Study On Reassortant Attenuated H1N1 Subtype Influenza Virus Vaccines

Influenza, a contagious, acute respiratory disease caused by an influenza virus, is a worldwide pandemic and hazardous infection which WHO declares to be strictly monitored. The three flu pandemics of the 20th century were devastating. Especially in the 1918/1919 "Spanish flu", more than 20 million people died which exceeded the total number of people killed during the World War I and most of them were young people. Since then, influenza has remained a serious cause of morbidity and mortality worldwide. It is reported that there are more than a billion influenza cases of severe illness during seasonal epidemics resulting in tens of billions economic loss in China. In the past decades, due to the seriously destroyed environment, the occurrences and developmental laws of human influenza had changed a lot and the frequency of morbidity increases which cause severe social impact and grievous economic loss.Influenza virus, a member of the family Orthomyxoviridae, is a negative sense RNA virus with eight genomic segments. Influenza viruses are divided into types A, B and C. Influenza A viruses are the principal causes of influenza in humans which cause serious complications and disorder of immune nerve-endocrine-network. Moreover, it imposes a huge burden on the human health and the national economy. Since human influenza A virus discovered, there are three kinds of subtype appeared. They are H1N1 subtype in 1918/1919, H2N2 subtype in 1957 and H3N2 subtype in 1968. Among them, H1N1 still is main subtype currently, seriously disasters and public health caused by it needs to be paid close attention. The antigens of the encoding influenza virus include HA, NA, NP and M polypeptides. As a result of seasonal antigenic variation of hemagglutinin(HA)and neuraminidase(NA)which decide the specificity of the pathogen, new virus strain appears annually, which pose a major obstacle to control influenza.To date, there is no effective therapeutic tool for influenza. Though influenza vaccines have been produced for over 50 years, vaccination is still the principal method of prophylaxis. Vaccines can be characterized as whole virus vaccines, split virus vaccines, surface antigen vaccines, DNA vaccines and live attenuated vaccines. Due to frequent antigenic shift, vaccine strain used in production needs to be changed annually. Commercially available trivalent inactivated vaccines suffer from limited efficacy in cross-protective immunity and routes of administration, given by intramuscular injection. Surface antigen vaccines can be used safely, but targeting mainly HA and NA antigen. They are not sufficiently cross-reactive to protect against antigenic variants, vaccine production is time-consuming and they are not fit for seasonal influenza. DNA vaccine developed late and aimed at protective antigen formulation. There are still some problems such as large antigen dose and gene integration with chromosomal which are difficult to solve. However, live attenuated vaccine is able to induce strong systemic immunity and humoral responses and local mucosal immune responses, but remains invariably virulence and potential rebound of toxicity. In view of the status quo of influenza vaccines, much attention has been given to the development of effective and neotype influenza vaccines. Recently, the reverse genetics provided a favourable opportunity for live attenuated vaccines. Efforts are currently under way to develop a live attenuated influenza vaccine which based on reverse genetics. Since 1999, Neumann and Hoffmann et al have developed an eight-plasmid DNA transfection system for the rescue of infectious influenza A virus entirely from cloned cDNA. The development opens the way for the study of live attenuated influenza vaccine. Rescuing recombinant influenza virus vaccine on the MDV-A background is a focus of influenza vaccine. In this dissertation, we used the eight-plasmid rescue system and selected 6 internal gene of influenza strain A/Ann Arbor/6/60(H2N2) as the genetic backbone for generation of recombinant H1N1 subtype influenza virus and 6+2 reassortants expressing the HA and NA from the 2006～2007 circulating strain A/New Caledonia/20/99(H1N1). Finally, we successfully established a cold-adapted H1N1 subtype influenza virus rescue system, this reverse genetics platform is strategically important for vaccine development and influenza prevention.The whole dissertation consists of three parts:1. Construction of a bi-directional transcription/expression vector pAD3000It is necessary to establish a rescue system for rescuing recombinant virus successfully, in which expression vector play a crucial role. To express effectively and improve the transcriptional and translational level of recombinant plasmids, the vector pHW2000 was modified to replace the bovine growth hormone (BGH) polyadenylation signals with a polyadenylation signal sequences derived from Simian virus 40(SV40) according to the cell lines used. The aim is to obtain higher efficiency of transfection and rescue as a basis for