Mpox vaccine

Abstract

Provided is a vaccine against viral infections belonging to the genus Orthopox, the vaccine comprising a non-replicating attenuated vaccinia virus DIs strain.

Description

Empox vaccine

[0001] The present invention relates to a vaccine for viral infections belonging to the genus Orthopox, particularly a vaccine for empox, which includes a non-replicating attenuated vaccinia virus DIs strain.

[0002] Empox is an acute exanthematous disease caused by infection with the empoxvirus, characterized primarily by fever and rash. The natural host of the empoxvirus is suspected to be rodents inhabiting Africa, and it is believed that humans become infected through bites from infected animals or through contact with the blood, bodily fluids, or skin lesions of infected animals. There are two main lineages of empoxvirus: clade I and clade II, with clade I exhibiting higher pathogenicity than clade II.

[0003] Traditionally, empox cases were mainly reported in central and western Africa, and outbreaks outside endemic areas were limited to travelers from those areas. However, since May 2022, there has been a sharp increase in human-to-human transmission cases of clade IIb empoxvirus, mainly in Europe and North America, with over 107,000 infections and more than 230 deaths worldwide. While most patients are men who have sex with men (MSM), cases of infection in children and pregnant women have also been reported. Recovery usually occurs spontaneously within 2 to 4 weeks, but it can become severe in children, pregnant women, and immunocompromised individuals.

[0004] Furthermore, while the Democratic Republic of the Congo (DRC) has long experienced an epidemic of empoxvirus clade I, 2023 saw the highest number of infections and deaths to date, with infections spreading within the DRC through sexual contact between men and women and same-sex partners, as well as through household transmission. In July 2024, the first cases of empoxvirus clade Ib were reported in Burundi, Rwanda, Uganda, and neighboring Kenya, all bordering the DRC. Moreover, cases of infection believed to be imported cases of empoxvirus clade I have been detected in Sweden and Thailand, raising concerns about a global spread of the infection, not just in the DRC and surrounding countries.

[0005] On the other hand, the vaccinia virus DIs strain is a highly attenuated vaccinia virus strain isolated by subculturing the vaccinia virus Dalian strain (DIE strain) in chicken egg embryos (Non-Patent Literature 1). This DIs strain can proliferate in chicken embryonic fibroblasts (CEF), but can hardly proliferate in other mammalian cells (Non-Patent Literature 2). This makes it possible to apply it to immunocompromised and immunosuppressed individuals, and it is thought to have advantages such as not leaving an inoculation mark.

[0006] In previous smallpox vaccine development, the DIs strain was comparable to other replicating vaccinia viruses at 1 × 10⁻¹⁰ 6 Doses below PFU levels did not induce a sufficient immune response, and therefore it was not approved as a smallpox vaccine.

[0007] Vaccinia virus vaccines, which are vaccines for smallpox, are believed to be effective in protecting against not only smallpox but also empox. Currently, two vaccinia virus vaccines are approved as empox vaccines: the modified vaccinia virus Ankara (MVA) strain "MVA-BN," which is a non-proliferating chicken fibroblast-adapted mutant strain, and the proliferating attenuated vaccinia virus LC16m8 strain "Dried Cell Culture Smallpox Vaccine LC16 "KMB"," a third-generation smallpox vaccine developed in Japan.

[0008] However, the gene region that ensures the non-proliferative and low-virulence properties of the MVA strain has not been fully elucidated (Non-Patent Literature 3). Furthermore, although the LC16m8 strain is a highly safe proliferating vaccinia virus strain, there is a possibility of the emergence of a reverting virus that can proliferate (Non-Patent Literature 4), and the possibility of it showing proliferation when inoculated into immunocompromised or immunosuppressed individuals cannot be ruled out.

[0009] Tagaya I, et al., A new mutant of dermovaccinia virus. Nature, 1961, vol. 192, p. 1187-1188Ishii K, et al., Structural analysis of vaccinia virus DIs strain: application as a new replication-deficient viral vector. Virology, 2002, vol. 302(2), p. 433-444Meisinger-Henschel C, et al., Introduction of the six major genomic deletions of Modified Vaccinia Virus Ankara (MVA) into the parental vaccinia virus is not sufficient to reproduce an MVA-like phenotype in cell culture and in mice. J. Virol. Vol. 84 (19), p. 9907-9919.Kidokoro M, et al., Genetically stable and fully effective smallpox vaccine strain constructed from highly attenuated vaccinia LC16m8. 2005, vol. 102 (11), p. 4152-4257

[0010] Given this situation, it is crucial that the empox vaccine, which targets a large number of unspecified recipients including immunosuppressed and immunocompromised individuals, possesses both efficacy and safety, making the development of further vaccines a challenge.

