WO2018095327A1 - 包含乙肝病毒样颗粒作为佐剂的疫苗组合物 - Google Patents

包含乙肝病毒样颗粒作为佐剂的疫苗组合物 Download PDF

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WO2018095327A1
WO2018095327A1 PCT/CN2017/112350 CN2017112350W WO2018095327A1 WO 2018095327 A1 WO2018095327 A1 WO 2018095327A1 CN 2017112350 W CN2017112350 W CN 2017112350W WO 2018095327 A1 WO2018095327 A1 WO 2018095327A1
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virus
vaccine composition
antigen
adjuvant
rsv
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PCT/CN2017/112350
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English (en)
French (fr)
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黄立民
黄任民
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黄立民
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Priority to CN201780072358.7A priority Critical patent/CN110099693A/zh
Priority to JP2019527541A priority patent/JP6902804B2/ja
Priority to EP17874747.3A priority patent/EP3545973A4/en
Priority to CA3044582A priority patent/CA3044582C/en
Priority to KR1020197016269A priority patent/KR102276200B1/ko
Priority to AU2017366407A priority patent/AU2017366407B2/en
Priority to US16/463,357 priority patent/US11116837B2/en
Publication of WO2018095327A1 publication Critical patent/WO2018095327A1/zh
Priority to US17/365,543 priority patent/US20210322544A1/en

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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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • A—HUMAN NECESSITIES
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P31/14—Antivirals for RNA viruses
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    • 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
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/02—Hepadnaviridae, e.g. hepatitis B virus
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    • A—HUMAN NECESSITIES
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    • 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
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • 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
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    • A61K2039/55588—Adjuvants of undefined constitution
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10123—Virus like particles [VLP]
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    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18571—Demonstrated in vivo effect
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to vaccine compositions, and methods for enhancing immunogenicity and improving immune responses to antigens.
  • the mucosal vaccine can be completed using an adjuvant and delivery system that adsorbs the vaccine antigen onto the mucosal surface of the mouth, intestine, nose, rectum, or vagina, and after adsorption, carries the vaccine antigen to the mucosa Related lymphoid tissue contact.
  • mucosal vaccines have advantages in providing effective induction of systemic immunity (production of IgG antibodies) and mucosal immunity (production of secretory IgA antibodies), and they are also inexpensive, easy to administer, and suitable for large-scale vaccination. .
  • aluminum salts As an adjuvant commonly used in immunology, aluminum salts have been used in vaccines since the 1930s. However, even though it is widely used throughout the world, aluminum salts are still relatively weak and only effective for certain diseases.
  • HBc VLP hepatitis B core virus-like particle
  • the recombinant HBc VLP may comprise an amino acid sequence which is identical to the amino acid sequence of SEQ ID NO: 1.
  • the adjuvant is an HBc VLP consisting of the amino acid sequence of SEQ ID NO: 1.
  • the antigen is an antigen derived from an infectious disease.
  • the antigen is derived from one of the group consisting of human immunodeficiency virus, varicella-zoster virus, herpes simplex virus type 1 , herpes simplex virus type 2 Human cytomegalovirus, dengue virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, Respiratory Syncytial Virus (RSV), Severe Acute Respiratory Syndrome (SARS) Virus ), human papillomavirus, influenza virus, Hib, meningitis virus, Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, enterotoxin Escherichia coli, Campylobacter, Streptococcus, Moraxella, Mycoplasma, Mycobacteria, Haemophilus, Plasmodium or Toxo
  • the present disclosure provides a method of vaccinating a subject comprising administering the vaccine composition described above to a mucosal surface of the subject.
  • the mucosal surface may be selected from the group consisting of respiratory mucosa, gastrointestinal mucosa, vaginal mucosa, nasal mucosa, rectal mucosa, and oral mucosa.
  • the present disclosure provides a recombinant HBc VLP.
  • the present disclosure provides a vaccine composition comprising an antigen from an infectious substance and an adjuvant as an adjuvant, an effective amount of recombination HBc VLP.
  • the vaccine composition of the present disclosure can induce an antibody response specific to the antigen in a subject and protect the subject from infection with the infectious substance without causing side effects.
  • the adjuvants, vaccine compositions, and methods of achieving immunization of the present disclosure are susceptible to large scale production and are more conducive to increasing the specificity of antibody identification and avoiding unnecessary reactions such as allergies.
  • Figures 1A through 1I are Results of SDS-PAGE analysis of recombinant protein and HBc.
  • Figures 1A, 1C, 1E, and 1G show the purified pairs, respectively Recombinant protein and Coomassie blue staining of HBc;
  • Figures 1B, 1D, and 1F show purified anti-His antibody pairs, respectively with Western blot analysis of recombinant proteins;
  • Figure 1H shows Western blot analysis of purified HBc using rabbit polyclonal anti-HBc antibody;
  • Figure 1I shows the use of murine monoclonal anti-RSV antibody against purified HBc and Western blot analysis of recombinant proteins.
  • Figure 2 shows a TEM image of purified HBc.
  • Figures 3A and 3B show a table of intranasal (IN) immunization schedules.
  • Figure 3A shows that mice of each group were immunized 4 times at weeks 0, 3, 6, and 9 using the candidate vaccine, and the mice received RSV challenge at week 12; and
  • Figure 3B shows the use of candidate vaccines at Each group of mice was immunized 3 times at 0, 3, and 6 weeks, and the mice were subjected to RSV challenge at week 9.
  • Another group included intramuscular (i.m) immunization with formalin-fixed RSV (FIRSV) prior to RSV challenge. Two days prior to RSV challenge, mouse serum, BALF, and spleen were collected from each group using the same dosing regimen.
  • FIRSV formalin-fixed RSV
  • Figures 4A through 4F show HR receiving 4 doses mixed or not mixed with HBc VLP 24 intranasal administration of mice Responsive to FIRSV-specific antibodies.
  • Mouse sera were collected from each group 2 days before RSV challenge.
  • Figures 4A to 4C show measured from serum, respectively Specific total IgG, IgGl, and IgG2a responses; and,
  • Figures 4D through 4F show FIRSV-specific total IgG, IgGl, and IgG2a responses measured from serum, respectively.
  • Figures 5A to 5K show that 4 doses are mixed or not mixed with CpG Intranasal administration of a mixture with HBc VLP in mice Responding to FIRSV-specific antibody responses and spleen cells.
  • Mouse serum, BALF, and spleen were collected 2 days prior to RSV challenge.
  • Figures 5A through 5D show measurements from serum, respectively Specific total IgG, IgG1, IgG2a response and IgG2a/IgG1 ratio;
  • Figures 5E to 5H show FIRSV-specific total IgG, IgG1, IgG2a response and IgG2a/IgG1 ratios measured from serum, respectively;
  • Figures 5I and 5J show detection from BALF, respectively of And FIRSV-specific secretory IgA (sIgA) response;
  • Figure 5K shows the level of IFN- ⁇ detected in the antigen re-stimulation experiment.
  • Figures 6A to 6H show that 4 doses are mixed or not mixed with CpG RSV F protein site in mice administered intranasally with a mixture of HBc VLP And site II specific antibody response. Two groups of mouse sera were collected 2 days before RSV.
  • Figures 6A to 6D show the sites measured from serum, respectively Specific total IgG, IgG1, IgG2a response and IgG2a/IgG1 ratio; and, Figures 6E to 6H show site II specific total IgG, IgG1, IgG2a response and IgG2a/IgG1 ratio measured from serum, respectively.
  • Figures 7A through 7E show that 3 doses are mixed or not mixed with HBc VLP or CpG. or Intranasal administration of mice Specific antibody response. Serum and BALF were collected 2 days prior to RSV challenge. Figures 7A, 7B, 7C, and 7E show measurements from serum, respectively Specific total IgG, IgG1, IgG2a and IgA responses; and, Figure 7D shows detection from BALF Specific sIgA response.
  • Figure 8 shows serum neutralization titers. Two days prior to RSV challenge, the control group or the sera of 4 doses of vaccinated mice administered intranasally were collected and tested for inhibition of RSV plaque formation.
  • Figure 9 shows the change in rat body weight after challenge. After RSV challenge, the control group or the vaccinated mice receiving 4 doses of intranasal administration were changed in body weight for 5 days. Body weight changes are expressed as a percentage of weight loss relative to Day 0.
  • Figure 10 shows lung histopathology. Five days after the RSV challenge, the lung tissues of the control group or the vaccinated mice receiving 4 doses of intranasal administration were collected for histological analysis.
  • the present disclosure provides a vaccine composition comprising an antigen and an adjuvant, wherein the adjuvant is a recombinant HBc VLP.
  • virus-like particle refers to a structure such as a virus, but is non-infectious because it lacks a viral genome.
  • non-infectious refers to the inability to enter a host cell.
  • virus-like particles lack replication or infectious components due to the lack of all or part of the viral genome, particularly the viral genome, and they are not replicable and lack pathogenicity.
  • the virus-like particle may be a viral capsid, such as a viral capsid of a corresponding virus coated with a lipid membrane considered to be a viral envelope.
  • viral capsid or “capsid” refers to a macromolecular component consisting of viral protein subunits.
  • virus-like particles are generally produced in large quantities by heterologous expression and can be easily purified.
  • the VLP can form spontaneously.
  • Methods for producing specific VLPs are known in the art.
  • the presence of the VLP can be detected using conventional techniques known in the art, such as by electron microscopy, X-ray crystallography, and the like.
