WO2024131664A1 - Vaccin contre le virus de l'herpès simplex et son utilisation - Google Patents

Vaccin contre le virus de l'herpès simplex et son utilisation Download PDF

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WO2024131664A1
WO2024131664A1 PCT/CN2023/139118 CN2023139118W WO2024131664A1 WO 2024131664 A1 WO2024131664 A1 WO 2024131664A1 CN 2023139118 W CN2023139118 W CN 2023139118W WO 2024131664 A1 WO2024131664 A1 WO 2024131664A1
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glycoprotein
seq
mrna
vaccine
hsv
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PCT/CN2023/139118
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Chinese (zh)
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诸兵
张莹
孙世超
华大威
张蕊
孙伟桢
张涛
张培培
唐莹
李超
唐纬坤
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南京奥罗生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/03—Herpetoviridae, e.g. pseudorabies virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention belongs to the field of biomedicine and virology, and specifically relates to a herpes simplex virus vaccine and application thereof.
  • Herpes simplex virus belongs to the alphaherpesviridae family of the herpesvirus genus. It is a double-stranded linear DNA virus in the herpesviridae family. HSV has two serotypes: HSV-1 and HSV-2. The genome sequence homology between the two is about 50%, and except for the specific glycoprotein gC, the other structures are highly similar. There are great differences in the transmission routes and epidemiological characteristics of the two subtypes of HSV.
  • HSV-1 is mainly transmitted through saliva and is the main pathogen of oral herpes; HSV-2 is mainly transmitted through sex and is the main pathogen of genital herpes; in addition, HSV-1 and HSV-2 can also be transmitted from mother to baby during delivery (mainly HSV-2), resulting in neonatal herpes, and HSV infection during pregnancy can also cause spontaneous abortion or fetal growth retardation.
  • Symptoms of HSV infection include the formation of blisters in the skin or mucous membranes of the mouth, lips and/or genitals.
  • HSV is a neuroinvasive virus that causes occasional recurrence events of viral reactivation in infected individuals. HSV is spread through contact with infected areas of the skin during the period of viral activation. HSV-1 is more associated with nongenital infection and is most often acquired during childhood through nonsexual contact.
  • HSV-2 is the cause of most genital herpes.
  • herpes simplex virus (HSV) is also a major risk factor for human immunodeficiency virus (HIV) infection.
  • WHO released the world's first HSV-2 infection survey report, which showed that 536 million people aged 15 to 49 were infected with HSV-2, and there were as many as 23 million new cases of infection each year.
  • studies have shown that the risk of HSV-2 seropositive people being infected with HIV-1 is three times higher than that of HSV-2 seronegative people. In areas with high prevalence of HSV-2, the attributable risk of HSV-2 to HIV infection is 47%.
  • Measures to prevent the spread of HSV-2 through sexual behavior include the use of condoms and antiviral treatment. The use of these measures can reduce the transmission of HSV-2 by about 50%, but it cannot fundamentally eliminate the spread of the virus. Whether from the perspective of individual protection or public health, the most effective and economical way to fight HSV-2 is to get vaccinated with HSV-2 vaccine.
  • nucleic acid vaccines can induce humoral and cellular immune responses, have a low effective dose, are simple to operate, can be effectively and quickly tested, are cost-effective and reproducible in large-scale production and separation, can be produced at a high frequency and easily separated, and are more temperature stable than conventional vaccines.
  • the mRNA technology is stable, has a long shelf life, is easy to store and transport, and is unlikely to require a cold chain. In recent years, researchers have been working on the application of mRNA technology in the field of disease prevention and treatment.
  • HSV herpes simplex virus
  • the present invention provides a herpes simplex virus vaccine and its application.
  • the present invention adopts a combined antigen method to prepare a vaccine, the antigens involved all adopt a truncated form of protein, and the corresponding nucleic acid coding sequence is sequence optimized, the expression efficiency of the protein and the stability of mRNA are improved, the mutation rate and the difficulty of LNP packaging are reduced, the stability of mRNA is improved and the immunogenicity is reduced.