[0011] The inventors of the present invention have conducted diligent studies to solve the above problems and have found that the non-replicating attenuated vaccinia virus DIs strain has a vaccine effect against empox, thereby completing the present invention. That is, the present invention is as follows: [1] A vaccine for viral infections belonging to the genus Orthopox, comprising the non-replicating attenuated vaccinia virus DIs strain. [2] The vaccine according to [1], wherein the virus belonging to the genus Orthopox is empox. [3] The vaccine according to [1], administered by subcutaneous injection, intramuscular injection, intradermal injection, epidermal scraping, or nasal administration. [4] 10 8 [1] The vaccine described in [1], administered in a dose of 5 to 100 μL of PFU. [5] A pharmaceutical composition for viral infections belonging to the genus Orthopox, comprising a non-replicating attenuated vaccinia virus DIs strain. [6] The pharmaceutical composition described in [5], wherein the virus belonging to the genus Orthopox is empox. [7] The pharmaceutical composition described in [5], administered by subcutaneous injection, intramuscular injection, intradermal injection, epidermal scraping, or nasal administration. [8] 10 8 [5] The pharmaceutical composition described in [9], administered in a dose of 5 to 100 μL of PFU. [9] A method for the prevention or treatment of a viral infection belonging to the genus Orthopox, comprising the step of administering a non-replicating attenuated vaccinia virus DIs strain.

[10] The method according to [9], wherein the virus belonging to the genus Orthopox is empox.

[11] The method according to [9], administered by subcutaneous injection, intramuscular injection, intradermal injection, epidermal scraping, or nasal administration.

[12] 10 8 The method according to [9], administered in doses of 5 to 100 μL of PFU.

[13] A non-replicating attenuated vaccinia virus DIs strain for use in the prevention or treatment of viral infections belonging to the genus Orthopox.

[0012] The present invention provides a vaccine against viral infections belonging to the Orthopox genus, comprising a non-replicating attenuated vaccinia virus DIs strain. The vaccine of the present invention exhibits particularly strong efficacy against empox.

[0013] This figure shows the analysis method for immunization induction against vaccinia virus and empoxvirus by the DIs strain. This figure shows the induction effect of Tfh cells and GC B cells in groups inoculated with the LC16m8 strain and the DIs strain. This figure shows the results of antibody titer measurement against each virus-derived antigen. This figure shows the results of antibody titer measurement against each virus-derived antigen. This figure shows the results of measurement of activated memory CD4-positive T cells (CD69+ in CD4+ CD44+ T cells) and memory CD8-positive T cells (CD69+ in CD8+ CD44+ T cells). This figure shows the results of neutralizing antibody titer measurement against empoxvirus. This figure shows the protective effect against empoxvirus infection in mice inoculated with the DIs strain. This figure shows the protective effect against empoxvirus infection in mice inoculated with the DIs strain. This figure shows the results of safety evaluation of the DIs strain using cynomolgus monkeys, a non-human primate model. This figure shows the results of quantitative PCR measurement of vaccinia virus DNA in the organs of cynomolgus monkeys. This figure shows the protective effect of the DIs strain against empoxvirus infection in cynomolgus monkeys. This figure shows the results of observing skin pathology in cynomolgus monkeys after empoxvirus infection.

[0014] This invention relates to an empox vaccine containing a non-replicating vaccinia virus DIs strain. 1. Overview Since the non-replicating attenuated vaccinia virus DIs strain (hereinafter also simply referred to as the "DIs strain") is non-replicating in mammals, the inventors considered that it would be possible to inoculate the DIs strain at a higher titer, thereby inducing higher immunity. Furthermore, 1 × 10 8 Since no skin lesions or viremia were observed when PFU's DIs strain was administered intravenously, we considered that the DIs strain could be a safe and effective empox vaccine. Furthermore, because the non-replicating vaccinia virus DIs strain has a large gene deletion region in its genome, making it less likely to undergo reversion to a highly virulent strain, the development of an empox vaccine using this strain is considered highly beneficial. Therefore, the inventors evaluated the immune-inducing effect and infection-protective effect against empox by inoculating with the DIs strain.

