Haiyan Chang

Inactivated SARS-CoV Vaccine Prepared from Whole Virus Induces a High Level of Neutralizing Antibodies in BALB/c Mice

We tested the ability of inactivated SARS-CoV vaccine to induce neutralizing antibodies in BALB/c mice. The inactivated vaccine was prepared by SARS-CoV virus propagation in Vero cells, with subsequent beta-propiolactone inactivation and Sepharose 4FF column chromatography purification. One hundred forty BALB/c female mice were divided into seven groups of 20 mice each. Of the seven groups, three groups were inoculated with 0.1, 1, and 3 microg of the vaccine without adjuvant while three other groups were inoculated at the same three dosages of vaccine with aluminum hydroxide as adjuvant, respectively. The remaining group was set up as a blank control. Each mouse was inoculated twice at an interval of 3 weeks. One week after the second immunization, mice sera were collected to detect serum neutralizing antibodies. An assay for determining neutralizing antibody titers was developed. The results can be summarized as follows: (1) higher dosages of vaccine induced higher levels of neutralizing antibody titer; (2) the level of neutralizing antibodies induced by the inoculation with aluminum hydroxide adjuvant was slightly higher than that without adjuvant, but the difference was not statistically significant.

A single immunization with HA DNA vaccine by electroporation induces early protection against H5N1 avian influenza virus challenge in mice

BackgroundDeveloping vaccines for the prevention of human infection by H5N1 influenza viruses is an urgent task. DNA vaccines are a novel alternative to conventional vaccines and should contribute to the prophylaxis of emerging H5N1 virus. In this study, we assessed whether a single immunization with plasmid DNA expressing H5N1 hemagglutinin (HA) could provide early protection against lethal challenge in a mouse model.MethodsMice were immunized once with HA DNA at 3, 5, 7 days before a lethal challenge. The survival rate, virus titer in the lungs and change of body weight were assayed to evaluate the protective abilities of the vaccine. To test the humoral immune response induced by HA DNA, serum samples were collected through the eye canthus of mice on various days after immunization and examined for specific antibodies by ELISA and an HI assay. Splenocytes were isolated after the immunization to determine the antigen-specific T-cell response by the ELISPOT assay.ResultsChallenge experiments revealed that a single immunization of H5N1 virus HA DNA is effective in early protection against lethal homologous virus. Immunological analysis showed that an antigen-specific antibody and T-cell response could be elicited in mice shortly after the immunization. The protective abilities were correlated with the amount of injected DNA and the length of time after vaccination.ConclusionA single immunization of 100 μg H5 HA DNA vaccine combined with electroporation was able to provide early protection in mice against homologous virus infection.

Vaccination with hemagglutinin or neuraminidase DNA protects BALB/c mice against influenza virus infection in presence of maternal antibody

BackgroundMaternal antibody is the major form of protection against disease in early life; however, its presence interferes with active immunization of offspring. In order to overcome the immunosuppression caused by maternal antibody, several immune strategies were explored in this paper using mouse model and influenza vaccines.ResultsThe results showed that: i) when the offspring were immunized with the same vaccine as their mothers, whether inactivated or DNA vaccine, the presence of maternal antibody inhibited offspring immune response and the offspring could not be protected from a lethal influenza virus infection; ii) when the offspring, born to mothers immunized with inactivated vaccine, were immunized with NA DNA vaccine, the interference of maternal antibody were overcome and the offspring could survive a lethal virus challenge; iii) when the offspring were immunized with different DNA vaccine from that for their mothers, the interference of maternal antibody were also overcome. In addition, high-dose inactivated vaccine in maternal immunization caused partial inhibition in offspring when the offspring were immunized with HA DNA vaccine, while lower dose caused no significant immunosuppression.ConclusionTo avoid the interference of maternal antibody in influenza vaccination of offspring, mothers and their offspring shall not be immunized with the same vaccine. If mothers are immunized with inactivated vaccine, NA DNA vaccine for the offspring shall be effective; and if mothers are immunized with HA (NA) DNA, NA (HA) DNA for the offspring shall be effective.

A single dose of DNA vaccine based on conserved H5N1 subtype proteins provides protection against lethal H5N1 challenge in mice pre-exposed to H1N1 influenza virus.