  • the first aspect of the present invention provides a herpes simplex virus glycoprotein or an immunogenic fragment thereof, which includes any one of glycoprotein B, glycoprotein C, glycoprotein D or glycoprotein E, and the amino acid sequences of the glycoprotein B, glycoprotein C, glycoprotein D or glycoprotein E are shown in SEQ ID NO: 50-53, respectively.
  • the N-terminus of the amino acid sequence of the glycoprotein B, glycoprotein C, glycoprotein D or glycoprotein E contains a signal peptide.
  • glycoprotein B amino acid sequence of glycoprotein B, glycoprotein C, glycoprotein D or glycoprotein E is as shown in SEQ ID NO: 5-8, or as shown in SEQ ID NO: 9-12, respectively.
  • a second aspect of the present invention provides a combination of herpes simplex virus glycoproteins or immunogenic fragments thereof, which include at least two of glycoprotein B, glycoprotein C, glycoprotein D or glycoprotein E, wherein the amino acid sequences of the glycoprotein B, glycoprotein C, glycoprotein D or glycoprotein E are respectively shown in SEQ ID NO: 50-53, or respectively shown in SEQ ID NO: 5-8, or respectively shown in SEQ ID NO: 9-12, or respectively shown in SEQ ID NO: 46-49.
  • the combination comprises glycoprotein C, glycoprotein D and glycoprotein E; or the combination comprises glycoprotein B, glycoprotein C, glycoprotein D and glycoprotein E.
  • the combination comprises glycoprotein C with an amino acid sequence as described in SEQ ID NO:51, glycoprotein D with an amino acid sequence as described in SEQ ID NO:52, and glycoprotein E with an amino acid sequence as described in SEQ ID NO:53; or,
  • the combination comprises glycoprotein C with an amino acid sequence as described in SEQ ID NO:6, glycoprotein D with an amino acid sequence as described in SEQ ID NO:7, and glycoprotein E with an amino acid sequence as described in SEQ ID NO:8; or,
  • the combination comprises glycoprotein C with an amino acid sequence as described in SEQ ID NO: 10, glycoprotein D with an amino acid sequence as described in SEQ ID NO: 11, and glycoprotein E with an amino acid sequence as described in SEQ ID NO: 12; or,
  • the combination comprises glycoprotein C with an amino acid sequence as described in SEQ ID NO:47, glycoprotein D with an amino acid sequence as described in SEQ ID NO:48, and glycoprotein E with an amino acid sequence as described in SEQ ID NO:49; or,
  • the combination comprises glycoprotein B with an amino acid sequence as described in SEQ ID NO:50, glycoprotein C with an amino acid sequence as described in SEQ ID NO:51, glycoprotein D with an amino acid sequence as described in SEQ ID NO:52, and glycoprotein E with an amino acid sequence as described in SEQ ID NO:53; or,
  • glycoprotein B with an amino acid sequence as described in SEQ ID NO:5
  • glycoprotein C with an amino acid sequence as described in SEQ ID NO:6
  • glycoprotein D with an amino acid sequence as described in SEQ ID NO:7
  • glycoprotein E with an amino acid sequence as described in SEQ ID NO:8; or
  • the combination comprises glycoprotein B with an amino acid sequence as described in SEQ ID NO:9, glycoprotein C with an amino acid sequence as described in SEQ ID NO:10, glycoprotein D with an amino acid sequence as described in SEQ ID NO:11, and glycoprotein E with an amino acid sequence as described in SEQ ID NO:12; or,
  • the combination comprises glycoprotein B having an amino acid sequence as described in SEQ ID NO:46, glycoprotein C having an amino acid sequence as described in SEQ ID NO:47, glycoprotein D having an amino acid sequence as described in SEQ ID NO:48 and glycoprotein E having an amino acid sequence as described in SEQ ID NO:49.
  • the third aspect of the present invention provides an isolated DNA encoding the herpes simplex virus glycoprotein or an immunogenic fragment thereof as described in the first aspect of the present invention, or encoding the amino acid sequence of the combination as described in the second aspect of the present invention.
