-T-1, and A35R-T-2 mRNA transfected into HEK293T cells.

[0173] Prepare the mRNAs corresponding to the fusion immunogens MHE, A35R, A35R-T-1, and A35R-T-2 according to Experimental Method II, and then use Lipofectamine. TM 3000 Reagent transfection reagent was mixed separately with mRNA molecules and transfected into HEK293T cells for validation. One day prior to transfection, HEK293T cells were seeded at a density of 200,000 cells / well in 12-well plates using DMEM complete medium. 1 μg of mRNA was transfected per well with Opti-MEM (Invitrogen) diluted with LIpofectamine. TM A mixture of 3000 mg / L was prepared at a mass-to-volume ratio of 1:2 and added dropwise to a 12-well plate. The plate was incubated at 37°C for 24 hours, and samples were collected for analysis. Flow cytometry was used for validation. The primary antibodies were specific antibodies against M1R, H3L, E8L, and A35R, and the secondary antibody was a FITC-conjugated fluorescent secondary antibody.

[0174] As shown in Figure 2, all candidate mRNA molecules were highly expressed after transfection into HEK293T cells. Specifically, the expression rates of M1R, H3L, and E8L in the fusion immunogen group MHE were 99.7%, 91.63%, and 98.6%, respectively, while the expression rate of A35R after transfection was as high as 91.7%. The expression efficiency of A35R in the A35R-T-1 and A35R-T-2 groups was lower, at 41.63% and 58.6%, respectively, presumably because the larger molecule size after fusion with the conserved T antigen had a certain impact on protein expression. The high level of protein expression indicates that the candidate mRNA molecules have good efficacy.

[0175] Example 3. Immunogenicity evaluation of MHE, A35R, A35R-T-1, and A35R-T-2 mRNA vaccines in BALB / c mice.

[0176] In this embodiment, the correctly expressed mRNA from Example 2 was selected and prepared into an mRNA cationic lipid nanoparticle vaccine for animal immunoassay evaluation. The specific antibody response against monkeypox virus in mice immunized with the vaccine was detected according to Experimental Method III described above. The levels and duration of neutralizing antibodies induced by various mRNA vaccines were evaluated. Blood samples were collected at different time points after vaccination (blood collection times are shown in Figure 3A), and the neutralizing capacity of different vaccine groups against monkeypox virus was analyzed using a dot-matrix neutralization assay.

[0177] The results are shown in Figures 3B-3D, where GMT represents the geometric mean titer of the antibody. All mRNA vaccine groups induced high titers of monkeypox virus-specific neutralizing antibodies: 3 weeks after the primary immunization, the geometric mean titer of neutralizing antibodies induced by the three mRNA vaccine groups reached 220 or higher; when two booster doses were administered 3 weeks later, the geometric mean titer of neutralizing antibodies in the three groups reached 16,000-18,000, an increase of at least 55-75 times. Over time, at 24 and 54 weeks after the booster doses, the antibody titers against monkeypox virus reached approximately 1,300-3,000. After two doses, up to week 54, the neutralizing antibody titer in the multivalent fusion immunogen MHE+A35R group (containing only the envelope protein group) decreased to 2060. However, the neutralizing antibody titer in the mRNA vaccine group containing conserved T-cell antigens, including the MHE+A35R-T-1 and MHE+A35R-T-2 groups, did not decrease significantly compared to week 24 after the booster. This further demonstrates the important role of virus-specific T-cell synergistic assistance and also proves that the technical solution of this application has achieved unexpected technical effects in providing long-term protection for up to one year.

[0178] Example 4. Evaluation of the protective effect of MHE, A35R, A35R-T-1, and A35R-T-2 mRNA vaccines against vaccinia virus challenge in BALB / c mice.

[0179] This embodiment further explores the protective effects of three mRNA vaccines against VAV in BALB / c mice. Intranasal challenge was then performed at week 54, with a viral dose of 5E6 PFU. Weight changes and mortality were monitored for 20 days post-challenge to assess the long-term protective effects of the three mRNA vaccines.