influenza vaccine. According to references, gene sequences derived from SV40 were amplified using pcDNA3.1 plasmid as a template. A desired 138bp fragment containing the SV40 polyadenylation signals was inserted into the vector pHW2000 which is modified via a series of reconstruction based on pcDNA3.0. The resulting plasmid, pAD3000, was sequenced and found to contain the SV40 polyadenylation site in the correct orientation, meanwhile, the full-length was analyzed by software. This is the base for the construction of eight-plasmid system for influenza virus.2. Construction of eight-plasmid system for influenza virusBased on functional bi-directional transcription/expression vector pAD3000, influenza virus strain A/AnnArbor/6/60 (H2N2), a cold-adapted (ca), temperature sensitive (ts), live attenuated was used as the master donor virus (MDV) for virus rescue, in which six internal gene fragments were fully synthesized. Meanwhile, five amino acid substitutions (PB1-391E,581G,661T,PB2-265S,NP-34G)have been artificially altered by human intervention and six pairs specific primers were designed according to references. The 2006～2007 circulating influenza virus strain A/New Caledonia/20/99 (H1N1) was propagated in 10-day-old embryonated chicken eggs and concentrated by density gradient centrifugation on sucrose. Total RNA was extracted from infected allantoic fluid. The HA and NA gene were amplified using the universal primer pairs according to Hoffmann. Expand High Fidelity PCR System was used and 8～10 clones were sequenced in order to confirm the authenticity of 5' and 3' ends. Finally, we spliced the 8 whole gene sequences containing the non-coding region of 3'and 5' ends by BLAST. cDNA fragments with BsmBI, BsaI and AarI of A/Ann Arbor/6/60 and A/New Caledonia/20/99 were cloned between the two BsmBl sites of the vector pAD3000. Then, all cloned were confirmed by full-length sequencing. Eight transcription /expression plasmids were obtained and named as pMDV-A-PB2, pMDV-A-PB1, pMDV-A-PA, pMDV-A-NP, pMDV-A-M, pMDV-A-NS, pMDV-A-HA, pMDV-A-NA, respectively. Finally, transcription and expression of vRNA and mRNA were realized by using one vector and pol I /pol II RNA polymerase of cells after transfection, which contribute to the construction of cold-adapted influenza virusrescue system.3. Preparation of reassortant H1N1 subtype influenza virus3.1 Validation of cold-adapted influenza virus rescue systemSome desired infectious H1N1 subtype recombinant viruses were produced by co-transfection a single gene segment from MDV-A together with the complementary seven segments from control A/PR/8/34 strain. In addition, HA and NA derived from strain A/New Caledonia/20/99 or A/PR/8/34 and other internal gene segments from A/Ann Arbor/6/60, in other words, there are eight kinds of 7+1 and a kind of 6+2 combinations. The results revealed that each of the 8 genomic segments cloned in pAD3000 was showed to be functionally expressed in a reassortant experiment and 6 internal gene backbone work synergialy. Meanwhile, it prepared for further screening and construction cold-adapted reassortant virus. The transfection reaction using the 8 A/PR/8/34 plasmids was used as a positive control. During these courses, progeny virus for eggs propagation is produced in 35mm dishes by optimizing rescue systems.Then, we identified and analyzed the partial biological properties of the recoveried viruses PB2/PR8, PR8 and PR8/rMDV-A. The rescued viruses had similar morphology to wild-type virus strain by electronic microscope. Amino acid substitutions were present in progeny virus by RT-PCR. The reassortant virus was identified by HA, HI and IFA experiment. It showed that reassortant virus propagation prefer to lower temperature such as 33 degree by PFU of PR8/rMDV-A. PR8/rMDV-A grew to high titer and weak virulence in embryonic eggs during passage of the virus and the cytoplasmic effect on infected MDCK cells were correlated with the strain A/New Caledonia/20/99, virulence and propagation in lower temperature were correlated with the master donor virus strain A/Ann Arbor/6/60. These results showed that the cold-adapted rescue system worked well.3.2 Generation and identification of H1N1 subtype attenuated influenza virus vaccineBased on the functional cold-adapted virus rescue system, we reassorted HA and NA gene of the 2006～2007 circulating influenza virus strain A/New Caledonia/20/99 (H1N1). A positive H1N1 subtype recombinant virus was obtained successfully by co-transfecting COS-1 cells. A HA titer of approximately 1:512～1:1024 were detected in the first passage of the rescued viruses. Virus titer was stable and the homology of HA and NA gene were above 99.9% following four passages. The virion morphology of the rescued viruses was verified to be identical to that of the wild-type PR8-strain. Preliminary identification was carried out and further study is in progress. The establishment of cold-adapted H1N1 subtype rescue system by RG will contribute to development of cold-adapted human influenza virus vaccine candidates and mucosal immune mechanisms against influenza.