  • the nucleotide sequence encoding glycoprotein E, glycoprotein B, glycoprotein C or glycoprotein D is shown as SEQ ID NO: 14-17, or as shown in SEQ ID NO: 18-21, or as shown in SEQ ID NO: 22-25, or as shown in SEQ ID NO: 26-29.
  • the fourth aspect of the present invention provides a recombinant expression vector comprising the isolated DNA described in the third aspect of the present invention.
  • the fifth aspect of the present invention provides a host cell, wherein the host cell comprises the recombinant expression vector as described in the fourth aspect of the present invention.
  • the host cell is a eukaryotic cell, such as a mammalian cell.
  • the sixth aspect of the present invention provides an isolated mRNA obtained by transcription from the nucleotides of the isolated DNA as described in the third aspect of the present invention.
  • sequence of the mRNA is as shown in any one of SEQ ID NO:30-45.
  • the seventh aspect of the present invention provides an isolated mRNA combination, which contains at least two of the mRNAs transcribed from nucleotides encoding glycoprotein B, glycoprotein C, glycoprotein D or glycoprotein E, wherein the glycoprotein B, glycoprotein C, glycoprotein D or glycoprotein E is as defined in the combination described in the second aspect of the present invention.
  • the combination of mRNAs comprises mRNAs obtained by transcribing nucleotides encoding glycoprotein C, glycoprotein D and glycoprotein E; or the combination of mRNAs comprises mRNAs encoding glycoprotein B, glycoprotein C, The mRNA obtained by transcription of nucleotides of glycoprotein D and glycoprotein E.
  • the mRNA obtained by transcribing the nucleotides encoding glycoprotein B is as shown in any one of SEQ ID NOs: 30-33
  • the mRNA obtained by transcribing the nucleotides encoding glycoprotein C is as shown in any one of SEQ ID NOs: 34-37
  • the mRNA obtained by transcribing the nucleotides encoding glycoprotein D is as shown in any one of SEQ ID NOs: 38-41
  • the mRNA obtained by transcribing the nucleotides encoding glycoprotein E is as shown in any one of SEQ ID NOs: 42-45.
  • the mRNA combination comprises the mRNA shown in SEQ ID NO:30, the mRNA shown in SEQ ID NO:34, the mRNA shown in SEQ ID NO:38 and the mRNA shown in SEQ ID NO:42; or,
  • the mRNA combination comprises the mRNA shown in SEQ ID NO:31, the mRNA shown in SEQ ID NO:35, the mRNA shown in SEQ ID NO:39 and the mRNA shown in SEQ ID NO:43; or,
  • the mRNA combination comprises the mRNA shown in SEQ ID NO:32, the mRNA shown in SEQ ID NO:36, the mRNA shown in SEQ ID NO:40 and the mRNA shown in SEQ ID NO:44; or,
  • the mRNA combination comprises the mRNA shown in SEQ ID NO:33, the mRNA shown in SEQ ID NO:37, the mRNA shown in SEQ ID NO:41 and the mRNA shown in SEQ ID NO:45; or,
  • the mRNA combination comprises the mRNA shown in SEQ ID NO:34, the mRNA shown in SEQ ID NO:38 and the mRNA shown in SEQ ID NO:42; or,
  • the mRNA combination comprises the mRNA shown in SEQ ID NO:35, the mRNA shown in SEQ ID NO:39 and the mRNA shown in SEQ ID NO:43; or,
  • the mRNA combination comprises the mRNA shown in SEQ ID NO:36, the mRNA shown in SEQ ID NO:40 and the mRNA shown in SEQ ID NO:44; or,
  • the mRNA combination includes mRNA shown as SEQ ID NO:37, mRNA shown as SEQ ID NO:41 and mRNA shown as SEQ ID NO:45.
  • the eighth aspect of the present invention provides a recombinant viral expression vector, which comprises the mRNA as described in the sixth aspect of the present invention or the mRNA combination as described in the seventh aspect of the present invention.
  • the viral expression vector is an oncolytic virus, adenovirus, adeno-associated virus, retrovirus, herpes simplex virus or lentivirus vector.
