The Preclinical Toxicology Studies On HSP65-MUC1 Tumor Vaccine, HIV DNA Vaccine And On HIV Recombinant Virus Vaccine

Vaccines are among the most widely used pharmaceuticals, providing significant benefits to human health through the eradication of smallpox and the protection against serious disease such as malaria. They are also used in the new treatment for cancer, autoimmunity, HIV, hypersensitivity and other diseases. New techniques involved in the production of vaccines are gene engineering and biochemical synthesis, by which the new type vaccines such as DNA vaccines, recombinant protein vaccines, synthetic peptide vaccines, recombinant virus and bacteria vaccines are produced.Preclinical toxicology study on vaccines has not been required in our country, and it is also an area in which many scientists worldwide are devoting their research interest. The toxicities, especially the immunotoxicities of vaccines, have been a hidden danger for the vaccination. In our country, new vaccines are continuously being developed, aiming at the initiation of clinical trials and entering the market as soon as possible. In order to conducting the preclinical safety evaluation of these new vaccines, the immunotoxicity and systemic toxicity for 3 selected types of vaccines, including heat shock protein-peptide complexes for tumor vaccine (recombinant heat shock protein 65-MUC1 fusion protein—HSP65-MUC1), HIV DNA vaccine (DNA vaccine) and recombinant smallpox virus vector HIV vaccine (virus vaccine), including a combination vaccine consisting of DNA and virus vaccine) were conducted. These common and special issues in the toxicity of these vaccines were expected to be found and the relevant method and techniques need to be established.The immunotoxicicty method established in this study includes: (1) Macropathology and histopathology study on the organs of immune system; (2) Lymphocyte proliferation test using CFSE stainning and flow cytometry in which radioactive elements are not utilized; (3) The examination of macrophage function; (4) Lymphocyte phenotyping by flow cytometry; (5) ELISA method to detect immunogenicity; (6) ELISA,intracellular staining and ELISPOT for T cell immune reaction (production of cytokine). For HIV DNA vaccine (DNA vaccine) and recombinant virus vector vaccine, a real time PCR method was established to detect the biodistribution of DNA (residual DNA). The general toxicity studies are body weights, food consumption, clinical signs, local tolerance, hematology, serum biochemistry, complete necropsy and histopathology.The toxicological study of HSP65-MUC1included:single dose toxicity study on C57BL/6 mice, repeated dose toxicity study on C57BL/6 mice and repeated dose toxicity study on rhesus monkeys. Given the number of doses in the proposed clinical trail is"n",we dosed mice by"n+1"and dosed monkeys by"n+4". The studies contained immunotoxicological and general toxicological components. The toxicological study of DNA vaccine was conducted together with the combination vaccines. BALB/c mice are used for a prime-boost regimen, which are same with that used in the proposed clinical trial. The study contained immunotoxicity, general toxicity and biodistribution studies. BALB/c mice were used in the toxicity study on virus vaccine and"n"and"n+1"regimen were designed respectively for immunotoxicity, general toxicity and biodistribution studies。The toxicology findings of these 3 tyeps of vaccines were as follows: (1) No HSP65-MUC1 related systemic or immunological toxicity was found in mice and monkeys; HSP65-MUC1 was found to be capable of inducing both humoral- immunity and the cell-mediated immunity; (2) A reversible local reaction was found induced by the virus vaccine; Some reversible changes were found in the study of combination vaccine and the virus vaccine; The immune reaction induced by the combination vaccine was stronger than that induced by individual vaccines; (3) DNA vaccine and the virus vaccine were found distributed only in small quantities in organs with the highest distribution at the injection sites and the level decreased disappeared with the passing of time.In conclusion, some method and a preliminary study system for the toxicological study of vaccines were established in this study: (1) Study platform and methods for immunotoxicity studies: Macropathology and histopathology study on the organs of immune system; Lymphocyte proliferation test using CFSE stainning and flow cytometry in which radioactive elements were not utilized; Lymphocyte phenotyping by flow cytometry ; Immunotoxicity studies related to humoral immunity and cell-mediated immunity; (2) Method for the monitoring of immunogenicity and cell-mediated immunity to capture the most sensitive phase of toxicity; (3) Three different method to detect the ability of lymphocyte to produceγ-IFN: ELISA method was used to detectγ-IFN level in the cell supernant which is convenient, and has lowest sensitivity in this study;Intracell staining was more sensitive than ELISA with a simple operation; ELISPOT was a most sensitive method forγ-IFN, but, with a complicated operation which every step needs to be controlled carefully. (4) Real time PCR to detect the biodistribution of HIV DNA vaccine and virus vaccine, an essential method for these kind of vaccines, which is useful for knowing the physiological disposition of the vaccines to help to predict the risk of integration. (5) The rationality of using a"n+1"regimen for dosing in animal studies was evaluated in the study of HSP65-MUC1 and virus vaccine;"n+x"was found to be well tolerated in the monkey study on HSP65-MUC1. This data can provide useful information for the clinical trials; A shortened study period and"n"(not"n+1") dosing regimen were found to induce the specific toxicity. (6) A longer recovery period should be maintained for the vaccines (at least 6-8 weeks), and for chemicals (usually 2 weeks).