  • the adjuvant may be a recombinant hepatitis B core antigen (HBcAg).
  • the adjuvant may be a recombinant HBcAg having an amino acid sequence, wherein the amino acid sequence is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 and has SEQ ID NO : 1 same function.
  • the recombinant HBcAg comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96% identical to the amino acid sequence of SEQ ID NO: 1. , 97%, 98%, or 99%.
  • sequence identity or, for example, encompasses “80% of sequence identity” It refers to the degree of consistency of a sequence from one nucleotide to one nucleotide or one amino acid to one amino acid throughout the comparison window.
  • the "percentage of sequence identity" can be calculated by comparing the two most optimally aligned sequences within the comparison window to determine the presence of identical nucleic acid bases in both sequences (eg, A, T, C, G, I) or a consistent amino acid residue (eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys And the number of positions of Met), the number of matching positions is obtained, the number of matching positions is divided by the total number of positions in the comparison window (ie, the window size), and the result is multiplied by 100 to obtain the percentage of sequence consistency.
  • a consistent amino acid residue eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys And the number of positions of Met
  • nucleotides and polypeptides typically, the polypeptide variant retains at least one biological activity or function of the reference polypeptide.
  • the adjuvant is a recombinant HBcAg consisting of the amino acid sequence of SEQ ID NO: 1, also referred to herein as "HBcAg148", which has been shown to form virus-like particles.
  • the present disclosure provides an adjuvant composition comprising HBcAg148 virus-like particles, wherein the HBcAg148 virus-like particles are inert empty capsids formed by self-assembly of capsid proteins from hepatitis B virus (HBV).
  • HBV hepatitis B virus
  • HBV is a small enveloped virus with a circular, partially double-stranded DNA genome. It is the leading cause of infectious liver disease worldwide. HBV infection affects approximately 2 billion people worldwide, and HBV infection in adults is generally transient.
  • HBcAg is an antigen that can be found on the surface of the nucleocapsid, the innermost layer of HBV. Because the assembly of HBcAg148 VLP does not incorporate genetic material, they are non-infectious.
  • the antigen is derived from an infectious disease including, but not limited to, human immunodeficiency virus, varicella-zoster virus, herpes simplex virus type 1, herpes simplex virus type 2, human giant Cell virus, dengue virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, RSV, SARS virus, human papilloma virus, influenza virus, Hib, meningitis virus, Salmonella, Neisseria, Baorou Spirochetes, Chlamydia, Bordetella, Enterotoxin E. coli, Campylobacter, Streptococcus, Moraxella, Mycoplasma, Mycobacterium, Haemophilus, Plasmodium or Toxoplasma, Stanworth Decapeptide.
  • infectious disease including, but not limited to, human immunodeficiency virus, varicella-zoster virus, herpes simplex virus type 1, herpes simplex virus type 2, human
  • the above antigen derived from an infectious disease means any substance that targets an immune response developed in the test organism.
  • the above antigen derived from an infectious disease may also be an immune response when contacted with an immunocompetent cell (eg, maturation of an immunocompetent cell, a cytokine) Targets for production, and production of antibodies.
  • an immunocompetent cell eg, maturation of an immunocompetent cell, a cytokine
  • the antigen can be derived from RSV.
  • RSV has been identified as the most common cause of lower respiratory tract infections in infants and young children.
  • RSV has three surface glycoproteins encoded by three consecutive genes (SH-GF): small hydrophobic glycoprotein (SH), glycoprotein (G), and fusion glycoprotein (F).
  • SH-GF small hydrophobic glycoprotein
  • G glycoprotein
  • F fusion glycoprotein
  • the primary target antigens for RSV vaccine development are RSV F and G, as each of these antigens produces neutralizing antibodies as well as T cell responses. F is particularly striking because of its considerable conservation in the RSV isolation group.
  • NT neutralizing
  • 5C4 shares these properties with two other antibodies isolated from immunized PBMCs, D25 and AM22, which have shown 100-fold greater potency than palivizumab in neutralizing RSV (McLellan, JS, et al) ., 2013).
  • the pre-fusion crystal structure of F protein and the crystal structure after fusion suggest that although site II and site IV are found in both structures, the site It appears to be specific for pre-fusion forms (McLellan, JS, et al., 2013).
  • the fusion peptide region of RSV F is located at the amino terminus of the F1 subunit (Collins, PL, et al., 1996), and the transmembrane segment contains two regions: 4,3-hydrophobic seven-membered repeat (HR),
  • HR 4,3-hydrophobic seven-membered repeat
  • the sequence motif of the coiled-coil structure is reminiscent (Chambers, P., et al., 1990; Singh, M., et al., 1999). These regions are indicated as HRN and HRC, respectively, and are separated by an intermediate domain of approximately 270 amino acids.
  • HRN and HRC form a hairpin-like structure of the trimer, and the HRC region is encapsulated in antiparallel to the coiled-coil formed by the HRN region (Baker, K.A., et al., 1999).
  • the antigen is a recombinant RSV F protein comprising an HRN region, an HRC region, and a site selected from the group consisting of At least one antigenic site of the group consisting of, site II and site IV.
  • the antigen can be represented by one of SEQ ID NO: 2 to SEQ ID NO: 4.
  • the vaccine composition can be used to induce an immune response to an infectious agent such as RSV in a subject.
  • a vaccine composition comprising a therapeutically effective amount of a recombinant RSV F protein as an antigen can be administered to a subject to elicit an immune response to RSV.
  • a vaccine composition comprising a therapeutically effective amount of a recombinant RSV F protein as an antigen is administered to the subject under conditions sufficient to prevent or alleviate RSV infection in a subject in need thereof Tester.
  • the vaccine composition is administered in an amount sufficient to elicit an immune response against the RSV antigen, such as the RSV F protein, in the subject.
  • the vaccine composition is suitable for mucosal vaccination and can be administered orally, intranasally, rectally or vaginally to the subject.
  • the adjuvant is present in the vaccine composition in an adjuvant effective amount of from 0.1 ⁇ g to 1,000 ⁇ g.
  • the vaccine composition comprises a mixture of an antigen and an adjuvant in a weight ratio of 10:1 to 1:10.
  • the vaccine composition comprises a weight ratio of antigen to adjuvant of from 5:1 to 1:5.
  • the HBc VLP in the vaccine composition is used as the sole adjuvant that effectively enhances the immune response to the antigen and/or modulates its development toward the desired immune response.
  • the vaccine composition can include an additional adjuvant. Additional adjuvants that can be used in the present disclosure can include, but are not limited to, CpG oligonucleotides.
  • the vaccine composition can further comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are suitable for administration of the vaccine compositions of the present disclosure.
  • Pharmaceutically acceptable carriers for use in the present disclosure may include, but are not limited to, preservatives, suspending agents, viscosity increasing agents, isotonic agents, buffering agents, and wetting agents.
  • the present disclosure further provides a method of preparing a vaccine composition comprising providing an adjuvant composition comprising HBcAg 148 VLP and a pharmaceutically acceptable carrier, and combining the adjuvant composition with a recombinant RSV F protein as an antigen.
  • the HBcAg148 VLP consists of the amino acid sequence of SEQ ID NO: 1.
  • the present disclosure provides a nucleic acid molecule encoding the above HBcAg148 VLP.
  • the nucleic acid molecule is codon optimized For expression in prokaryotic cells.
  • the prokaryotic cell is an E. coli cell.
  • the nucleic acid molecule comprises a nucleic acid sequence which is identical to the nucleic acid sequence of SEQ ID NO: 5.
  • the degree of coincidence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
  • the nucleic acid molecule is codon optimized for expression in eukaryotic cells.
  • the eukaryotic cell is a yeast cell or a mammalian cell.
  • the mammalian cell is a human cell.
  • the present disclosure further provides a method of inducing a mucosal immune response and a systemic immune response by the vaccine compositions disclosed above.
  • the vaccine composition is administered to a subject in need thereof to induce a mucosal immune response and a systemic immune response in the subject, wherein the mucosal immune response is the production of an antigen-specific IgA antibody, and the Systemic immune responses are the production of antigen-specific IgG antibodies and the production of antigen-specific cell-mediated immunity.
  • the present disclosure provides a method of vaccinating a subject, the method comprising administering the vaccine composition disclosed above to the subject.
  • the vaccination method comprises administering the vaccine composition by any conventional route known in the field of vaccines, for example, via mucosa (eg, ocular, intranasal, pulmonary, buccal, stomach, small intestine, rectum,
  • mucosa eg, ocular, intranasal, pulmonary, buccal, stomach, small intestine, rectum
  • the vaginal or urinary tract surface is administered parenterally (e.g., subcutaneously, intradermally, intramuscularly, intravenously, or intraperitoneally), or externally (e.g., via a transdermal delivery system such as a patch).
  • the vaccine composition is administered to the mucosal surface of the subject.
  • the mucosal surface may be selected from the group consisting of respiratory mucosa, gastrointestinal mucosa, vaginal mucosa, nasal mucosa, rectal mucosa, and oral mucosa.
  • a full length cDNA sequence of the RSV F protein with the optimal codon for E. coli expression was synthesized. Using this sequence as a PCR template, four gene fragments of the RSV F protein were amplified, including HRN and sites. Nucleotides 457 to 633 (SEQ ID NO: 6), nucleotides 760 to 849 containing site II (SEQ ID NO: 7), nucleotides 1264 to 1314 containing site IV (SEQ ID NO: 8), and nucleotides 1426 to 1560 (SEQ ID NO: 9) containing the C-terminal ⁇ -helix (HRC).