  • the ninth aspect of the present invention provides a cell comprising the isolated mRNA as described in the sixth aspect of the present invention, the mRNA combination as described in the seventh aspect of the present invention, or the recombinant viral expression vector as described in the eighth aspect of the present invention.
  • the cells are mammalian cells, such as moDC cells, Hek293 cells, Hela cells or NIH-3T3 cells.
  • the tenth aspect of the present invention provides a vaccine comprising: the herpes simplex virus saccharide as described in the first aspect of the present invention; A protein or an immunogenic fragment thereof, a combination as described in the second aspect of the invention, an isolated DNA as described in the third aspect of the invention, an isolated mRNA as described in the sixth aspect of the invention or an mRNA combination as described in the seventh aspect of the invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is a liposome.
  • the vaccine described in the present invention can be a protein, polypeptide vaccine (with or without sugar modification) or a nucleic acid vaccine.
  • the nucleic acid vaccine can be a DNA vaccine or an RNA vaccine or an RNA vaccine.
  • the RNA vaccine can be an mRNA vaccine.
  • the eleventh aspect of the present invention provides a pharmaceutical composition, which comprises the herpes simplex virus glycoprotein or an immunogenic fragment thereof as described in the first aspect of the present invention, the combination as described in the second aspect of the present invention, the isolated DNA as described in the third aspect of the present invention, the recombinant expression vector as described in the fourth aspect of the present invention, the host cell as described in the fifth aspect of the present invention, the isolated mRNA as described in the sixth aspect of the present invention, the mRNA combination as described in the seventh aspect of the present invention, the recombinant virus expression vector as described in the eighth aspect of the present invention, the cell as described in the ninth aspect of the present invention, or the vaccine as described in the tenth aspect of the present invention.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the twelfth aspect of the present invention provides a herpes simplex virus glycoprotein or an immunogenic fragment thereof as described in the first aspect of the present invention, the combination as described in the second aspect of the present invention, the isolated DNA as described in the third aspect of the present invention, the recombinant expression vector as described in the fourth aspect of the present invention, the host cell as described in the fifth aspect of the present invention, the isolated mRNA as described in the sixth aspect of the present invention, the mRNA combination as described in the seventh aspect of the present invention, the recombinant virus expression vector as described in the eighth aspect of the present invention, the cell as described in the ninth aspect of the present invention, or the vaccine as described in the tenth aspect of the present invention, or the pharmaceutical composition as described in the eleventh aspect of the present invention for use in the preparation of drugs for regulating immunity.
  • the immune regulation is the treatment and/or suppression of herpes disease.
  • the herpes disease is a disease caused by herpes simplex virus, such as herpetic whitlow, herpes encephalitis, eczema herpeticum or neonatal herpes.
  • the thirteenth aspect of the present invention provides a method for regulating immunity, which comprises administering to a patient in need thereof a therapeutically effective amount of the herpes simplex virus glycoprotein or an immunogenic fragment thereof as described in the first aspect of the present invention, the combination as described in the second aspect of the present invention, the isolated DNA as described in the third aspect of the present invention, the recombinant expression vector as described in the fourth aspect of the present invention, the host cell as described in the fifth aspect of the present invention, the isolated mRNA as described in the sixth aspect of the present invention, the mRNA combination as described in the seventh aspect of the present invention, the recombinant virus expression vector as described in the eighth aspect of the present invention, the cell as described in the ninth aspect of the present invention, or the vaccine as described in the tenth aspect of the present invention, or the pharmaceutical composition as described in the eleventh aspect of the present invention.
  • the immune regulation is the treatment and/or suppression of herpes disease.
  • the herpes disease is a disease caused by herpes simplex virus, such as herpetic whitlow, herpetic encephalitis, Eczema herpeticum or neonatal herpes.