[0015] In this invention, 1 × 10 8 Wild-type mice inoculated twice with PFU's DIs strain at 4-week intervals induced binding and neutralizing antibodies against empoxvirus, confirming its protective effect against empox infection. Furthermore, safety and protective effect against empoxvirus infection were also confirmed in cynomolgus monkeys, a non-human primate model. Based on these results, we considered the possibility that the non-replicating vacciniavirus DIs strain could become an empox vaccine with high safety and efficacy, and thus completed the present invention.

[0016] 2. Non-reproducing attenuated vaccinia virus DIs strain The non-reproducing attenuated vaccinia virus DIs strain is a highly attenuated vaccinia virus strain isolated by subculturing the Dalian strain of vaccinia virus (DEI strain) in chicken egg embryos (Tagaya I, et al., A new mutant of dermovaccinia virus. Nature, 1961, vol. 192, p. 1187-1188). It can proliferate in chicken embryonic fibroblasts (CEF) but is non-reproducing in other mammalian cells, making it highly safe. The DIs strain can be prepared according to the above-mentioned known method (Tagaya I, et al, Nature, 1961, vol. 192, p. 1187-1188), or it can be obtained from the National Institute of Infectious Diseases. To maintain the DIs strain, it should be subculturized and grown on chicken fibroblasts or developing chicken egg paraalucleate, and stored under refrigeration or cryopreservation. It can be thawed and used at the time of use.

[0017] 3. Vaccine against viruses belonging to the genus Orthopoxvirus and pharmaceutical composition for preventing or treating the infectious disease The present invention relates to a vaccine against an infectious disease caused by a virus belonging to the genus Orthopoxvirus, and further relates to a pharmaceutical composition containing the above Vaccinia virus DIs strain. The genus Orthopoxvirus includes variola virus, smallpox virus, vaccinia virus, cowpox virus, etc., and many of these viruses show serological cross-reactions. Therefore, the virus that exhibits the effect as a vaccine in the present invention is the above virus. In a preferred embodiment, the vaccine of the present invention is particularly a vaccine against variola. Therefore, the present invention provides a pharmaceutical composition as a preventive and therapeutic agent for an infectious disease caused by a virus belonging to the genus Orthopoxvirus, particularly variola infection.

[0018] Examples of infectious diseases caused by viruses belonging to the genus Orthopoxvirus include smallpox, variola, cowpox, horsepox, camelpox, etc. The pharmaceutical composition of the present invention can be introduced into a living body by any known method, for example, injection such as intramuscular, intraperitoneal, intradermal or subcutaneous, epidermal abrasion administration, intranasal (nasal cavity) administration, inhalation from the oral cavity or lungs, oral administration, but subcutaneous injection, intramuscular injection, intradermal injection, epidermal abrasion administration or intranasal administration is preferred.

[0019] In addition, the pharmaceutical composition of the present invention can be mixed with known pharmaceutically acceptable carriers such as excipients, bulking agents, binders, lubricants, etc., buffers, isotonic agents, chelating agents, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, etc. The pharmaceutical composition of the present invention can be administered orally or parenterally according to the form, such as oral dosage forms such as tablets, capsules, powders, granules, pills, liquids, syrups, etc., and parenteral dosage forms such as injections, external preparations, suppositories, eye drops, etc.

[0020] The dosage is appropriately selected according to the type of active ingredient, administration route, administration subject, patient's age, weight, sex, symptoms and other conditions. As a single dose of the virus, in the case of oral administration, it is about 1×10 3 ~1×10 9 PFU (plaque forming units), preferably 1×10 5~1 x 10 8 The amount is about PFU, and in the case of parenteral administration it is 1 × 10⁶ 3 ~1 x 10 9 Approximately PFU, preferably 1 × 10 5 ~1 x 10 8 The PFU level is about the same. The virus can be administered once a day, or it can be administered in several doses with intervals of several days between doses (2, 3, 4, 5, 6 doses, etc.).

