Transcutaneous Immunization With Inactivated Highly Pathogenic Human Avian Influenza Vaccine

As of June 2, 2009, highly pathogenic human avian influenza(HPAI) has spread to 15 countries and areas, and has officially been reported 433 cases of influenza A (H5N1) infection all over the world, including 262 deaths with the mortality 60%.China government has reported 38 laboratory confirmed human cases, including 25 death, and the mortality is 65.8%.Except that, as poultry and wild birds migrate from one terrain to another, at present, highly pathogenic human avian influenza virus has been found in Asia, Middle East, Europe, North America and part of Africa, whose genotype and antigenicity change continuously, and the range of this virus'mammal host has been extended. Above all, the situation continues to evolve rapidly, so World Health Organization (WHO) required all the countries prepared enough vaccines to prevent the pandemic influenza and did the research on the new effective vaccines as soon as possible.Vaccination is a potent means to prevent highly pathogenic human avian influenza. Presently, the research on highly pathogenic human avian influenza vaccines is concentrated on inactivated whole virus vaccine, split vaccine, attenuated vaccine, and so on.Among these, inactivated whole virus vaccine and split vaccine are approved, which is proved safe, efficient and steady. However, these vaccines are vaccinated by injection, and induce robost influenza virus specific neutralizing antibody, but this antibody cannot neutralize the other subtypes of influenza virus, moreover, the protection for the old and young is not satisfactory, and the way to inoculate vaccine is not so convenient and proper for large scale vaccination. Transcutaneous immunization which emerged recently may solve all these problems in the future.Transcutaneous immunization, one method of needle-free vaccination, plays an important role in preventing pandemic influenza, such as accelerating the rate of vaccination, better safety and compliance, no special doctor or nurse in great need of, and so on. Furthermore, the effectiveness of transcutaneous immunization has been proved, but its mechanism is still not verified. One theory suggested as follows: a lot of antigen presenting cells in the skin, especially Langerhans cells, ingest the antigen and progress it, then present it to T lymphocytes and B lymphocytes, which stimulates the systemic and mucosal immune response. Though the quantity of Langerhans cells accounts for 2% in skin cells, their surface area is about 25% of that of the skin. The density and function of Langerhans cells are the basis for transcutaneous immunization.In this study, choose inactivated whole virus vaccine prepared from WHO recommended H5N1 strain (A/Vietnam/H5N1-PR8/CDC-RG) as the antigen. The adjuvants are cholera toxin, the B subunit of cholera toxin, Escherichia coli heat-labile enterotoxin, MF59, CpG oligodeoxynucleotide 1826 and CpG oligodeoxynucleotide 2006, and the permeation enhancers are ethanol, propylene glycol, dimethylsulfoxide, retinoic acid and oleic acid. Immune Balb/c mouse transcutaneously, and evaluate their safety and effectiveness, then screen potent adjuvants and permeation enhancers, and observe immune responses after inoculate the vaccine. But because the heat-labile toxin is hard to acquire in China, at the end it is prepared by our laboratory.The research was carried out in three parts:In the first part, screen the effective adjuvant. The antigen was inactivated whole virus vaccine provided by Hualan Biotechnology Corporation, and the permeation enhancer was oleic acid. Firstly, mix 50μg antigen with different adjuvants (cholera toxin, the B subunit of cholera toxin, Escherichia coli heat-labile enterotoxin, MF59, CpG ODN1826 and CpG ODN 2006),adjust the concentration of antigen into 1.0μg/μl,then immune Balb/c mouse transcutaneously, and detect the titers of influenza virus specific serum IgG antibody and lung, nasal and vaginal IgG, sIgA antibody, at the same time, the titers of hemagglutination inhibition (HAI) were also detected in this study. It appeared that heat-labile enterotoxin and cholera toxin were potent trancutaneous adjuvants in mouse model with inactivated highly pathogenic human avian influenza vaccine. Secondly, prepare Escherichia coli heat-labile enterotoxin (rLT).The genes of LTA and LTB were cloned from the E. coli E24377A genome, and inserted this gene into plasmids pET-28a and pET-22b.Then the proteins were induced and purified. After that, LTA and LTB were mixed in the molar ratio 1:5 to form rLT, and purified again. The characteristics of rLT was confirmed by SDS-PAGE, WB, GM-ELISA, CHO cytotoxicity assay and MALDI-TOF-MS. Except that, we detected the adjuvanticity of rLT in transcutaneous immunization with inactivated highly pathogenic human avian influenza vaccine. It was proved that the protein we gained at last was rLT, and it was a potent adjuvant for transcutaneous immunization.The second part was screening effective permeation enhancer. The 50μg antigen and the 10μg recombinant LT as the adjuvant was the same as above, the only difference was permeation enhancers. Ethanol, propylene glycol, dimethylsulfoxide, ratinoic acid and oleic acid were used in this study. After immunization, the titers of influenza virus specific IgG and sIgA antibody in serum, lung lavage, nasal lavage and vaginal lavage of Balb/c mouse were tested. The titers of HAI were also detected. We concluded that the potent permeation enhancers were oleic acid, dimethysulfoxide and retinoic acid,which were proved safe by skin pathology. But because of DMSO's stimulating smell, it was not used widely in the experiments.The third part was to observe immune responses after inoculating this vaccine transcutaneously. Firstly, to find out the proper dose of antigen and adjuvant (rLT). Different doses of antigen(5μg, 10μg, 25μg, 50μg, 100μg)were formulated with different doses of rLT(0μg, 1μg, 5μg, 10μg, 25μg, 50μg, 100μg), and transcutaneously immune Balb/c mouse with oleic acid as permeation enhancer. The titers of influenza virus specific serum IgG and HAI were detected, and the titers of IgG, sIgA antibody in lung lavage, nasal lavage and fecal lavage were detected, too.The result showed the potent dose for antigen and adjuvant was antigen 50μg and rLT 10μg. Subsequently, compare transcutaneous immune response with that of the other vaccination methods.The results showed that influenza virus specific serum IgG titers and HAI titers in the transcutaneous group were higher than that in the PBS and HA groups, but lower than the injection group and the nasal group;mucosal immune respone in transcutaneous group was almost the same as that in the nasal group;besides,the results of ELISPOT assay showed transcutaneous immunization with inactivated highly pathogenic human avian influenza vaccine can mainly induce Th2 cell immune response.Above all, transcutaneous immunization with inactivated highly pathogenic human avian influenza vaccine can not only induce humoral immune response, but also induce mucosal immune response and cellular immune response.Conclusion: In this study, we do preliminary research on the adjuvant, permeation enhancer,antigen and transcutaneous immune response in mouse model with inactivated highly pathogenic human avian influenza vaccine.According to our research,in this model,the potent transcutaneous adjuvants were cholera toxin and heat-labile toxin, and there were two permeation enhancers(oleic acid and retinoic acid) which were proved to be effective and safe. Besides, the potent dose of antigen and adjuvant for transcutaneous immunization was antigen 50μg and rLT 10μg. Furthermore, prepare heat-labile enterotoxin successfully and the results of experiments showed it was toxic and with good adjuvanticity.Except that, this research showed transcutaneous immunization not only can induce potent humoral immune response,but also can induce mucosal immune response and cellular immune response.