  • the present invention provides a herpes simplex virus glycoprotein or an immunogenic fragment thereof as described in the first aspect of the present invention, a combination as described in the second aspect of the present invention, an isolated DNA as described in the third aspect of the present invention, a recombinant expression vector as described in the fourth aspect of the present invention, a host cell as described in the fifth aspect of the present invention, an isolated mRNA as described in the sixth aspect of the present invention, an mRNA combination as described in the seventh aspect of the present invention, a recombinant virus expression vector as described in the eighth aspect of the present invention, a cell as described in the ninth aspect of the present invention, or a vaccine as described in the tenth aspect of the present invention, or a pharmaceutical composition as described in the eleventh aspect of the present invention for use in regulating immunity.
  • the immune regulation is the treatment and/or suppression of herpes disease.
  • the herpes disease is a disease caused by herpes simplex virus, such as herpetic whitlow, herpes encephalitis, eczema herpeticum or neonatal herpes.
  • the herpes simplex virus (HSV) described in the present invention is HSV-2 type.
  • the glycoprotein described in the present invention is HSV-2 type glycoprotein.
  • the glycoprotein B, glycoprotein C, glycoprotein D, and glycoprotein E described in the present invention are also called gB, gC, gD, and gE, respectively.
  • the reagents and raw materials used in the present invention are commercially available.
  • the present invention adopts the method of combining antigens to prepare HSV-2 mRNA vaccine.
  • the HSV-2 mRNA vaccine in the present invention comprises a combination of four glycoproteins selected from gB, gC, gD and gE derived from HSV-2.
  • the 4 HSV-2 antigens gB, gC, gD and gE involved in the vaccine of the present invention all adopt the truncated form of protein, cut off the intracellular region of the corresponding protein, and only retain the extracellular B cell epitope region.
  • the nucleic acid sequence of the corresponding encoded polypeptide is optimized and sequence-modified (each U in the mRNA molecule is modified by 1-methyl-pseudouridine), and the nucleic acid modified by optimization and pseudouridine improves the expression efficiency of protein, improves the stability of mRNA, reduces the difficulty of LNP encapsulation, and reduces immunogenicity.
  • the HSV-2 mRNA vaccine prepared by the combined antigen method provided by the present invention can significantly increase the HSV-2 neutralizing antibody titer in the serum samples of the subjects, can successfully induce CD4 + and CD8 + T cell response reactions in the subjects, and can significantly and successfully increase the survival rate of the subjects.
  • Fig. 1 is a schematic diagram of an mRNA in vitro transcription vector
  • Figure 2a and Figure 2b respectively show the neutralizing antibody titer and CD4 + and CD8 + T cell responses in mice after immunization with G1-G5mRNA-LNP;
  • Figure 3a and Figure 3b are respectively the survival rate and body weight changes of mice immunized with G1-G5 mRNA-LNP after vaginal challenge;
  • FIG4 shows the HSV-2 vaginal titer in mice immunized with G1-G5 mRNA-LNP after vaginal challenge
  • Figure 5 shows the clinical symptom scores of genital herpes in mice immunized with G1-G5mRNA-LNP after vaginal challenge
  • Figure 6 shows the results of detecting the viral copy number in mouse sacral ganglion samples using the qPCR method after G1-G5mRNA-LNP immunization.
  • the mRNA expressing HSV-2 glycoproteins B, C, D, and E is based on the mRNA encoding the amino acids of glycoprotein B (gB2) of HSV-2 333 strain (SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 9), glycoprotein C (gC2) (SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 10), glycoprotein D (gD2) (SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO: mRNA is prepared by using DNA coding sequences of gB2 (SEQ ID NOs: 14, 18, 22 and 26), gC2 (SEQ ID NOs: 15, 19, 23 and 27), gD2 (SEQ ID NOs: 16, 20, 24 and 28), and gE2 (SEQ ID NOs: 17, 21, 25 and 29) and glycoprotein E (gE2) amino acids (SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 12).
  • SEQ ID NOs: 1-4 are the original amino acid sequences of HSV-2gB, gC, gD, and gE proteins;
  • SEQ ID NOs: 5-8 are truncated amino acid sequences of HSV-2gB, gC, gD, and gE proteins;
  • SEQ ID NOs: 9-12 are truncated original amino acid sequences of HSV-2gB, gC, gD, and gE proteins with the signal peptide replaced with LAMP1;
  • SEQ ID NO: 13 is the lamp1 protein signal peptide.