[0021] The DIs strain of the present invention can be used as a preventive or therapeutic vaccine for viral infections belonging to the Orthopox genus, particularly empox infection, but it is preferable to measure the antibody titer or cellular immune activity as a vaccine in advance. For example, the antibody titer against the DIs strain of the present invention can be obtained by inoculating mice, rabbits, etc. with these virus strains, collecting serum or plasma over time, and measuring the ELISA titer against the virus-derived antigen protein. This allows confirmation of viral gene expression and the presence or absence of an immune response in the inoculated individuals. In the present invention, an increase in antibody titer against the viral antigen protein was observed in mouse serum inoculated with the DIs strain of the present invention starting two weeks after inoculation. In the present invention, the dosage and administration when using the virus as a vaccine can be the same as the dosage and administration when using it as a pharmaceutical composition.

[0022] Furthermore, cellular immune activity can be measured by inoculating mice with the DIs strain of the present invention, isolating spleen cells from the immunized mice, and determining whether vaccinia virus-specific CD4-positive and CD8-positive cells are induced or activated using FACS or ELISPOT methods. In this invention, activated T cells were detected when spleen cells derived from animals immunized with the DIs strain of the present invention were co-cultured with vaccinia virus and empoxvirus protein. This indicates that immunization with the DIs strain of the present invention induces cellular immunity specific to viral antigen proteins in animals such as mice.

[0023] From the above, it has been confirmed that the DIs strain of the present invention induces humoral and cellular immunity, particularly against Empox. Furthermore, the DIs strain of the present invention has, or is expected to have, an elimination effect (a disease-preventing effect through immune response) against all reported genotypes of Empox.

[0024] The present invention will be described in more detail below with reference to examples. However, the scope of the present invention is not limited to these examples.

[0025] Method: In this invention, 1 × 10⁶ mice were introduced to wild-type mice, namely C57BL / 6J mice and BALB / c mice. 8 PFU's DIs strain was administered twice at 4-week intervals via epidermal inoculation (ss) or intradermal inoculation (id). One week after the second DIs inoculation, plasma, lymph node, and splenocytes were collected, and antibody and cellular immunity induction were evaluated. Flow cytometry using lymphocytes collected from lymph nodes was used to measure the induction of follicular helper T cells (Tfh), which play a crucial role in the induction of antigen-specific B cells, and the induction of germinal center B (GC B) cells that produce IgG. In addition, the binding antibody titers in plasma were measured by ELISA using vaccinia virus-derived antigens (L1R, B5R, A27L) and empoxvirus-derived antigens (M1R, L1R, I1L, H3L, B6R, A35R, A33R, A29L), and the antigen-specific T cells in the spleen were measured by flow cytometry.

[0026] For the measurement of antigen-specific T cells, we also used vaccinia virus strains LC16m8 and DIs. Furthermore, we administered 1 × 10⁶ cells to CAST / EiJ mice, which are wild-type mice susceptible to empoxvirus infection. 8 PFU's DIs strain was administered epidermally twice at 4-week intervals, followed by intranasal infection with clade I or clade II empoxvirus two weeks after the second dose. Daily weight changes after empoxvirus infection and infectious viral load in lung tissue 9 days after infection were measured to evaluate the protective effect against empoxvirus infection.

[0027] Next, the safety of the DIs strain and its protective effect against smallpox virus were evaluated using cynomolgus monkeys, a non-human primate model. Individuals intravenously administered with the LC16m8 strain and the Lister strain, which are replicating vaccinia viruses, at 1×10 8 PFU were used as the control group. Ten days after intravenous administration of 1×10 8 PFU of the DIs strain, skin symptoms were observed, various organs were collected, and viral dissemination in the body was measured by PCR to evaluate safety. In addition, groups inoculated intradermally twice with 1×10 8 PFU of the DIs strain at 4-week intervals, a group inoculated intradermally once with 1×10 8 PFU of the LC16m8 strain, and a non-vaccinated group were intravenously inoculated with smallpox virus, and the protective effect was evaluated by measuring the amount of virus in plasma and observing skin symptoms after smallpox virus infection.

