Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification
Reexamination Certificate
1998-12-22
2001-04-24
LeGuyader, John L. (Department: 1633)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
C435S320100, C424S189100, C424S202100, C424S227100, C424S228100
Reexamination Certificate
active
06221664
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to vaccines, and/or immunogenic composition methods for their preparation and uses therefore. More particularly, the present invention comprises a method of preparing complex vaccines and/or immunogenic composition composed of antigen-antibody and recombinant DNA(s), especially microbial source antigen(s) and its(their) antibody and recombinant DNA(s) as immunogen(s) or adjuvant(s), which can be used for prevention or treatment against infectious diseases.
BACKGROUND OF THE INVENTION
Vaccines have been used to prevent infectious diseases, and the successful global elimination of smallpox was attained only through vaccination. However, not all preventive vaccines are as effective as vaccinia; therefore developing measures to improve the quality of vaccines, as well as decreasing the number of inoculations of vaccines to be given are necessary. Aside from preventive vaccines, therapeutic vaccines used for active immunization of patients with chronic bacterial or viral diseases (Tuberculosis, Herpes simplex, Viral hepatitis B, AIDS etc.) have also been developed. There are two categories of therapeutic vaccines, namely specific and non-specific therapeutic vaccines. Immunization with non-specific therapeutic vaccines (e.g. BCG,
Corynebacterium parvum
etc.) can induce non-specific host immune responses, especially cell-mediated immune responses, which showed some effects, but were usually not potent enough to terminate chronic infections. Since specific therapeutic vaccines can induce specific humoral and cellular immune responses in infected hosts, therefore specific therapeutic vaccines are more preferable. Presently, specific therapeutic vaccines can be grouped as: (1). Inactivated or killed whole microbial vaccines. Usually specific strains of microbes were selected and were inactivated or killed for preparation. (2). Recombinant vaccines expressing specific microbial antigen(s). Recombinant technologies were employed to express one or more microbial antigen(s). (3). Chimeric vaccines: Chimeric genes constructed with genes coding for specific microbial antigens fused with genes coding for cytokines (interferons or interleukin-2 etc.) were expressed as fusion proteins and used for active immunization. (4). Synthetic peptide vaccines: Epitopes from microbes were selected and were either used for preparation of synthetic peptides or used for preparation of chimeric peptides (with adjuvants or cytokines added) for active vaccination. (5). Microbial antigen-antibody complex: Appropriate ratio of microbial antigen and antibody complex was used to generate more potent immune responses. (6). Nucleic acid (DNA or polynucleotide, or recombinant DNA vaccine) vaccines: Fragments of genes coding for the protective antigens of microbes were cloned into expression vectors and these recombinant-vector nucleic acid vaccines were used for immunization. Significant enhanced humoral, and especially cellular immune response could be generated by this approach of immunization. Appropriate method of recombinant DNA vaccine delivery into tissues (muscles, skin) is critical, in order to induce effective immune responses. In addition, minimizing the amount of nucleic acid used for induction of effective immune responses is desirable.
Davis H L et al. (
Human Molecular Genetics
1993;2:1857) demonstrated that by using a vector DNA which contains the encoding gene fragment of hepatitis B surface antigen for DNA-based immunization in mice, secretion of hepatitis B surface antigen and high levels of circulating antibody was detected. A number of publications appeared in the past few years in regard to various microbial nucleic acid (or DNA) vaccination. Among these, Macini M et al (
Proc. Natl. Acad. Sci. USA
1996; 93:12496) reported by DNA-based immunization in a transgenic mouse model of the hepatitis B surface antigen chronic carrier state, resulted in complete clearance of circulating HBsAg, which raised the possibility of designing effective ways for treatment of chronic hepatitis B. In PCT WO 95/11307 (Nucleotide vector, composition containing such vector and vaccine for immunization against hepatitis) and PCT WO 95/20660 (Immunization by inoculation of DNA transcription unit) etc., different composition and routes of nucleic acid immunization have been employed; however, preparation of antigen-antibody-recombinant DNA complex vaccine and its immunogenicity has not been mentioned in such literature.
Enhanced immune responses were shown by immunization using microbial antigen complexed to specific antibodies, as reported in Venezuelan Equine Encephalomyelitis Virus (Houston W E et al.
J Inf Dis
1977;155:600), tetanus toxoid (Stoner R D et al.
J Immunol
1963;91:761), hepatitis B surface antigen (Chang T S.
Immunology Today
1985;6:245). In addition, mice immunized with solid-matrix-antibody-antigen complexes of multivalent antigens (Herpes simplex virus glycoprotein D, influenza virus HA protein, simian virus HN proteins etc.) showed vigorous humoral and cellular immune responses (Randall R E et al.
J Virol
1989;63:1808). Such published manuscripts have described only the use of polyclonal or monoclonal animal antibodies complexed to antigens to induce immune responses. Immune responses were improved, but there was no description of using the complex for treatment of chronic persistent infections. One of the present inventors patented (in China) and published in the literature using hepatitis B vaccine complexed to human specific immunoglobulins against Hepatitis B vaccine (HBIG) to treat chronic hepatitis B patients (Wen Y M et al.
Lancet
1995;345:8964). To date, no antigen-antibody complex including incorporation of recombinant plasmid DNA as a complex vaccine has been described.
SUMMARY OF THE INVENTION
This invention provides a vaccine and/or immunogenic composition composed of antigen(s) of microbial source, its(their) specific antibody(s) and recombinant plasmid DNA(s) from the gene or genes of microbes or other sources as immunogen or adjuvant. Immunization with this complex vaccine can significantly enhance host humoral and cellular immune responses, and thus can be used to prevent or treat subjects who are immune tolerant or immune defective against specific microbial infections. This complex vaccine can be used to treat chronic persistent infected patients or asymptomatic carriers.
DETAILED DESCRIPTION
The technology of this invention is to use one or more natural or recombinant antigen(s) and its (their) specific antibody(s) and recombinant plasmid DNA containing one or more gene(s) coding for microbial proteins and other proteins to prepare a complex vaccine and/or immunogenic composition. Specifically, microbial protein antigen and its specific antibody or high titer immunoglobulin (animal or human source) were separately diluted and titrated by chessboard method, to assay for the best combination ratio of antigen-antibody complex. According to the property of the specific antigen protein used and the affinity of the antibody used, antigen and antibody were incubated at 37° C. for 2-4 hours, and spinned at 12,000 rpm to separate the precipitate. The remnant antigen and antibody in the supernatant can be very little and absorbence (A) assayed by Enzyme-linked immuno-sorbent assay (ELISA) can both be less than 0.4, in the appropriate antigen-antibody complex tube. After removal of the supernatant, the same amount of antigen as titrated in the appropriate ratio study was further added to the precipitate to ensure proper amount of antigen in excess, and subsequently, recombinant plasmid DNA (pCMV HBs) was added. This recombinant plasmid DNA contains eukaryotic cell expression element, such as pcDNAI/Amp (Invitrogen product), VR1012 (Vical product), PCI
eo (Promega product) etc. After digestion with restriction enzyme EcoRI, Hepatitis B virus surface antigen (HBsAg) encoding gene or the core gene of Hepatitis C virus is inserted to construct the recombinant plasmid DNA. In addition, recombinant plasmid DNA can be constructed with gene(
He Lifang
Qu Di
Wen Yumei
LeGuyader John L.
Merchant & Gould P.C.
Shanghai Medical University
Stroup Carrie
LandOfFree
Composite vaccine which contains antigen, antibody and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Composite vaccine which contains antigen, antibody and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Composite vaccine which contains antigen, antibody and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2471256