  • the four PCR amplicons are linked by partial overlapping PCR and ligated by glycine-rich linkers such as GSGS, GGGS, GGSG, SGSG and GG to form the constructed gene (named Then, the gene was inserted into the NcoI-XhoI restriction site of pET28b labeled with 6-His at the carboxy terminus to obtain Plasmid.
  • glycine-rich linkers such as GSGS, GGGS, GGSG, SGSG and GG
  • HBc plasmid construction process and The plasmids are similar, but the differences are as follows.
  • the plasmid obtained above was transformed into E. coli BL21 (DE3) competent cells for protein expression.
  • the primers used in the PCR of Examples 1 and 2 are represented by SEQ ID NO: 10 to SEQ ID NO: 29 and are shown in Table 1.
  • Recombinant RSV F protein-6His and HBc-6His were expressed in transformed E. coli BL21 (DE3) obtained from Examples 1 and 2, respectively, and purified using nickel affinity chromatography. Elution (using 500 mM imidazole, 50 mM NaH 2 PO 4 , 300 mM NaCl pH 8.0) protein by dialysis against a volume of 200 volumes of dialysis buffer (from 350 mM, 150 mM to 0 mM imidazole, 1 x PBS) Buffer exchange was performed for 12 hours per step. The dialyzed protein-6His was concentrated to a concentration of about 1 mg/mL using a centrifugal concentrator (10,000 MWCO, Sartorius). The molecular size and purity of the protein was determined by SDS-PAGE.
  • Example 4 Transmission electron microscopy (TEM) image of recombinant HBc VLP
  • HBc VLP forms virus-like particles (Fig. 2).
  • the RSV A2 strain was obtained from the ATCC. Propagation of the virus was carried out in HEp-2 cell ATCC. Cells up to 80% confluently grown in 100 mm petri dishes (Thermo Scientific) were seeded with RSi A2 at a moi of 0.2 (multiplicity of infection). Adsorption of the virus was carried out in a serum-free Dulbecco's Modified Eagle's medium (DMEM) in a 37 ° C CO 2 incubator. After 2 hours, the medium was replaced with DMEM supplemented with 2% fetal bovine serum, and the culture dish was further cultured for 48 to 72 hours. The supernatant containing the virus was separated from the cell debris by centrifugation at 3,000 rpm for 10 min. The virus was then concentrated by a centrifugal concentrator (100,000 MWCO, Sartorius).
  • DMEM Dulbecco's Modified Eagle's medium
  • the RSV virus titer was determined by plaque assay.
  • the HEp-2 cell fusion monolayer in a 12-well plate was washed with 1 x PBS, and then the cells were infected with RSV A2 virus at various dilutions (10 -3 to 10 -7 ). After 2 hours of virus adsorption, the supernatant was removed and the monolayer of cells was washed with 1 x PBS and then covered with DMEM + 2% fetal bovine serum + 0.3% agarose. After 5 days of culture in a CO 2 incubator at 37 ° C, the cells were fixed with 10% formalin and plaque quantified using 0.05% crystal violet staining.
  • Pathogen-free C57BL/6J female mice (6 to 8 weeks old) were randomized into several groups and immunized on days 0, 21, 42 and 63 by intranasal (in) route using candidate vaccines (Fig. 3A). or immunization (FIG. 3B) at 0, 21 and day 42 and day 84 mice order (FIG. 3A) or day 63 (FIG. 3B) was 1 ⁇ 10 6 pfu RSV challenge.
  • candidate vaccines include: HBc VLP+ HBc VLP+ +CpG(TCGTCGTTTTCGGCGCGCGCCG, SEQ ID NO.
  • HBc VLP+ HBc VLP+ +CpG HBc VLP+ And HBc VLPs+ +CpG.
  • a control group a group vaccinated only with HBc VLP, and a group immunized intramuscularly (im) with 1 x 10 5 pfu of formalin-fixed RSV (FIRSV).
  • mice serum, tracheal alveolar lavage fluid (BALF) and spleen were collected from each group using the same dosing regimen.
  • BALF tracheal alveolar lavage fluid
  • Example 6 Evaluation of antibody responses elicited by candidate vaccines
  • Antibody responses to serum and BALF collected from immunized mice as disclosed in Example 5 were tested by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • a dilution curve was made for each sample and the endpoint titer was calculated as the reciprocal of the dilution that produced the optimal concentration, which was greater than the background value (1/50 dilution of pooled pre-immune sera, or pooled 1/5 dilution against BALF) 0.1U high.
  • An IgG titer of less than 50 (negative sample) or a secretory IgA (sIgA) titer of less than 5 was arbitrarily designated as 50 or 5.
  • FIG. 4A to 4F the figure shows the results of immunogenicity evaluation of candidate vaccines, wherein each group of mice used 4 doses of i) (10 ⁇ g); ii) (50 ⁇ g); iii) HBc (10 ⁇ g)+ (50 ⁇ g); or iv) HBc (10 ⁇ g) as a negative control was immunized by intranasal route.
  • the results show that the use Intranasal immunization of the mixture produces significantly higher Specific total IgG, IgG1 and IgG2a ( Figures 4A to 4C). FIRSV-specific total IgG and IgG1 were also induced but not significantly higher ( Figures 4D and 4E).
  • each group of mice was intranasally immunized with 4 doses of the following reagents: i) (10 ⁇ g); ii) HBc VLP (50 ⁇ g)+ (10 ⁇ g); iii) (10 ⁇ g) + CpG (20 ⁇ g); iv) HBc VLP (25 ⁇ g) + (10 ⁇ g) + CpG (20 ⁇ g) (labeled HBc+ in the figure) +CpG-1);v)HBc VLP(50 ⁇ g)+ (10 ⁇ g) + CpG (20 ⁇ g) (labeled HBc+ in the figure) +CpG-2); or vi) HBc VLP (50 ⁇ g).
  • Serum and BALF were analyzed by indirect ELISA.
  • Figures 5A to 5C and 5I show that administration of HBc Mixture can cause significantly higher serum Specific total IgG, IgG1, IgG2a and lung Specific sIgA. Furthermore, the use of CpG as an adjuvant did not enhance these humoral responses.
  • mice Since protective immunity against RSV requires an efficient Th1 bias response and IFN- ⁇ production, we tested splenocytes from immunized mice for The ability of recombinant proteins to respond to stimulation in vitro. Determined from 48h by the murine IFN- ⁇ ELISA kit (BioLegend) T cell expansion of the culture is stimulated. The production of Ag-specific IFN- ⁇ from the culture supernatant of these cells was analyzed. As shown in Figure 5K, from use Spleen cells of immunized mice, shown in There was no IFN- ⁇ secretion after stimulation. In contrast, from use with Splenocytes of mice in the immunized group, shown in Significantly higher levels of IFN- ⁇ secretion after stimulation. It is noteworthy that there was no IFN- ⁇ secretion in the mice or the control group immunized with HBc.
  • Figures 7A through 7E show that three doses of intranasal administration are mixed or not mixed with HBc VLP or CpG. or In vivo Specific antibody response.
  • Each group of mice was intranasally immunized with 3 doses of the following reagents: i) HBc VLP (25 ⁇ g); ii) (50 ⁇ g); iii) HBc VLP (25 ⁇ g)+ (25 ⁇ g); iv) HBc VLPs (25 ⁇ g)+ (25 ⁇ g) + CpG (20 ⁇ g); v) HBc VLP (25 ⁇ g) + (50 ⁇ g); vi) HBc VLP (25 ⁇ g)+ (50 ⁇ g) + CpG (20 ⁇ g); vii) (50 ⁇ g); viii) HBc VLP (25 ⁇ g)+ (50 ⁇ g); ix) HBc VLP (25 ⁇ g)+ (50 ⁇ g) + CpG (20 ⁇ g); x) (50 ⁇ g);
  • FIG. 7A through 7E the figure shows that by using HBc as an adjuvant, with Can cause significantly higher serum Specific total IgG, IgG1, IgG2a, IgA, and lung Specific sIgA, of which The highest endpoint titer was observed in the group.
  • HBc HBc
  • CpG a mixture of HBc and CpG as an adjuvant
  • Neutralizing antibodies are important functional components in the immune response induced by vaccines.
  • Figure 8 shows that from Group of serum can reduce plaque formation by about 10% from The serum of the group reduced plaque formation by about 25%, while the group receiving CpG as an adjuvant reduced the plaque by about 17% to 35%.
  • mice receiving intramuscular FIRSV showed the highest weight loss (approximately 23%) while using Mice immunized showed approximately 10% weight loss.
  • Mice immunized with different doses of HBc (0, 25, 50 ⁇ g), ie group, group, Groups of mice showed approximately 8%, 4%, and 0% weight loss on day 4 post challenge.
  • the present disclosure provides better protection against mouse weight loss and provides rapid recovery from initial weight loss following challenge with live RSV. These proofs provide protection against antiviral immunity caused by the antigen of the present application against the live RSV A2 strain.
  • lung tissue was collected from individual mice for histological analysis.
  • lung samples were fixed in 10% neutral buffered formalin for 24 hours, embedded in paraffin blocks, cut into 5 ⁇ m thick sections, and stained with hematoxylin and eosin (H&E).