[0028] Results: The results are shown in Figures 1 to 12. Figure 1: shows the analytical method for immune induction against vaccinia virus and smallpox virus by the DIs strain. Using the group inoculated intradermally once with 2×10 5 PFU of the LC16m8 strain as the control group, C57BL / 6J mice and BALB / c mice were inoculated epidermally or intradermally twice with 1×10 8 PFU of the DIs strain at 4-week intervals, and samples were taken 1 week after the second inoculation. Antigen-specific binding antibodies (IgG) were measured by ELISA using plasma, and the induction of follicular helper T cells (Tfh), which play an important role in the induction of antigen-specific B cells, and the induction of germinal center B (GC B) cells that produce IgG were measured by flow cytometry using lymphocytes prepared from lymph nodes. The antigen-specific T cell response was analyzed by flow cytometry using immune cells prepared from the spleen.

[0029] Figure 2: Compared with the group inoculated once with the LC16m8 strain, which is the control group, in mice inoculated twice with the DIs strain, both epidermally inoculated and intradermally inoculated individuals could induce good Tfh cells and GC B cells. The same effect was observed in both C57BL / 6J mice and BALB / c mice.

[0030] Figure 3: In the measurement of binding antibodies (IgG: immunoglobulin G) by ELISA using plasma, in C57BL / 6J mice inoculated with the DIs strain (1×10 8 PFU), in both the epidermal inoculation group (s.s.) and the intradermal inoculation group (i.d.), an increase in antibody titers against vaccinia virus-derived antigens (L1R, B5R, A27L) and smallpox virus-derived antigens (M1R, I1L, H3L, B6R, A35R) was observed 4 weeks after the first inoculation (Day 28), and a further increase in antibody titers was recognized by inoculating the DIs strain a second time (Day 35). On the other hand, in the group inoculated once with the LC16m8 strain (2×10 5 PFU), the antibody titer was lower compared to the DIs strain double-inoculation group.

[0031] Figure 4: The same test using BALB / c mice also showed results similar to the antibody induction seen in C57BL / 6J mice (Figure 3).

[0032] Figure 5: Memory CD4-positive T cells (CD69+ in CD4+ CD44+ T cells) and memory CD8-positive T cells (CD69+ in CD8+ CD44+ T cells) activated by flow cytometry were measured after restimulating splenocytes with vaccinia virus-derived antigens (L1R, B5R, A27L), or smallpox virus-derived antigens (M1R, L1R, I1L, H3L, B6R, A35R, A33R, A29L), the LC16m8 strain, or the DIs strain for 18 hours. Activation of memory CD4-positive T cells and memory CD8-positive T cells was hardly observed with restimulation with vaccinia virus-derived antigens (L1R, B5R, A27L), or smallpox virus-derived antigens (M1R, L1R, I1L, H3L, B6R, A35R, A33R, A29L), or the LC16m8 strain, but significant activation of memory CD4-positive T cells and memory CD8-positive T cells was observed in both C57BL / 6J mice and BALB / c mice when restimulated with the DIs strain.

[0033] Figure 6: The neutralizing antibody titer against smallpox virus was measured at 1×10 8The measurements were performed using serum from mice that had received two doses of PFU's rDIs-S vaccine at 4-week intervals. In serum obtained from mice inoculated with rDIs-S, neutralizing antibody titers of 256-fold or higher were detected against all three empoxvirus strains (clade I, clade IIa, and clade IIb).

[0034] Figure 7: To evaluate the protective effect against empoxvirus infection in mice inoculated with the DIs strain, 1 × 10⁶ mice were inoculated into wild-type mice susceptible to empoxvirus infection (CAST / EiJ mice). 8 PFU's DIs strain was administered epidermally twice at 4-week intervals, and two weeks after the second dose, participants were infected intranasally with either clade I (Zr-599 strain) or clade IIb (TK-006 strain) of empoxvirus. When infected intranasally with clade I (Zr-599 strain), rapid weight loss was observed from 6 days after infection in the unvaccinated group (PBS(-) vaccinated group), but no weight loss was observed in the DIs strain vaccinated group. Similarly, when infected intranasally with clade IIb (TK-006 strain), weight loss was observed in the unvaccinated group (PBS(-) vaccinated group), but no weight loss was observed in the DIs strain vaccinated group.