[0180] We continuously observed the changes in body weight and survival of mice in different groups. We found that the body weight of mice in the control group continued to decrease. In the unvaccinated group, all mice in the space-time LNP control group died on day 7 after infection. The multivalent fusion immunogen MHE+A35R group with only the envelope protein also all died within 6-7 days (Figure 4B). During the 20-day observation period, the body weight of the MHE+A35R-T-1 and MHE+A35R-T-2 mRNA inoculation groups decreased to some extent and gradually recovered starting 8 days after infection. The MHE+A35R-T-1 mRNA inoculation group showed a protection efficiency of 40% against lethal vaccinia virus, while the MHE+A35R-T-2 inoculation group achieved a protection efficiency of up to 80% (Figures 4A and 4B). This further illustrates the key role of virus-specific T cells in virus clearance. These data provide preliminary evidence for the MHE+A35R-T-2 vaccine as a broad-spectrum anti-orthozvovirus vaccine.

[0181] Example 5. Evaluation of the protective effect of MHE, A35R, and A35R-T-2 vaccines against monkeypox virus challenge in BALB / c mice.

[0182] Given that in Examples 2, 3, and 4, only the fusion immunogen mRNA vaccine (MHE+A35R) with envelope protein neutralizing antibody epitopes could induce a high antibody response in BALB / c mice, and the mRNA vaccine (MHE+A35R-T-2) with conserved T-cell antigen could provide protection against vaccinia virus challenge for up to one year, this example further explores the protective effect against monkeypox virus in different groups of mice.

[0183] Following the third protocol of the animal immunization protocol in Experimental Method III, BALB / c mice aged 6-8 weeks were used as a challenge model. The immunization strategy is shown in Figure 5A. Intranasal challenge was performed on day 11 after the first injection. The challenge dose of monkeypox virus was 5E5 FFU, with a volume of 40 μL. Weight changes and mortality were monitored for 14 days post-challenge to assess the protective efficacy of different vaccine groups against monkeypox virus.

[0184] The results are shown in Figures 5B and 5C. Continuous observation of body weight changes and survival in different groups revealed that, compared to the VCV-positive control group, both the MHE+A35R mRNA vaccine group and the MHE+A35R-T-2 mRNA vaccine group completely protected mice from lethal monkeypox virus infection. In terms of body weight changes, the MHE+A35R-T-2 mRNA vaccine group promoted faster weight recovery in mice within the group; furthermore, the body weight change in the MHE+A35R-T-2 mRNA vaccine group was smaller, further demonstrating the important role of the fusion-conserved T-cell antigen in viral clearance.

[0185] As can be seen from the above embodiments, the immunogen composition MHE+A35R-T-2, which integrates envelope protein and conserved T-cell antigen, provided by the present invention encodes multiple protective epitopes of MPXV through two mRNA molecules. This allows it to induce the production of high-titer, long-lasting neutralizing antibodies against monkeypox virus and / or vaccinia virus, and to provide effective protection against lethal vaccinia virus challenge in mice for up to one year. This provides a fundamental guarantee for addressing the current prevalence of co-infection of monkeypox virus and HIV, as well as the potential widespread transmission caused by the Clade I b monkeypox virus outbreak outside of Africa.

[0186] All references to this application are incorporated herein by reference as if each reference were individually incorporated herein by reference. Furthermore, it should be understood that after reading the foregoing teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

[0187] Appendix: Sequence numbers, sequence names, and specific sequences of nucleic acid molecules used in each step of the examples:

Claims

1. An immunogen composition comprising: (1) One or more primary immunogens derived from RNA polymerases and / or DNA polymerases expressed in the early and / or mid-stages following orthopoxvirus infection, and which induce a cellular immune response; and (2) One or more second immunogens derived from the antigenic peptides of intracellular mature viral particles (IMV) and / or extracellular envelope particles (EEV) of orthopoxvirus, and which induce humoral immune responses.

2. The immunogen composition as described in claim 1, The first immunogen includes one or more selected from F8L, F4L, A6R, A25R, L6R, L4R proteins or their immunogenic fragments or homologous proteins; and / or The antigenic peptides of the intracellular mature viral particles (IMV) mentioned above include M1R, H3L, and E8L proteins or their immunogenic fragments or homologous fragments; and / or The antigenic peptides of the extracellular envelope particles (EEVs) include the A35R protein or its immunogenic fragments or homologous proteins.