  • the present disclosure provides a purified HBcAg148 protein, and a nucleic acid molecule encoding the HBcAg148 protein having an optimized codon for E. coli expression.
  • the purified HBcAg148 protein has been confirmed to form virus-like particles by TEM.
  • the efficacy of HBcAg148 in enhancing the immune response in vivo can be observed. It is shown by the present disclosure that HBcAg148 will elevate serum total IgG, IgGl and IgG2a responses against RSV, and this adjuvant effect is similar to the CpG motif.
  • the vaccine compositions of the present disclosure comprising recombinant HBc VLPs as adjuvants can induce systemic and mucosal antibody responses specific for the antigen.
  • Mice immunized with the vaccine compositions of the present disclosure were shown to be protected against the antigen without causing lung disease.
  • the vaccine composition of the present disclosure does not overly stimulate lymphocytes in a mouse model compared to FIRSV and provides a potentially safe RSV vaccine candidate.

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Abstract

一种疫苗组合物以及诱导系统性免疫应答和黏膜免疫应答的方法，其中，该疫苗组合物包含抗原和作为佐剂的乙肝核心病毒样颗粒(HBc VLP)。该疫苗组合物适用于给药至受试者的黏膜表面，且在引起对抗感染的保护性免疫应答中有效。

Description

包含乙肝病毒样颗粒作为佐剂的疫苗组合物
相关申请的交叉引用
本申请主张在2016年11月22日提交的美国专利临时申请案第62/425,079号的优先权，该临时申请案通过引用而以其整体并入本文。
技术领域
本发明涉及疫苗组合物，及提升免疫原性和改善对抗原的免疫应答的方法。
背景技术
到2021，疫苗佐剂的全球市场预计从2016年的4.670亿美元上升至7.694亿美元。驱动这一市场成长的主要因素是感染性疾病和动物传染病的大流行、许多政府机构对免疫计划的关注提高，以及对抗现有疾病和新兴疾病的改善且长效的免疫作用的关注度日益增长。
近年来，因为黏膜表面是大多数感染性物质进入的关口且对于动物在进入的关口处即发展出强效保护性抗体和细胞介导的免疫应答非常重要，对于通过黏膜表面传送的疫苗存在普遍的关心。黏膜疫苗可使用佐剂和传送系统完成，该佐剂和传送系统会将疫苗抗原吸附至口腔、肠、鼻、直肠、或阴道的黏膜表面上，且在吸附之后，将疫苗抗原带至与黏膜相关淋巴组织接触。因此，黏膜疫苗在提供有效诱导系统性免疫(IgG抗体的产生)和黏膜免疫(分泌性IgA抗体的产生)的益处方面有优势，并且它们还廉价、容易给药且适用于大规模的疫苗接种。
作为一种免疫学常用的佐剂，自1930年代以来，铝盐已经被用于疫苗中。然而，即使在全世界广泛使用，铝盐仍然相对疲弱且仅对某些疾病生效。
因此，对于具有能穿透黏膜上皮细胞且诱导保护性和长效性免疫应答的佐剂的黏膜疫苗存在需求。
发明内容
有鉴于前述，本公开提供一种包含抗原和佐剂的疫苗组合物，其中，该佐剂是重组乙肝核心病毒样颗粒(HBc VLP)，本文中也简称为“HBc”。
该重组HBc VLP可包含氨基酸序列，该氨基酸序列与SEQ ID NO:1的氨基酸序列
(MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTV)一致程度为至少80％且具有与SEQ ID NO:1相同的功能。本公开的一具体实施例中，该佐剂是由SEQ ID NO:1的氨基酸序列组成的HBc VLP。
本公开的一具体实施例中，该抗原是源自感染性疾病的抗原。本公开的另一具体实施例中，该抗原源自下列所组成的群组中的一种：人类免疫缺陷病毒、水痘-带状疱疹病毒、1型单纯性疱疹病毒、2型单纯性疱疹病毒、人类巨细胞病毒、登革病毒、甲肝病毒、乙肝病毒、丙肝病毒、戊肝病毒、呼吸道合胞体病毒(Respiratory Syncytial Virus,RSV)、严重急性呼吸道合胞体病毒(Severe Acute Respiratory Syndrome(SARS)Virus)、人乳头状瘤病毒、流感病毒、Hib、脑膜炎病毒、沙门氏菌、奈瑟氏菌(Neisseria)、包柔氏螺旋体(Borrelia)、衣原体(Chlamydia)、博戴氏杆菌(Bordetella)、肠毒素大肠杆菌、弯曲菌(Campylobacter)、链球菌、莫拉克斯氏菌(Moraxella)、支原体(Mycoplasma)、分枝杆菌(Mycobacteria)、嗜血杆菌(Haemophilus)、疟原虫或弓浆虫(Toxoplasma)、Stanworth十肽(Stanworth decapeptide)。
根据本公开的又一态样，本公开提供一种对受试者接种疫苗的方法，包含将上述的疫苗组合物给药至该受试者的黏膜表面。
本公开的另一具体实施例中，该黏膜表面可选自呼吸道黏膜、胃肠黏膜、阴道黏膜、鼻黏膜、直肠黏膜和口腔黏膜所组成的群组。
本公开提供一种重组HBc VLP。另外，本公开提供一种疫苗组合物，其包含来自感染性物质的抗原和作为佐剂的佐剂有效量的重组 HBc VLP。本公开的疫苗组合物可在受试者体内诱导对该抗原为特异性的抗体应答，并保护受试者免于该感染性物质的感染且不造成副作用。如此，本公开的佐剂、疫苗组合物及实现免疫的方法容易实现大规模的生产，且更有助于增加抗体鉴别的特异性并避免不必要的反应如过敏。
附图说明
通过阅读下述具体实施例的详细说明并参照附图，可更完整地理解本公开，其中：
图1A至1I是
Figure PCTCN2017112350-appb-000001
重组蛋白和HBc的SDS-PAGE分析结果。图1A、1C、1E、和1G分别显示对经纯化的
Figure PCTCN2017112350-appb-000002
重组蛋白以及HBc的考马斯亮蓝染色；图1B、1D、和1F分别显示使用抗His抗体对经纯化的
Figure PCTCN2017112350-appb-000003
和
Figure PCTCN2017112350-appb-000004
重组蛋白的蛋白质印迹分析；图1H显示使用兔多克隆抗HBc抗体对经纯化的HBc的蛋白质印迹分析；以及，图1I显示使用鼠单克隆抗RSV抗体对经纯化的HBc和
Figure PCTCN2017112350-appb-000005
重组蛋白的蛋白质印迹分析。
图2显示经纯化的HBc的TEM图像。
图3A和3B显示鼻内(IN)免疫接种程序表。图3A显示，使用候选疫苗在第0、3、6、和9周对各组小鼠免疫接种4次，并令小鼠在第12周接受RSV激发；而图3B显示，使用候选疫苗在第0、3、和6周对各组小鼠免疫接种3次，并令小鼠在第9周接受RSV激发。另包括一组为在RSV激发前使用经福尔马林固定的RSV(FIRSV)进行肌肉内(i.m)免疫接种。在RSV激发前2天，从使用相同投药方案的各组别收集小鼠血清、BALF和脾脏。
图4A至4F显示接受4剂与HBc VLP混合或不混合的HR
Figure PCTCN2017112350-appb-000006
24的鼻内给药的小鼠体内的
Figure PCTCN2017112350-appb-000007
和FIRSV特异性抗体应答。在进行RSV激发前2天，从各组收集小鼠血清。图4A至4C分别显示从血清测量的
Figure PCTCN2017112350-appb-000008
特异性总IgG、IgG1、和IgG2a应答；以及，图4D至4F分别显示从血清测量的FIRSV特异性总IgG、IgG1、和IgG2a应答。
图5A至5K显示接受4剂与CpG混合或不混合的
Figure PCTCN2017112350-appb-000009
与HBc VLP混合物的鼻内给药的小鼠体内的
Figure PCTCN2017112350-appb-000010
和FIRSV特异性抗体应 答和脾细胞再刺激。在进行RSV激发前2天，收集小鼠血清、BALF、和脾脏。图5A至5D分别显示从血清测量的
Figure PCTCN2017112350-appb-000011
特异性总IgG、IgG1、IgG2a应答和IgG2a/IgG1比；图5E至5H分别显示从血清测量的FIRSV特异性总IgG、IgG1、IgG2a应答和IgG2a/IgG1比；图5I和5J分别显示从BALF检测的