  • mRNA concentration was determined by NANODROP TM , and the integrity and purity of mRNA were analyzed by capillary electrophoresis.
  • lipid nanoparticles By PNI Ignite TM mixed mRNAs containing different sequences into four lipid buffer systems in proportion to assemble lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • four mRNA-LNPs were prepared, and the four mRNA-LNPs respectively contained gD (SEQ ID NO: 39), gCDE (SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43), gBCDE (SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43) and Luciferase mRNA (as a control).
  • mice 75 SPF-grade BALB/c female mice (5-6 weeks old) were selected (mice were purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd., laboratory animal quality certificate number 20220009002112) and kept in an ABSL-2 facility. The care and use of mice followed the established protocol of the cooperative laboratory. The mice were randomly divided into 5 groups, 15 in each group, and they needed to spend no less than 3 days of environmental adaptation before the start of the experiment;
  • Immunization groups (G3-G5 groups): Immunization was performed on the 0th and 28th days of the experiment, respectively. Each time, 20 ⁇ g of sample was injected into the thigh muscle of the mice, 50 ⁇ L on each side.
  • Blank control group (G1: normal saline group): an equal volume of normal saline was injected into the bilateral thigh muscles, 50 ⁇ L on each side.
  • Negative control group (G2: Fluc group): 20 ⁇ g of mRNA-LNP labeled with firefly luciferase was injected into bilateral muscles, 50 ⁇ L on each side.
  • the three vaccination groups were:
  • Group G3 modified gD mRNA-LNP (SEQ ID NO: 39), Group G4: modified gCDE mRNA-LNP (SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43), Group G5: modified gBCDE (SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43)): 20 ⁇ g of different test mRNA-LNPs were injected into bilateral intramuscularly, 50 ⁇ L on each side.
  • Health monitoring During the experiment, the animals were observed twice a week to record their weight, health, survival status, and general clinical lesions at the injection site. Any abnormal symptoms would be recorded;
  • mice in each group were injected subcutaneously with 3 mg of medroxyprogesterone;
  • Immunogenicity On day 55, 5 untreated mice in each group were killed, and serum and spleen tissues were collected (serum samples were used for neutralizing antibody detection, and spleen tissues were used for the expression detection of IFN- ⁇ and TNF- ⁇ in CD4+ and CD8+ T cells);
  • mice in each group were inoculated with 10 ⁇ L (1.0E+04 PFU/mouse) of HSV-2 virus by vaginal instillation;
  • vaginal lavage fluid from 10 challenged mice in groups 1-5 was collected (for virus titer detection).
  • sacral ganglia were collected and quickly frozen in dry ice (for virus copy number detection).
  • Example 4 Mouse immunogenicity study (neutralizing antibody titer, T cell response)
  • mice in each group that were not challenged were euthanized by CO2 inhalation, and serum and spleen tissue were collected.
  • Plaque reduction test was used to detect the level of neutralizing antibodies in serum
  • flow cytometry was used to detect the expression levels of IFN- ⁇ and TNF- ⁇ in CD4+ and CD8+ T cells in spleen tissue, in order to study the effect of HSV vaccine containing chemically modified mRNA encoding one HSV protein or a combination of HSV proteins in mice. Immunogenicity, the specific experimental steps are as follows:
  • mice On the 56th day, sera from 5 mice were collected from each group, heat-inactivated at 56°C for 30 minutes, and then diluted tenfold and then fivefold with experimental culture medium, for a total of 5 dilutions. ;
  • Mouse spleen was ground and filtered through a 70 ⁇ m cell strainer to obtain a single cell suspension. Red blood cells were lysed with 1 ⁇ RBC and spleen cells were obtained after washing. The cells were counted using a cell counter. Based on the counting results, the cells were diluted and inoculated into a 96-well U-bottom cell culture plate at 1 ⁇ 106/well/100 ⁇ L.