BackgroundHighly pathogenic avian influenza virus subtype H5N1 infects humans with a high fatality rate and has pandemic potential. Vaccination is the preferred approach for prevention of H5N1 infection. Seasonal influenza virus infection has been reported to provide heterosubtypic immunity against influenza A virus infection to some extend. In this study, we used a mouse model pre-exposed to an H1N1 influenza virus and evaluated the protective ability provided by a single dose of DNA vaccines encoding conserved H5N1 proteins.ResultsSPF BALB/c mice were intranasally infected with A/PR8 (H1N1) virus beforehand. Six weeks later, the mice were immunized with plasmid DNA expressing H5N1 virus NP or M1, or with combination of the two plasmids. Both serum specific Ab titers and IFN-γ secretion by spleen cells in vitro were determined. Six weeks after the vaccination, the mice were challenged with a lethal dose of H5N1 influenza virus. The protective efficacy was judged by survival rate, body weight loss and residue virus titer in lungs after the challenge. The results showed that pre-exposure to H1N1 virus could offer mice partial protection against lethal H5N1 challenge and that single-dose injection with NP DNA or NP + M1 DNAs provided significantly improved protection against lethal H5N1 challenge in mice pre-exposed to H1N1 virus, as compared with those in unexposed mice.ConclusionsPre-existing immunity against seasonal influenza viruses is useful in offering protection against H5N1 infection. DNA vaccination may be a quick and effective strategy for persons innaive to influenza A virus during H5N1 pandemic.

Comparison of adjuvant efficacy of chitosan and aluminum hydroxide for intraperitoneally administered inactivated influenza H5N1 vaccine.

A safe and effective adjuvant is important to develop vaccines against highly pathogenic avian influenza virus. Chitosan, a derivative from the natural amino polysaccharide chitin, has been proved to be an effective adjuvant for inactivated influenza virus vaccine. In this study, protective immunity in mice provided by chitosan-adjuvanted inactivated H5N1 vaccine was compared with that from an aluminum hydroxide-adjuvanted one. Mice were injected intraperitoneally once or twice with various dosages of inactivated vaccine alone or in combination with an adjuvant (chitosan or aluminum hydroxide). To test the immunization effect, mice were challenged with a lethal dose of H5N1 virus. The results showed that the adjuvanted vaccines were more effective than adjuvant-free ones in inducing humoral immune responses and protecting mice against lethal challenge. Chitosan was comparable to the alum adjuvant in efficacy. These findings indicated that chitosan might be a candidate adjuvant for parenteral administration of inactivated influenza vaccines.

Characterization of H5N1 influenza A viruses isolated from domestic green-winged teal

Two avian influenza virus strains, A/domestic green-winged teal/Hunan/67/2005 (H5N1) (D-GWT/67) and A/domestic green-winged teal/Hunan/79/2005 (H5N1) (D-GWT/79), were isolated from healthy domestic green-winged teals (Anas crecca) in Hunan Province, South China. Genomic analysis showed that both isolates were reassortants. The hemagglutinin (HA) genes of the two isolates were closely related to that of an H5N1 strain isolated from tree sparrow (A/tree sparrow/Henan/1/04). The neuraminidase (NA) genes and the internal protein genes of both isolates were closely related to those from A/chicken/Shantou/4231/2003-like (H5N1) viruses, with exception of the matrix (M) gene of D-GWT/79, which was closely related to that of the H7N3 strain A/mallard/Netherlands/12/2000 isolated from wild mallard duck. The virulence of the two isolates was examined in chickens, ducks, and mice. Both strains were found to be highly pathogenic in chickens and ducks, but showed low pathogenicity in mice. These findings contribute to the realization that domestic green-winged teals carrying the H5N1 virus may play an important role in transmitting the virus among birds.

Electroporation at Low Voltages Enables DNA Vaccine to Provide Protection against a Lethal H5N1 Avian Influenza Virus Challenge in Mice

Objective: Electroporation has been proved to enhance the efficacy of intramuscular delivery of DNA. However, the process of electroporation causes pain and discomfort to the patient receiving the treatment. Higher the electroporation voltages inflict greater pain, and this limits the circumstances in which the technique can be used clinically. The voltages generally used for electroporation in animals range from 100 to 1,200 V/cm. We studied the effect of DNA vaccination when electroporation was performed at lower voltages. Methods: BALB/c mice were immunized twice by electroporation, at a 3-week interval, with H5N1 virus hemagglutinin (HA) DNA. One week after the booster had been given, the mice were challenged with a lethal dose of mouse-adapted H5N1 virus. The immune effects of HA DNA were evaluated by the survival rate, lung virus titer, bodyweight change and antibody titer of the mice. Results: The higher the voltage used, the more able were the mice to survive the challenge. However, a significant degree of protection could also be achieved with a voltage as low as 5 V. When electroporation was performed at a voltage of 10 V, an immunization amount of 5 µg is enough for HA DNA to provide effective protection. Conclusion: Low-voltage electroporation can induce immunity and protect mice effectively. There is, therefore, the potential to reduce the voltages currently used for DNA electroporation.