Figure PCTCN2017112350-appb-000012
和FIRSV特异性分泌性IgA(sIgA)应答；以及，图5K显示在抗原再刺激实验中检测的IFN-γ的水平。
图6A至6H显示接受4剂与CpG混合或不混合的
Figure PCTCN2017112350-appb-000013
与HBc VLP混合物的鼻内给药的小鼠体内的RSV F蛋白位点
Figure PCTCN2017112350-appb-000014
和位点II特异性抗体应答。在进行RSV前2天，收集各组的小鼠血清。图6A至6D分别显示从血清测量的位点
Figure PCTCN2017112350-appb-000015
特异性总IgG、IgG1、IgG2a应答和IgG2a/IgG1比；以及，图6E至6H分别显示从血清测量的位点II特异性总IgG、IgG1、IgG2a应答和IgG2a/IgG1比。
图7A至7E显示接受3剂与HBc VLP或CpG混合或不混合的
Figure PCTCN2017112350-appb-000016
或
Figure PCTCN2017112350-appb-000017
的鼻内给药的小鼠体内的
Figure PCTCN2017112350-appb-000018
特异性抗体应答。在进行RSV激发前2天，收集血清和BALF。图7A、7B、7C、和7E分别显示从血清测量的
Figure PCTCN2017112350-appb-000019
特异性总IgG、IgG1、IgG2a和IgA应答；以及，图7D显示从BALF检测的
Figure PCTCN2017112350-appb-000020
特异性sIgA应答。
图8显示血清中和效价。在进行RSV激发前2天，收集对照组或接受4剂鼻内给药的接种疫苗的小鼠的血清，并测试对于RSV噬斑形成的抑制。
图9显示鼠体重在激发后的改变。在进行RSV激发后，监控对照组或接受4剂鼻内给药的接种疫苗的小鼠的体重改变5天。体重改变以相对于第0天体重减轻的百分比表示。
图10显示肺组织病理学。在进行RSV激发5天后，收集对照组或接受4剂鼻内给药的接种疫苗的小鼠的肺组织，进行组织学分析。
具体实施方式
使用下述具体实施例来例示性说明本公开。基于本公开的揭露和说明，该领域技术人员可设想本公开的其它优点。本公开也可如不同的具体实施例中所揭示的予以实施或应用。可对上述实施例作成修饰 及/或变更，以在不同态样和应用中实施本公开，而不抵触本公开的精神和范畴。
本文中，除非语境中明确排除，单数形式“一”和“该”包括复数个对象。因此，例如，所提及的“一抗原”包括抗原的混合物；所提及的“一药学可接受的载剂”包括两种或多种此类载剂的混合物等等。如此，术语“一”、“一个或多个”、以及“至少一个”可在本文中互换地使用。
此外，本文中使用的“及/或”被用于对两种特定特征或成分中的一或二者的详细说明。因此，本文中，在语句“A及/或B”中使用的术语“及/或”意图包括“A及B”、“A或B”、“(仅有)A”和“(仅有)B”。
本公开提供一种包含抗原和佐剂的疫苗组合物，其中，该佐剂是重组HBc VLP。
本文中，术语“病毒样颗粒”(VLP)是指像是病毒的结构，但因为其缺乏病毒基因组而是非感染性的。本文中，术语“非感染性的”是指不能进入宿主细胞内。典型地，由于病毒样颗粒缺少全部或部分的病毒基因组，特别是该病毒基因组的复制性和感染性组分，它们不能复制且缺乏致病性。该病毒样颗粒可以是病毒衣壳，如包覆有被认为是病毒包膜的脂质膜的相应病毒的病毒衣壳。术语“病毒衣壳”或“衣壳”是指，由病毒蛋白次单元构成的大分子组件。另外，病毒样颗粒通常可通过异源表达而大量生产且可轻易地纯化。
一旦在适宜的表达系统内进行该蛋白的重组表达，则VLP可自发形成。生产特定VLP的方法是该领域已知的。VLP的存在可使用该领域已知的传统技术检测，如通过电子显微镜、X射线晶体学等检测。
本公开的一具体实施例中，该佐剂可以是重组乙肝核心抗原(HBcAg)。本公开的另一具体实施例中，该佐剂可以是具有一氨基酸序列的重组HBcAg，其中，该氨基酸序列与SEQ ID NO:1的氨基酸序列的一致程度为至少80％且具有与SEQ ID NO:1相同的功能。本公开的又一具体实施例中，该重组HBcAg包含一氨基酸序列，该氨基酸序列与SEQ ID NO:1的氨基酸序列的一致程度为至少约80％、85％、90％、95％、96％、97％、98％、或99％。
本文中，术语“序列一致性”或，例如，包含“80％的序列一致” 是指，在整个比较窗口中，序列以一个核苷酸对一个核苷酸或一个氨基酸对一个氨基酸为基准的一致性程度。因此，可通过下述计算“序列一致性的百分比”：比较在比较窗口内两个最适宜地对准的序列，确定两个序列中出现一致的核酸碱基(如，A、T、C、G、I)或一致的氨基酸残基(如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置数，得到匹配位置数，该匹配位置数除以比较窗口内位置的总数(即，窗口尺寸)，结果再乘以100，得到序列一致性的百分比。所包括的是与本文中揭示的任何参考序列(见，如，序列表)具有至少约80％、85％、90％、95％、97％、98％、99％或100％序列一致性的核苷酸和多肽，典型地，该多肽变体保持该参考多肽的至少一种生物学活性或功能。
本公开的一具体实施例中，该佐剂是由SEQ ID NO:1的氨基酸序列组成的重组HBcAg，本文中也称为“HBcAg148”，已经证实其形成病毒样颗粒。
本公开提供包含HBcAg148病毒样颗粒的佐剂组合物，其中，该HBcAg148病毒样颗粒是由来自乙肝病毒(HBV)的衣壳蛋白自组装而形成的惰性空衣壳。HBV是具有环状的、部分双链的DNA基因组的小包膜病毒。它是全世界感染性肝病的主要肇因。HBV感染影响全世界大约20亿人，且成年人的HBV感染一般是短暂的。HBcAg是可在核衣壳即HBV最内层的表面上发现的抗原。因为HBcAg148VLP的组装不并入遗传物质，所以它们是非感染性的。
本公开的一具体实施例中，该抗原源自感染性疾病，包括但不限于，人类免疫缺陷病毒、水痘-带状疱疹病毒、1型单纯性疱疹病毒、2型单纯性疱疹病毒、人类巨细胞病毒、登革病毒、甲肝病毒、乙肝病毒、丙肝病毒、戊肝病毒、RSV、SARS病毒、人乳头状瘤病毒、流感病毒、Hib、脑膜炎病毒、沙门氏菌、奈瑟氏菌、包柔氏螺旋体、衣原体、博戴氏杆菌、肠毒素大肠杆菌、弯曲菌、链球菌、莫拉克斯氏菌、支原体、分枝杆菌、嗜血杆菌、疟原虫或弓浆虫、Stanworth十肽。
上述源自感染性疾病的抗原是指，被测试生物体内发展出的免疫应答作为靶标的任何物质。上述源自感染性疾病的抗原也可以是当与免疫活性细胞接触时的免疫应答(如，免疫活性细胞的成熟、细胞因子 的产生、和抗体的产生)的靶标。
本公开的一具体实施例中，该抗原可源自RSV。如本文中所述，RSV已经被识别为婴幼儿下呼吸道感染的最常见肇因。RSV具有由三个连续基因(SH-G-F)编码的三种表面糖蛋白：小疏水性糖蛋白(SH)、依附糖蛋白(G)和融合糖蛋白(F)。RSV疫苗研发的主要靶标抗原是RSV F和G，因为这些抗原各自能生成中和抗体以及T细胞应答。由于其在RSV隔离群中可观的保守性，F尤其引人注目。历史上，存在两种已知的发现于RSV F的融合前和融合后两种构型上与中和(NT)活性相关联的主要抗原性位点。它们最初通过结合至鼠科动物单克隆抗体(mAb)的1129(位点II)而被界定(Beeler,J.A.et al.,1989；Arbiza,J.,et al.,1992)和101F(位点IV)(Wu,S.J.,et al.,2007)。位点II作为帕利珠单抗(palivizumab)的靶标而为人所知，帕利珠单抗可减少高风险婴儿群体的重度RSV疾病。McLellan等人(McLellan,J.S.,et al.,2013)分离了鼠抗体，5C4，它有效中和RSV但显示不结合至融合后的F蛋白。5C4与从经免疫接种的PBMCs、D25和AM22分离的其它两种抗体分享这些性质，该抗体在中和RSV方面已经显示了比帕利珠单抗大100倍的效力(McLellan,J.S.,et al.,2013)。D25和AM22以位点
Figure PCTCN2017112350-appb-000021
为靶标，该位点是位于融合前RSV F三聚体表面上的亚稳态抗原性位点(Spits,H.,et al.,2010；Beaumont,T.,et al.,2012)。F蛋白的融合前晶体结构和融合后晶体结构暗示，虽然在两种结构上都发现了位点II和位点IV，位点
Figure PCTCN2017112350-appb-000022
似乎是对融合前形式有特异性(McLellan,J.S.,et al.,2013)。
RSV F的融合肽区域位于F1亚基的氨基端(Collins,P.L.,et al.,1996)，而该跨膜段含有两个区域：4,3-疏水性七元重复序列(HR)、令人联想到卷曲螺旋结构的序列基序(Chambers,P.,et al.,1990；Singh,M.,et al.,1999)。这些区域分别指示为HRN和HRC，且通过约270个氨基酸的中介结构域分隔。HRN和HRC形成三聚体的发夹样结构，且该HRC区域以与由HRN区域形成的卷曲螺旋反平行的方式封装(Baker,K.A.,et al.,1999)。
本公开的一具体实施例中，该抗原是重组RSV F蛋白，其包含HRN区域、HRC区域、以及选自由位点
Figure PCTCN2017112350-appb-000023
、位点II和位点IV所组成群组的至少一个抗原性位点。本公开的另一具体实施例中，该抗原可由SEQ  ID NO:2至SEQ ID NO:4中之一表示。
