Construction Of Influenza A H1N1Variant Virus Vaccine Candidates

Influenza is a contagious, acute respiratory disease caused by an influenza virus. Each year, seasonal epidemics of influenza cause serious illness and death throughout the world. In the United States, the annual burden of disease is estimated to be25million to50million cases of influenza, resulting in an average of225,000hospitalizations. Over the past three decades, the estimated number of influenza-associated deaths per year in the United States has ranged from3349to48,614. The majority of deaths (>90%) occur among elderly persons, usually those with chronic underlying health conditions. The World Health Organization uses these estimates to extrapolate a likely global disease burden from influenza of up to1billion infections,3million to5million cases of severe disease, and between300,000and500,000deaths annually.1Pandemics of influenza with varying rates of illness and death have occurred throughout history; the most notable was the1918-1919pandemic, which claimed an estimated50million to100million lives worldwide.First isolated from humans in1933, influenza viruses contain8single-stranded RNA segments encoding11proteins. There are three types of influenza viruses:A, B, and C, with types A and B causing annual human epidemics. A novel virus can emerge in humans either through direct interspecies transmission or as a result of molecular exchanges between influenza viruses that already infect humans, because the influenza virus genome is segmented, co-infection of a single host cell with two or more different influenza viruses can result in a reassortment (or shuffle) of their genetic material. The antigenic shift can lead to a pandemic if the resulting progeny virus contains an HA protein to which humans have no preexisting immunity, if it has an efficient replication-competent set of internal genes, and if it can readily spread from human to human-as was the case with the2009H1N1virus.Another key feature of the influenza virus is its error-prone polymerase, which results in an accumulation of genetic mutations that are selected for in hemagglutinin (HA) and to a lesser extent neuraminidase (NA)-the major surface glycoproteins of the virus. This antigenic drift of the HA protein renews our susceptibility to influenza viruses and is the basis for frequent updating of the composition of seasonal influenza vaccines. The influenza A H1N1virus had become the seasonal influenza virus, which circulate in humans, so we perform the follow research for the potential antigenic variation of influenza A H1N1virus.In our study, a strain of influenza A H1N1virus was isolated from the throat swab specimens of human case, and molecular evolutionary analysis showed that this isolated strain was highly homologous with that of2009influenza pandemic virus and belonged to the cluster2genotype. Our lab study shows that the mutation at haemagglutinin(HA) of influenza virus A/Changchun/01/2009(H1N1) had caused antigenic drift under antibody pressure selected.In order to constructed the influenza vaccine candidate for variant virus(UI-182), we constructed the H1N1 reassortant virus (PR8-XD) by reverse genetics, using published sequences of A/PR/8/34(H1N1) to generate all genes encoding the internal proteins. The HA and NA genes were cloned from influenza A H1N1virus(A/Changchun/01/2009(H1N1)). We constructed vaccine candidate strain PR8-XD-Mu by replaced the hemagglutinin (HA) gene of influenza virus PR8-XD with the mutations amino acids at the hemagglutinin (HA) gene of influenza virus UI-182.We evaluated the immunogenicity and vaccine efficacy of the PR8-XD and PR8-XD-Mu viruses in mouse model. Groups of6week old female Kunming mice were injected intramuscularly with the vaccine PR8-XD or PR8-XD-Mu. The titers of haemagglutinin inhibition(HI) antibodies had been detected two weeks after the first immunization And the titers of haemagglutinin inhibition(HI) antibodies rising significantly after the four weeks of immunization. Four weeks post vaccination, we challenged the mice intranasal with a lethal dose(100MLD50)of the viruses UI-182. In both of the PR8-XD and PR8-XD-Mu vaccinated groups, mice were completely protected against the virus challenge. and the protection rate reached to100%.Lyophilized erythrocytes obtained by Hydroformylation and Lyophilisation. Erythrocytes keep remaining morphous after rehydration. the Lyophilized erythrocytes can instead of fresh red blood cells for the hemagglutinin (HA) and hemagglutination inhibition (HI) test, and lyophilized Erythrocytes still stabled after8month at4℃.In summary, We has isolated the influenza A H1N1virus(A/Changchun/01/2009(H1N1)), found that this isolated strain was highly homologous with that of2009influenza pandemic and belonged to the cluster2. The influenza vaccine strains PR8-XD and the variation of the candidate strains PR8-XD-Mu constructed by reverse genetics technology. The vaccines have show good immunogenicity and efficacy in mouse infected model. The recombinant virus could be used as a candidate vaccine strain for variant H1N1virus. At the same time, The Lyophilized erythrocytes can instead of fresh red blood cells for the hemagglutinin (HA) and hemagglutination inhibition (HI) test, and lyophilized Erythrocytes still stabled after8month at4℃, which has an important significance for promoting the development and application of red blood cell agglutination indicating system diagnostic techniques.