[0035] Figure 8: Furthermore, organs were collected 9 days after empoxvirus infection, and the amount of infectious empoxvirus in lung tissue was measured by plaque assay. In Zr-599 strain infection, the PBS(-) inoculated group showed 1 × 10⁶ levels. 8 Although high viral loads exceeding PFU / g organ were detected, all four animals in the DIs strain inoculation group were below the detection limit. Similar results were obtained in the TK-006 strain infection, with the PBS(-) inoculation group showing 1 × 10⁶ viral loads. 6 Infectious viral loads exceeding PFU / g organ were detected, but in the DIs strain inoculated group, all four mice showed levels below the detection limit. These results indicate that the DIs strain exhibits a preventive effect against clade I and clade IIb mouse models of empoxvirus infection.

[0036] Figure 9: Using the non-human primate model, the crab-eating monkey, 1 × 10 8The safety of intravenous administration of PFU's DIs strain was evaluated. For comparison, the replicating attenuated vaccinia virus strains LC16m8 and Lister were administered at 1 × 10⁻⁶ doses. 8 Intravenous administration was performed using PFU. In the groups inoculated with the LC16m8 strain and the Lister strain, skin conditions caused by vaccinia virus proliferation were observed 10 days after inoculation, but no skin conditions were observed in the group inoculated with the non-proliferating DIs strain.

[0037] Figure 10: Furthermore, various organs were collected 10 days after intravenous inoculation with vaccinia virus, and vaccinia virus DNA in the organs was measured by quantitative PCR to investigate vaccinia virus transmission in the body. In individuals inoculated with the Lister strain, vaccinia virus DNA was detected in the intestinal tract, ovaries, lymph nodes, and skin lesions, but in individuals inoculated with the DIs strain, it was below the detection limit in all measured organs. Together with the results in Figure 9, we believe that non-proliferative activity in cynomolgus monkeys has been confirmed.

[0038] Figure 11: The protective effect of DIs strains against empoxvirus infection was evaluated in cynomolgus monkeys. 1 × 10 8 In the group that received two intradermal inoculations of PFU's DIs strain at 4-week intervals, 1 × 10⁶ 8 In a study comparing a group that received one intradermal inoculation of PFU's LC16m8 strain with a group that did not receive the vaccine, the results were 5 × 10⁶ 7 PFU's empoxvirus was administered intravenously. In individuals that received two intradermal inoculations with the DIs strain, no vaccinia virus DNA was detected in the blood after inoculation. However, in the group inoculated with the LC16m8 strain, vaccinia virus DNA was detected in the blood of 2 out of 3 individuals one week after inoculation, indicating transient viremia. On the other hand, in the groups inoculated with either the DIs strain or the LC16m8 strain, empoxvirus DNA was below the detection limit in the blood after intravenous infection with empoxvirus, but elevated levels of empoxvirus DNA were observed in all 3 individuals in the unvaccinated group.

[0039] Figure 12: Observation of skin conditions 10 days after empoxvirus infection revealed that skin lesions were observed in all three unvaccinated monkeys, but no skin lesions attributable to empoxvirus infection were observed in the groups vaccinated with the DIs strain or the LC16m8 strain. These results indicate that the DIs strain is non-proliferative and highly safe in cynomolgus monkeys, while also being able to prevent empoxvirus infection.

Claims

1. A vaccine against viral infections belonging to the Orthopox genus, including non-replicating attenuated vaccinia virus DIs strains.

2. The vaccine according to claim 1, wherein the virus belonging to the genus Orthopox is empox.

3. The vaccine according to claim 1, administered by subcutaneous injection, intramuscular injection, intradermal injection, epidermal scraping, or nasal administration. 4.10 8 The vaccine according to claim 1, administered in a dose of 5 to 100 μL of PFU.

5. A pharmaceutical composition for viral infections of the Orthopox genus, containing a non-replicating, attenuated vaccinia virus DIs strain.

6. The pharmaceutical composition according to claim 5, wherein the virus belonging to the genus Orthopox is empox.

7. The pharmaceutical composition according to claim 5, administered by subcutaneous injection, intramuscular injection, intradermal injection, epidermal scraping, or nasal administration. 8.10 8 The pharmaceutical composition according to claim 5, administered in a dose of 5 to 100 μL of PFU.

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