一具体实施例中，该疫苗组合物可用来诱导受试者体内对感染性物质如RSV的免疫应答。因此，若干具体实施例中，可将该包含治疗有效量的重组RSV F蛋白作为抗原的疫苗组合物给药至受试者，以引起对RSV的免疫应答。
本公开的另一具体实施例中，在足以预防或缓解有需要的受试者体内的RSV感染的条件下，将包含治疗有效量的重组RSV F蛋白作为抗原的疫苗组合物给药至该受试者。该疫苗组合物以足以引起该受试者体内的对抗RSV抗原如RSV F蛋白的免疫应答的量给药。本公开的一具体实施例中，该疫苗组合物适用于黏膜疫苗接种，且可口服、鼻内、经直肠或经阴道给药至该受试者。
本公开的一具体实施例中，该佐剂以0.1μg至1,000μg的佐剂有效量存在于该疫苗组合物中。本公开的另一具体实施例中，该疫苗组合物包含重量比为10:1至1:10的抗原与佐剂的混合物。本公开的又一具体实施例中，该疫苗组合物中包含的抗原与佐剂的重量比为5:1至1:5。
本公开的一具体实施例中，疫苗组合物中的HBc VLP用作为有效增强对抗原的免疫应答及/或调节其朝向所希望的免疫应答发展的唯一佐剂。本公开的另一具体实施例中，该疫苗组合物可包含额外的佐剂。可用于本公开的额外的佐剂可包括，但不限于，CpG寡核苷酸。
本公开的一具体实施例中，该疫苗组合物可进一步包含药学可接受的载剂。本文中，“药学可接受的载剂”包括可适用于本公开疫苗组合物的给药的任何和全部溶剂、分散介质、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。可用于本公开的药学可接受的载剂可包括，但不限于，防腐剂、悬浮剂、增粘剂、等渗剂、缓冲剂、和湿润剂。
本公开进一步提供一种制备疫苗组合物的方法，该方法包含提供包含HBcAg148VLP和药学可接受的载剂的佐剂组合物，以及将该佐剂组合物与作为抗原的重组RSV F蛋白合并。本公开的一具体实施例中，该HBcAg148VLP由SEQ ID NO:1的氨基酸序列组成。
根据本公开的又一态样，本公开提供一种编码上述HBcAg148VLP的核酸分子。本公开的一具体实施例中，该核酸分子是经密码子优化 以用于在原核细胞内表达。另一具体实施例中，该原核细胞是大肠杆菌细胞。再一具体实施例中，该核酸分子包含核酸序列，该核酸序列其与SEQ ID NO:5的核酸序列
(ATGGACATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTCAGAGATCTTCTAGACACCGCCTCAGCTCTGTATCGAGAAGCCTTAGAGTCTCCTGAGCATTGCTCACCTCACCATACTGCACTCAGGCAAGCCATTCTCTGCTGGGGGGAATTGATGACTCTAGCTACCTGGGTGGGTAATAATTTGGAAGATCCAGCATCCAGGGATCTAGTAGTCAATTATGTTAATACTAACATGGGTTTAAAGATCAGGCAACTATTGTGGTTTCATATATCTTGCCTTACTTTTGGAAGAGAGACTGTACTTGAATATTTGGTCTCTTTCGGAGTGTGGATTCGCACTCCTCCAGCCTATAGACCACCAAATGCCCCTATCTTATCAACACTTCCGGAAACTACTGTT)的一致程度为至少约80％、85％、90％、95％、96％、97％、98％、或99％。
本公开的一具体实施例中，该核酸分子是经密码子优化以用于在真核细胞内表达。另一具体实施例中，真核细胞是酵母细胞或哺乳动物细胞。又一具体实施例中，该哺乳动物细胞是人细胞。
本公开进一步提供一种通过上文揭示的疫苗组合物诱导黏膜免疫应答和系统性免疫应答的方法。将该疫苗组合物给药至有此需要的受试者，从而诱导该受试者体内的黏膜免疫应答和系统性免疫应答，其中，该黏膜免疫应答是抗原特异性IgA抗体的产生，而该系统性免疫应答是抗原特异性IgG抗体的产生和抗原特异性细胞介导免疫的产生。
根据本公开的又一态样，本公开提供一种对受试者接种疫苗的方法，该方法包括将上文揭示的疫苗组合物给药至该受试者。疫苗接种方法包含通过疫苗领域中已知的任何传统途径给药该疫苗组合物，该途径为，例如，经由黏膜(如，眼部、鼻内、肺部、口腔、胃部、小肠、直肠、阴道、或泌尿道)表面、经由肠胃外(如，皮下、皮内、肌肉内、静脉内、或腹膜内)途径、或外用给药(如，经由透皮传送系统如贴剂)。
本公开的一具体实施例中，将该疫苗组合物给药至该受试者的黏膜表面。本公开的另一具体实施例中，该黏膜表面可选自呼吸道黏膜、胃肠道黏膜、阴道黏膜、鼻黏膜、直肠黏膜和口腔黏膜所组成的群组。
已经使用多个实施例来例示性说明本公开。下述实施例不应视为限制本公开的范畴。
[实施例]
实施例1：重组HBc VLP表达载体的构建
合成了具有用于大肠杆菌(Escherichia coli(E.coli))表达的最优密码子的HBc蛋白的全长度cDNA序列(Genomics BioSci&Tech)。使用这一序列作为PCR模板，扩增了HBc的核苷酸1至444(SEQ ID NO:5)，随后将其插入以6-His在C端标记的pET28a的NcoI-XhoI限制位点，以获得重组HBc VLP质粒。将所得质粒转化至大肠杆菌BL21(DE3)感受态细胞内用于蛋白质表达。
实施例2：重组RSV嵌合体F蛋白表达载体的构建
合成了具有用于大肠杆菌表达的最优密码子的RSV F蛋白的全长度cDNA序列(Genomics BioSci&Tech)。使用这一序列作为PCR模板，扩增了RSV F蛋白的4种基因片段，包括含有HRN和位点
Figure PCTCN2017112350-appb-000024
的核苷酸457至633(SEQ ID NO:6)、含有位点II的核苷酸760至849(SEQ ID NO:7)、含有位点IV的核苷酸1264至1314(SEQ ID NO:8)、以及含有该C端α-螺旋(HRC)的核苷酸1426至1560(SEQ ID NO:9)。
通过部分重叠PCR链接这4种PCR扩增子，并通过富甘氨酸链接基如GSGS、GGGS、GGSG、SGSG和GG连接，以形成所构建的基因(命名为
Figure PCTCN2017112350-appb-000025
)，随后，该将该基因插入以6-His在羧基端标记的pET28b的NcoI-XhoI限制位点内，以获得
Figure PCTCN2017112350-appb-000026
质粒。
Figure PCTCN2017112350-appb-000027
和HBc质粒的构建过程与
Figure PCTCN2017112350-appb-000028
质粒类似，但区别如下。
对于
Figure PCTCN2017112350-appb-000029
质粒的构建，扩增了RSV F蛋白的以SEQ ID NO:6和SEQ ID NO:9表示的基因片段。随后，将这两种PCR扩增子插入以6-His在C端标记且通过富甘氨酸链接基(linker)而连接的pET28a的NcoI/BamHI和EcoRI/XhoI限制位点内，以获得
Figure PCTCN2017112350-appb-000030
质粒。
对于
Figure PCTCN2017112350-appb-000031
质粒的构建，扩增了RSV F蛋白的以SEQ ID NO:6和SEQ ID NO:9表示的基因片段。再者，通过PCR创建分别含有 NheI/BamHI、BamHI/EcoRI、或EcoRI/HindIII限制位点的三种位点
Figure PCTCN2017112350-appb-000032
片段。随后，将这5种PCR扩增子插入以6-His在C端标记且通过富甘氨酸链接而连接的pET28a的NcoI NheI/BamHI/EcoRI/HindIII/XhoI限制位点内，以获得
Figure PCTCN2017112350-appb-000033
质粒。
将上述所得质粒转化至大肠杆菌BL21(DE3)感受态细胞内进行蛋白质表达。
实施例1和2的PCR中使用的引物以SEQ ID NO:10至SEQ ID NO:29表示，显示于表1中。
表1：引物序列
Figure PCTCN2017112350-appb-000034
Figure PCTCN2017112350-appb-000035
实施例3：重组蛋白表达和纯化
分别在从实施例1和2中获得的经转化的大肠杆菌BL21(DE3)中表达重组RSV F蛋白-6His和HBc-6His，并使用镍亲和色谱纯化。通过1体积的样品对200体积的透析缓冲液(从350mM、150mM至0mM咪唑，1×PBS中)的梯度透析，对洗脱的(使用500mM咪唑、50mMNaH2PO4、300mM NaCl pH 8.0)蛋白进行缓冲液交换，每一步骤进行12小时。使用离心浓缩机(10,000MWCO,Sartorius)将经透析的蛋白-6His浓缩至约1mg/mL的浓度。通过SDS-PAGE测定该蛋白的分子大小和纯度。
通过使用指向为抗His标签的抗体的免疫印迹分析，检测了同一移动性的条带，结果显示于图1B,1D、和1F中。通过使用指向为抗HBc和RSV的抗体的免疫印迹分析，检测了同一移动性的条带，结果分别显示于图1H和1I中。对考马斯蓝染色的凝胶的光密度扫描显示，纯化的蛋白
Figure PCTCN2017112350-appb-000036
和HBc相当于总蛋白的90％以上(图1A、1C、1E、和1G)，其纯度足以进行免疫接种。
实施例4：重组HBc VLP的透射电镜(TEM)图像
在室温下，令PBS中的8μg的纯化HBc VLP在铜网格(300目)上吸附3分钟。随后，使用滤纸轻柔地吸干网格。以1％乙酸双氧铀水溶液染色30秒后，移除过量的液体。使用JEM-1400电子显微镜在80kV检查网格。