Genotype Diversity of H9N2 Viruses Isolated from Wild Birds and Chickens in Hunan Province, China

Three H9N2 avian influenza viruses were isolated from the Dongting Lake wetland, among which one was from fresh egret feces, the other two were from chicken cloacal swabs in poultry markets. Phylogenetic analyses suggested that eight genes of the egret-derived H9N2 virus might come from Korean-like or American-like lineages. The two poultry-derived H9N2 viruses were reassortants between the CK/BJ/94-like and G1-like viruses. Except the PB1 genes (90.6%), the nucleotide sequence of other internal genes of the two viruses exhibited high homology (>95%). In addition, they also exhibited high homology (96–98.3%) with some genes of the H7N9 virus that caused an epidemic in China in 2013. Nucleotide sequence of the poultry-derived and egret-derived H9N2 viruses shared low homology. Infection studies showed that the egret-derived H9N2 virus was non-pathogenic to both mice and chickens, and the virus was unable to infect chickens even through 8 passages continuously in the lung. On the other hand, the chickens infected by poultry-derived viruses showed obvious clinical symptoms and even died; the infected mice showed no noticeable clinical symptoms and weight loss, but viruses could be detected in their lungs. In conclusion, for the egret-derived H9N2 virus, it would take a long adaptation process to achieve cross-species transmission in poultry and mammals. H9N2 viruses isolated at different times from the same host species in the same geographical region presented different evolutionary status, and virus isolated from different hosts in the same geographical region exhibited genetic diversity. Therefore, it is important to continue the H9N2 virus surveillance for understanding their evolutionary trends so as to provide guidance for disease control and prevention.

Computational screen and experimental validation of anti-influenza effects of quercetin and chlorogenic acid from traditional Chinese medicine.

The Influenza A virus is a great threat for human health, while various subtypes of the virus made it difficult to develop drugs. With the development of state-of-art computational chemistry, computational molecular docking could serve as a virtual screen of potential leading compound. In this study, we performed molecular docking for influenza A H1N1 (A/PR/8/34) with small molecules such as quercetin and chlorogenic acid, which were derived from traditional Chinese medicine. The results showed that these small molecules have strong binding abilities with neuraminidase from H1N1 (A/PR/8/34). Further details showed that the structural features of the molecules might be helpful for further drug design and development. The experiments in vitro, in vivo have validated the anti-influenza effect of quercetin and chlorogenic acid, which indicating comparable protection effects as zanamivir. Taken together, it was proposed that chlorogenic acid and quercetin could be employed as the effective lead compounds for anti-influenza A H1N1.

Efficacy of inactivated vaccine against H5N1 influenza virus infection in mice with type 1 diabetes.

We sought to determine susceptibility to highly pathogenic avian influenza (HPAI) H5N1 virus and to explore immune protection of inactivated H5N1 vaccine in streptozotocin-induced type 1 diabetic mice. Susceptibility of diabetic mice to an H5N1 virus was evaluated by comparing the median lethal dose (LD(50)) and the lung virus titers with those of the healthy after the viral infection. To evaluate the influence of diabetes on vaccination, diabetic and healthy mice were immunized once with an inactivated H5N1 vaccine and then challenged with a lethal dose of H5N1 virus. The antibody responses, survival rates, lung virus titers and body weight changes were tested. Mice with type 1 diabetes had higher lung virus titers and lower survival rates than healthy mice after H5N1 virus infection. Inactivated H5N1 vaccine induced protective antibody in diabetic mice, but the antibody responses were postponed and weakened. In spite of this, diabetic mice could be protected against the lethal virus challenge by a single dose of immunization when the amount of the antigen increased. These results indicated that type 1 diabetic mice were more susceptible to H5N1 influenza virus infection than healthy mice, and can be effectively protected by inactivated H5N1 vaccine with increased antigen.