3. The immunogenic composition of claim 1, wherein the first immunogen comprises one or more selected from F8L, A25R, L6R proteins or their immunogenic fragments or homologous proteins.

4. The immunogen composition of claim 1, wherein the first immunogen comprises a conserved T-cell antigen.

5. The immunogen composition of claim 1, wherein the first immunogen comprises the full length or a portion thereof of the amino acid sequence shown in SEQ ID NO:9, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequence shown in SEQ ID NO:

9.

6. An expression vector composition comprising one or more genetic expression vectors, said one or more genetic expression vectors comprising: A genetic expression vector (a) containing a nucleic acid sequence encoding one or more first immunogens, Genetic expression vector (b), which contains a nucleic acid sequence encoding one or more second immunogens, and / or A genetic expression vector (c) comprising a nucleic acid sequence encoding one or more first immunogens and one or more second immunogens; Wherein, the first immunogen originates from RNA polymerase and / or DNA polymerase expressed in the early and / or mid-stages after orthopoxvirus infection, inducing a cellular immune response; and / or The second immunogen is derived from the antigenic peptides of intracellular mature viral particles (IMV) and / or extracellular envelope particles (EEV) of orthopoxvirus, which induce humoral immune responses.

7. The expression vector composition of claim 6, wherein... The genetic expression vector (b) includes a genetic expression vector (b') containing a nucleic acid sequence encoding at least one intracellular mature viral particle (IMV) antigenic peptide derived from orthopoxvirus. The genetic expression vector (b) further includes a genetic expression vector (b”) containing a nucleic acid sequence encoding at least one antigenic peptide derived from an extracellular envelope particle (EEV) of orthopoxvirus, and / or The genetic expression vector (c) includes a genetic expression vector (c') containing a nucleic acid sequence encoding at least one first immunogen and at least one antigenic peptide derived from extracellular envelope particles (EEVs).

8. The expression vector composition of claim 6, wherein the genetic expression vector (c) comprises, from the 5' end to the 3' end, a nucleic acid sequence encoding a second immunogen, a linker sequence, and a first immunogen.

9. The expression vector composition of claim 6, wherein the first immunogen comprises a conserved T-cell antigen.

10. The expression vector composition of claim 8, wherein the linkage sequence comprises a rigid connector, an IRES linkage sequence, a shearable connector, or a flexible connector; preferably, the linkage sequence is a rigid connector.

11. The expression vector composition of claim 7, wherein... The genetic expression vector (b') contains nucleic acid sequences encoding M1R protein, H3L protein, and E8L protein, or immunogenic fragments thereof, or homologous fragments thereof; and / or The genetic expression vector (b”) contains a nucleic acid sequence encoding the A35R protein or its immunogenic fragment or homologous fragment; and / or The genetic expression vector (c') contains, from the 5' end to the 3' end, a nucleic acid sequence encoding the A35R protein or its immunogenic fragment or its homologous fragment, a linker sequence, and a conserved T-cell antigen. Optionally, the connection sequence further includes a rigid joint, an IRES connection sequence, a shearable joint, or a flexible joint; preferably, the connection sequence is a rigid joint. Optionally, the coding sequence contained in the genetic expression vector has undergone codon optimization and RNA secondary structure optimization.

12. The expression vector composition of claim 6, wherein the genetic expression vector comprises an mRNA vector, a DNA plasmid vector, a recombinant viral vector, and a recombinant bacterial vector.

13. Use of the immunogen composition of any one of claims 1 to 5 or the expression vector composition of any one of claims 6 to 12 in the preparation of a pharmaceutical composition for the prevention and / or treatment of orthopoxvirus infection in a subject.

14. A biological product comprising the immunogen composition as described in any one of claims 1 to 5 or the expression vector composition as described in any one of claims 6 to 12.

15. The biological product of claim 14, wherein it is a prophylactic and / or therapeutic biological product for the prevention and / or treatment of orthopoxvirus; said biological product induces a long-lasting immune protective response against orthopoxvirus.

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