  • the serum neutralizing antibody titers of the blank control group (G1) and the negative control group (G2) were both below the detection limit, and the serum neutralizing antibody titers of the test vaccine groups (G3-G5) were all higher than those of the blank group (see Figure 2a).
  • G1 and G2 groups were almost unable to induce CD4+ T and CD8+ T cells to produce the tested cytokines.
  • G3, G4 and G5 groups were able to induce CD4+ T and CD8+ T cells to produce IFN- ⁇ and TNF- ⁇ cytokines, and the induction efficiency of G5 group was stronger than that of G3 and G4 groups (see Figure 2b).
  • mice were able to effectively induce neutralizing antibodies and T cell immune responses in mice, among which the G5 group containing a combination of four antigens had a better immune effect.
  • mice were subcutaneously injected with 3 mg/dose medroxyprogesterone.
  • mice were inoculated with 1.0E+04 PFU/mouse HSV-2 virus by vaginal instillation (10 mice per group).
  • mice in the G1 and G2 groups began to die on days 63 and 64 (days 7 and 8 after virus inoculation), respectively, and all died on days 65 and 66 (days 9 and 10 after virus inoculation), respectively, with a final animal survival rate of 0%.
  • the mice in the G3-G5 groups did not die during the experiment, and the final survival rate was 100% (see Figure 3a).
  • mice in the G1 group did not decrease, and mice in the G2-G5 groups experienced a transient slowdown in weight gain within 3 days after LNP injection, which then returned to normal.
  • the weight of mice in the G1 and G2 groups began to decrease significantly from day 61 (5 days after virus inoculation), and then continued to decrease until death.
  • the weight of mice in the G3-G5 groups remained basically stable after the virus attack, with no significant decrease (see Figure 3b).
  • mice were able to effectively improve the survival rate of mice and reduce the impact of the virus challenge experiment on the body weight of mice.
  • Example 6 HSV-2 vaginal titer after vaginal challenge
  • mice were subcutaneously injected with 3 mg/dose medroxyprogesterone.
  • mice were inoculated with 1.0E+04 PFU/mouse HSV-2 virus by vaginal instillation.
  • Vaginal lavage fluid samples were collected on the 58th and 60th days, and the plaque reduction test was used to detect the virus titer level in the vaginal lavage fluid (10 mice in each group).
  • Plaque reduction test was used to detect the virus titer level of vaginal lavage fluid samples.
  • Virus sample dilution The vaginal lavage fluid sample was thawed in a 37°C water bath and centrifuged for 20 seconds to obtain the supernatant, which was then diluted 10-fold using experimental culture medium and then diluted 5 times in a gradient manner.
  • Infect cells Remove the culture medium from the cell plate, add 0.5 ml of experimental culture medium, add 0.5 ml/well of the diluted solution to the 6-well plate with cells and shake it on a shaker for 5-10 minutes, set up a cell control. After mixing, place it in a 37°C cell culture incubator and incubate for 2 hours to allow the virus to fully adsorb.
  • Plaque staining and counting On the third day, cells were fixed with paraformaldehyde at room temperature for 4 h, then removed, rinsed with water, and then 0.5 ml/well of 0.5% crystal violet solution was added. The cells were shaken on a shaker for 15 min, and then the crystal violet was rinsed with water and dried. Scan the well plate and read the number of plaques therein to calculate the virus titer in the sample, which is expressed as Log 10 (number of plaques per ml vaginal lavage fluid sample).
  • the results are shown in Figure 4.
  • the average virus titers in the vaginal lavage fluid of mice in group G1 on days 58 and 60 were 4.638Log10 (PFU/mL) and 3.663Log10 (PFU/mL), respectively.
  • the average virus titers in the vaginal lavage fluid of mice in group G2 on days 58 and 60 were 4.563Log10 (PFU/mL) and 3.739Log10 (PFU/mL), respectively, and the virus titer levels were comparable to those in group G1.
  • Example 7 Genital herpes disease scores after intravaginal challenge
  • mice were subcutaneously injected with 3 mg/dose of medroxyprogesterone.