已经通过TEM证实，HBc VLP形成病毒样颗粒(图2)。
实施例5：动物免疫分析
1.RSV A2株原液的制备
从ATCC获得RSV A2株。在HEp-2细胞ATCC中实施病毒的繁殖。使用RSV A2以0.2的m.o.i(感染复数)接种在100mm有盖培养皿(Thermo Scientific)中生长的高达80％融合度的细胞。在37℃的CO2培养箱内，在不含血清的达尔博克改良伊戈尔培养基(Dulbecco's Modified Eagle's medium(DMEM))中进行病毒的吸附。2小时后，将培养基替换为以2％胎牛血清补充的DMEM，将给培养皿再培养48至72小时。通过以3,000rpm离心10min，从细胞碎片分离出含有该病毒的上清液。随后通过离心浓缩机(100,000MWCO,Sartorius)浓缩病毒。
2.RSV噬斑试验
通过噬斑试验测定RSV病毒效价(titer)。以1×PBS洗涤12孔板中的HEp-2细胞融合单层，随后以多种稀释度(10-3至10-7)的RSV A2病毒感染该细胞。进行2小时的病毒吸附后，移除上清液，以1×PBS洗涤细胞的单层，之后以DMEM+2％胎牛血清+0.3％琼脂糖覆盖。于37℃在CO2培养箱中培养5天后，细胞以10％福尔马林固定，并使用 0.05％结晶紫染色进行噬斑定量。
3.疫苗给药和RSV激发(challenge)
将无病原体的C57BL/6J雌性小鼠(6至8周龄)随机分为若干组，通过鼻内(i.n)途径使用候选疫苗在第0、21、42和63天进行免疫接种(图3A)或在第0、21和42天进行免疫接种(图3B)，并在第84天(图3A)或第63天(图3B)令小鼠经1×106p.f.u RSV激发。候选疫苗包括：
Figure PCTCN2017112350-appb-000037
HBc VLP+
Figure PCTCN2017112350-appb-000038
HBc VLP+
Figure PCTCN2017112350-appb-000039
+CpG(TCGTCGTTTTCGGCGCGCGCCG，SEQ ID NO.30)(基龙米克斯生物科技公司，台湾)；
Figure PCTCN2017112350-appb-000040
HBc VLP+
Figure PCTCN2017112350-appb-000041
HBc VLP++CpG；
Figure PCTCN2017112350-appb-000043
HBc VLP+
Figure PCTCN2017112350-appb-000044
以及HBc VLPs+
Figure PCTCN2017112350-appb-000045
+CpG。还包括对照组、仅使用HBc VLP进行免疫接种的组、和使用1×105p.f.u福尔马林固定的RSV(FIRSV)进行肌肉内(i.m)免疫接种的组。
在RSV激发前的2天，从使用相同给药方案的各组别中收集小鼠血清、气管肺泡灌洗液(BALF)和脾脏。对于RSV激发，使用1.5％异氟烷麻醉动物，随后通过1×106p.f.u RSV的鼻内接种令其感染。进行RSV激发后，监控小鼠的体重改变5天。最终，牺牲小鼠，收集个体的肺进行病毒负载和组织病理学实验。
实施例6：对由候选疫苗引起的抗体应答的评估
通过酶联免疫吸附试验(ELISA)测试从如实施例5中揭示的免疫接种小鼠收集的血清和BALF的抗体应答。简而言之，在4℃，以50μL的纯化
Figure PCTCN2017112350-appb-000046
(10μg/ml)涂覆96孔板过夜。以2％BSA将该板在37℃封闭1小时，使用血清样品在试验稀释剂(1％BSA、0.05％Tween 20，在1×PBS中)中的逐级稀释液(10-2至5.12×10-4)或BALF的逐级稀释液(10-1至1.28×10-3)在室温培养2小时。对每一样品做稀释物曲线，且将终点效价计算为产生最优浓度的稀释物的倒数，该最优浓度比背景值(对汇集的前免疫血清进行1/50稀释，或对汇集的对照BALF进行1/5稀释)高0.1U。将低于50(阴性样品)的IgG效价或低于5的分泌性IgA(sIgA)效价任意地指定为50或5。
参考图4A至4F，该图显示候选疫苗的免疫原性评估结果，其中， 各组小鼠分别使用4剂的i)
Figure PCTCN2017112350-appb-000047
(10μg)；ii)
Figure PCTCN2017112350-appb-000048
(50μg)；iii)HBc(10μg)+
Figure PCTCN2017112350-appb-000049
(50μg)；或作为阴性对照的iv)HBc(10μg)经鼻内途径进行免疫接种。使用间接ELISA分析所收集的最终血清的
Figure PCTCN2017112350-appb-000050
特异性或FIRSV特异性IgG应答。结果表明，使用
Figure PCTCN2017112350-appb-000051
混合物进行鼻内免疫接种会生成显著更高的
Figure PCTCN2017112350-appb-000052
特异性总IgG、IgG1和IgG2a(图4A至4C)。FIRSV特异性总IgG和IgG1也被诱导但不是显著更高(图4D和4E)。这些结果证实，HBc VLP可提升
Figure PCTCN2017112350-appb-000053
特异性体液应答。
参考图5A至5J，为了评估HBc VLP的剂量-应答关系，分别使用4剂的下述试剂对各组小鼠进行鼻内免疫接种：i)
Figure PCTCN2017112350-appb-000054
(10μg)；ii)HBc VLP(50μg)+
Figure PCTCN2017112350-appb-000055
(10μg)；iii)
Figure PCTCN2017112350-appb-000056
(10μg)+CpG(20μg)；iv)HBc VLP(25μg)+
Figure PCTCN2017112350-appb-000057
(10μg)+CpG(20μg)(图中标注为HBc+
Figure PCTCN2017112350-appb-000058
+CpG-1)；v)HBc VLP(50μg)+
Figure PCTCN2017112350-appb-000059
(10μg)+CpG(20μg)(图中标注为HBc+
Figure PCTCN2017112350-appb-000060
+CpG-2)；或vi)HBc VLP(50μg)。通过间接ELISA分析血清和BALF。图5A至5C和5I显示，投予HBc
Figure PCTCN2017112350-appb-000061
混合物可引起显著更高的血清
Figure PCTCN2017112350-appb-000062
特异性总IgG、IgG1、IgG2a以及肺
Figure PCTCN2017112350-appb-000063
特异性sIgA。此外，使用CpG作为佐剂并未提升这些体液应答。
由于对抗RSV的保护性免疫需要有效的Th1偏差应答和IFN-γ的产生，我们测试了来自经免疫接种小鼠的脾细胞对于
Figure PCTCN2017112350-appb-000064
重组蛋白体外刺激的应答能力。通过鼠IFN-γELISA试剂盒(BioLegend)测定来自48h 
Figure PCTCN2017112350-appb-000065
刺激培养物的T细胞扩增。分析来自这些细胞的培养物上清液的Ag特异性IFN-γ的产生。如图5K所示，来自使用
Figure PCTCN2017112350-appb-000066
进行免疫接种的小鼠的脾细胞，显示在
Figure PCTCN2017112350-appb-000067
刺激后没有IFN-γ分泌。相比之下，来自使用
Figure PCTCN2017112350-appb-000068
和
Figure PCTCN2017112350-appb-000069
进行免疫接种的组中小鼠的脾细胞，显示在
Figure PCTCN2017112350-appb-000070
刺激后显著更高水平的IFN-γ分泌。值得注意的是，在使用HBc进行免疫接种的鼠体内或对照组中没有IFN-γ分泌。
参考图6A至6H，为了检测对抗RSV F蛋白抗原性位点的特异性应答，使用RSV F位点
Figure PCTCN2017112350-appb-000071
、II和IV肽涂覆ELISA板(10μg/mL)。图6A至6H显示，仅有接受
Figure PCTCN2017112350-appb-000072
混合物的小鼠可生成显著更高 的位点
Figure PCTCN2017112350-appb-000073
特异性和位点II特异性总IgG、IgG1和IgG2a。所有组中均未检测出位点IV特异性IgG(数据未显示)。
此外，图7A至7E显示，接受3剂鼻内给药的与HBc VLP或CpG混合或不混合的
Figure PCTCN2017112350-appb-000074
或
Figure PCTCN2017112350-appb-000075
的小鼠体内的
Figure PCTCN2017112350-appb-000076
特异性抗体应答。各组小鼠分别使用3剂的下述试剂进行鼻内免疫接种：i)HBc VLP(25μg)；ii)
Figure PCTCN2017112350-appb-000077
(50μg)；iii)HBc VLP(25μg)+
Figure PCTCN2017112350-appb-000078
(25μg)；iv)HBc VLPs(25μg)+
Figure PCTCN2017112350-appb-000079
(25μg)+CpG(20μg)；v)HBc VLP(25μg)+
Figure PCTCN2017112350-appb-000080
(50μg)；vi)HBc VLP(25μg)+
Figure PCTCN2017112350-appb-000081
(50μg)+CpG(20μg)；vii)
Figure PCTCN2017112350-appb-000082
(50μg)；viii)HBc VLP(25μg)+
Figure PCTCN2017112350-appb-000083
(50μg)；ix)HBc VLP(25μg)+
Figure PCTCN2017112350-appb-000084
(50μg)+CpG(20μg)；x)
Figure PCTCN2017112350-appb-000085
(50μg)；xi)HBc VLP(25μg)+
Figure PCTCN2017112350-appb-000086
(50μg)；或xii)HBc VLP(25μg)+
Figure PCTCN2017112350-appb-000087
(50μg)+CpG(20μg)。通过间接ELISA分析血清和BALF。
参考图7A至7E，该图显示，通过使用HBc作为佐剂，
Figure PCTCN2017112350-appb-000088
Figure PCTCN2017112350-appb-000089
和
Figure PCTCN2017112350-appb-000090
可引起显著更高的血清
Figure PCTCN2017112350-appb-000091
特异性总IgG、IgG1、IgG2a、IgA以及肺
Figure PCTCN2017112350-appb-000092
特异性sIgA，其中，在
Figure PCTCN2017112350-appb-000093
组中观察到了最高的终点效价。此外，通过使用HBc与CpG的混合物作为佐剂，
Figure PCTCN2017112350-appb-000094
也可引起更高的血清
Figure PCTCN2017112350-appb-000095
特异性总IgG、IgG1、IgG2a、IgA以及肺
Figure PCTCN2017112350-appb-000096
特异性sIgA。
实施例7：候选疫苗在保护小鼠对抗RSV感染上的效果
为了评估投予4次该候选疫苗时的效力，使用如实施例5和图5A至6H中揭示的活体RSV A2株激发的经免疫接种小鼠。
1.进行RSV激发后的血清中和效价
中和抗体是由疫苗诱导的免疫应答中的重要功能性组分。图8显示，来自
Figure PCTCN2017112350-appb-000097
组的血清可减少约10％的噬斑形成，来自
Figure PCTCN2017112350-appb-000098
组的血清减少约25％的噬斑形成，而接受CpG作为佐剂的组则可减少约17％至35％的噬斑。
2.进行RSV激发后的小鼠体重改变
小鼠在激发感染后的体重改变是评价疫苗保护性效力的最重要的指标。参考图9，接受肌肉注射FIRSV的小鼠显示最高的体重减轻(约 23％)，而使用
Figure PCTCN2017112350-appb-000099
进行免疫接种的小鼠显示约10％的体重减轻。使用不同剂量的HBc(0、25、50μg)进行免疫接种的小鼠，即
Figure PCTCN2017112350-appb-000100
组、
Figure PCTCN2017112350-appb-000101
组、
Figure PCTCN2017112350-appb-000102
组的小鼠，分别在激发后第4天显示约8％、4％和0％的体重减轻。
因此，本公开提供更好的保护以预防小鼠体重减轻，并提供从活体RSV激发后的初始体重减轻快速的恢复。这些证明，由本申请案的抗原引起的抗病毒性免疫提供保护，用于对抗活体RSV A2病毒株。
3.进行RSV激发后的肺组织病理学
在进行激发5天后，从个体小鼠收集肺组织用于组织学分析。对于组织学分析，肺样本在10％中性缓冲福尔马林中固定24小时，包埋在石蜡块中，切割为厚度为5μm的切片，且使用苏木精和曙红(H&E)染色。
参考图10，在对照组或经疫苗接种的小鼠体内观察到了肺组织病理学的改变。经FIRSV免疫接种的小鼠显示严重水平的组织病理学。
在1960年代后期，当在临床试验中使用Lot 100FIRSV疫苗时，接种疫苗的儿童在感染时发展出增强的呼吸道疾病(Kim,H.W.,et al.,1969)。另外，目前的研究显示，FIRSV在激发RSV的小鼠体内诱发了显著的肺部肺泡炎(alveolitis)和血管周炎(perivascusculitis)。相比之下，如图10所示，如气管和肺泡周边的某种程度的浸润所证明的，接受与CpG混合或不混合的
Figure PCTCN2017112350-appb-000103
混合物组的小鼠，显示了中度的肺部病理学。再者，在仅使用重组
Figure PCTCN2017112350-appb-000104
进行免疫接种的小鼠体内，未观察到明显的肺部病理学改变。
本公开提供经纯化的HBcAg148蛋白、以及编码该HBcAg148蛋白的具有用于大肠杆菌表达的优化密码子的核酸分子。已经通过TEM证实，该经纯化的HBcAg148蛋白形成病毒样颗粒。再者，通过对小鼠同时经鼻内投予RSV候选疫苗和佐剂HBcAg148，可观察到HBcAg148在提升体内免疫应答中的效力。通过本公开表明，HBcAg148将会提升对抗RSV的血清总IgG、IgG1和IgG2a应答，且这一佐剂效应与CpG基序类似。
因此，这些结果表明，本公开的包含重组HBc VLP作为佐剂的疫苗组合物可诱导对于该抗原为特异性的系统性和黏膜抗体应答。使用本公开的疫苗组合物进行免疫接种的小鼠，显示被保护对抗该抗原而不造成肺病。再者，与FIRSV相比，在小鼠模型中，本公开的疫苗组合物并不过度刺激淋巴细胞，且提供了一种潜在的、安全的RSV候选疫苗。
上文中，已经使用例示性优选具体实施例详细揭示了本公开。但应理解，本公开的范畴并不限于所揭露的具体实施例。相反，本公开意图覆盖多种修饰和类似的调整。因此，权利要求的范畴应根据最广泛的诠释而定，从而涵盖所有此类修饰和类似的调整。

Claims (20)

  1. 一种疫苗组合物，其包含抗原和佐剂，其特征在于，该佐剂是乙肝核心病毒样颗粒(HBc VLP)，该颗粒具有与SEQ ID NO:1的氨基酸序列具有至少80％一致的氨基酸序列且具有与SEQ ID NO:1相同的功能。
  2. 如权利要求1所述的疫苗组合物，其特征在于，该佐剂是由SEQ ID NO:1的氨基酸序列组成的HBc VLP。
  3. 如权利要求1所述的疫苗组合物，其特征在于，该抗原是源自感染性疾病的抗原。
  4. 如权利要求1所述的疫苗组合物，其特征在于，该抗原源自下列所组成的群组中的一种：人类免疫缺陷病毒、水痘-带状疱疹病毒、1型单纯性疱疹病毒、2型单纯性疱疹病毒、人类巨细胞病毒、登革病毒、甲肝病毒、乙肝病毒、丙肝病毒、戊肝病毒、呼吸道合胞体病毒(RSV)、严重急性呼吸道合胞体病毒(SARS病毒)、人乳头状瘤病毒、流感病毒、Hib、脑膜炎病毒、沙门氏菌、奈瑟氏菌、包柔氏螺旋体、衣原体、博戴氏杆菌、肠毒素大肠杆菌、弯曲菌、链球菌、莫拉克斯氏菌、支原体、分枝杆菌、嗜血杆菌、疟原虫或弓浆虫、Stanworth十肽。
  5. 如权利要求4所述的疫苗组合物，其特征在于，该抗原源自RSV。
  6. 如权利要求5所述的疫苗组合物，其特征在于，该抗原是由SEQ ID NO:2至SEQ ID NO:4中之一表示的重组RSV F蛋白。
  7. 如权利要求1所述的疫苗组合物，其特征在于，该佐剂以0.1μg至1,000μg的佐剂有效量存在。
  8. 如权利要求1所述的疫苗组合物，其特征在于，该抗原和佐剂 的重量比为10:1至1:10。
  9. 如权利要求8所述的疫苗组合物，其特征在于，该抗原和佐剂的重量比为5:1至1:5。
  10. 如权利要求1所述的疫苗组合物，其适用于黏膜疫苗接种。
  11. 如权利要求1所述的疫苗组合物，其适用于口服、鼻腔、直肠或阴道使用。
  12. 如权利要求1所述的疫苗组合物，其诱导黏膜免疫应答和系统性免疫应答，该黏膜免疫应答是抗原特异性分泌性IgA抗体的产生，而该系统性免疫应答是抗原特异性IgG抗体的产生和抗原特异性细胞介导性免疫的产生。
  13. 一种对受试者接种疫苗的方法，包含给药如权利要求1所述的疫苗组合物至该受试者的黏膜表面。
  14. 如权利要求13所述的方法，其特征在于，该黏膜表面选自呼吸道黏膜、胃肠黏膜、阴道黏膜、鼻黏膜、直肠黏膜和口腔黏膜所组成的群组。
  15. 如权利要求13所述的方法，其特征在于，该受试者是人或动物。
  16. 一种多肽，由SEQ ID NO:1的氨基酸序列组成。
  17. 一种核酸分子，其编码如权利要求16所述的多肽。
  18. 如权利要求17所述的核酸分子，其密码子被优化以用于在原核细胞或真核细胞内表达。
  19. 如权利要求18所述的核酸分子，其特征在于，该原核细胞是大肠杆菌细胞，而该真核细胞是酵母细胞或哺乳动物细胞。
  20. 如权利要求19所述的核酸分子，其特征在于，该核酸分子包含与SEQ ID NO:5的核酸序列具有至少80％一致的核酸序列。
PCT/CN2017/112350 2016-11-22 2017-11-22 包含乙肝病毒样颗粒作为佐剂的疫苗组合物 WO2018095327A1 (zh)

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