  • mice were inoculated with 1.0E+04 PFU/mouse HSV-2 virus by vaginal instillation (10 mice in each group).
  • virus inoculation from the 56th to the 84th day
  • the animal status was observed once a day and clinical scores were performed: erect hair, arched back, vaginal redness and swelling, vaginal ulceration, paralysis. The occurrence of any one symptom was scored as 1 point, and the clinical score was the cumulative sum of all items.
  • mice in groups G1 and G2 began to show clinical symptoms from day 60 (day 4 after virus inoculation), and the symptoms worsened over time.
  • mice in groups G1 and G2 were 4.8 and 4.4 points on day 65 (9 days after virus inoculation), and all died on days 65 and 66, respectively.
  • Mice in groups G3-G5 began to show mild clinical symptoms only on days 60 and 61, which then disappeared, with the highest clinical symptom score of 0.2 points (see Figure 5).
  • Example 8 qPCR detection of viral copy number in sacral ganglion samples
  • mice were subcutaneously injected with 3 mg/dose medroxyprogesterone.
  • mice were inoculated with 1.0E+04 PFU/mouse HSV-2 virus by vaginal instillation (10 mice per group). Sacral ganglia samples were collected on the 83rd and 84th days or on the day of mouse death, and the qPCR method was used to detect the number of viral copies in the sacral ganglia samples.
  • DNA extraction of sacral ganglia Extraction of total DNA from mouse sacral ganglia: The experimental steps refer to the instructions of QIAamp DNA Miniprep Kit (QIAGEN, 51304). The method is briefly described as follows: Sacral ganglia tissue was treated with 20 ⁇ L proteinase K and 200 ⁇ L AL digestion. Then, DNA was precipitated with 200 ⁇ L anhydrous ethanol. Transferred to a spin column, washed with 500 ⁇ L AW1 and AW2, and eluted with 80 ⁇ L AE buffer. The extracted DNA was quantified with Nanodrop (Thermo), then diluted to 10ng/ ⁇ L, and 2 ⁇ L of DNA was used for qPCR.
  • Nanodrop Thermo
  • qPCR detection The HSV-2 virus copy number in the mouse sacral ganglion samples was quantified according to the qPCR standard curve, expressed as HSV-2 virus copy number/10 ng sacral ganglion DNA.
  • Quantitative PCR steps Prepare the qPCR reaction mixture as shown in Table 5 below, add the sample and standard, and perform the PCR reaction. Reaction conditions: 95°C, 10 minutes; 95°C, 15 seconds, 60°C, 1 minute, 40 cycles.
  • the average copy number of HSV-2 virus in the sacral ganglia of mice in group G1 was 3.327Log10 (copies/10ng tissue DNA).
  • the average copy number of HSV-2 virus in the sacral ganglia of mice in group G2 was 3.123Log10 (copies/10ng tissue DNA), which was equivalent to that in group G1.
  • the copy number of HSV-2 virus in the sacral ganglia of mice in groups G3-G5 was below the detection limit.

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Abstract

L'invention concerne un vaccin à ARNm contre le virus de l'herpès simplex et son utilisation. Le vaccin contre le virus de l'herpès simplex est un vaccin à ARNm et comprend une séquence d'ARNm codant pour une ou plusieurs parmi une glycoprotéine B, une glycoprotéine C, une glycoprotéine D, ou une glycoprotéine E. Le vaccin à ARNm est préparé de manière à combiner des antigènes. Les antigènes impliqués ont tous la forme d'une protéine tronquée, et une optimisation de séquence est effectuée sur des séquences de codage d'acide nucléique correspondantes, de telle sorte que l'efficacité d'expression de protéine et la stabilité d'ARNm sont améliorées, le taux de mutation d'ARNm et la difficulté d'enveloppement avec LNP sont réduits, la stabilité d'ARNm est améliorée, et l'immunogénicité est réduite.
PCT/CN2023/139118 2022-12-19 2023-12-15 Vaccin contre le virus de l'herpès simplex et son utilisation WO2024131664A1 (fr)

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