Reverse genetic platform for inactivated and live-attenuated influenza vaccine

Abstract

Influenza vaccine strains have been traditionally developed by annual reassortment between vaccine donor strain and the epidemic virulent strains. The classical method requires screening and genotyping of the vaccine strain among various reassortant viruses, which are usually laborious and time-consuming. Here we developed an efficient reverse genetic system to generate the 6:2 reassortant vaccine virus from cDNAs derived from the influenza RNAs. Thus, cDNAs of the two RNAs coding for surface antigens, haemagglutinin and neuraminidase from the epidemic virus and the 6 internal genes from the donor strain were transfected into cells and the infectious viruses of 6:2 defined RNA ratio were rescued. X-31 virus (a high- growth virus in embryonated eggs) and its cold-adapted strain X-31 ca were judiciously chosen as donor strains for the generation of inactivated vaccine and live-attenuated vaccine, respectively. The growth properties of these recombinant viruses in embryonated chicken eggs and MDCK cell were indistinguishable as compared to those generated by classical reassortment process. Based on the reverse genetic system, we generated 6+2 reassortant avian influenza vaccine strains corresponding to the A/Chicken/Korea/ MS96 (H9N2) and A/Indonesia/5/2005 (H5N1). The results would serve as technical platform for the generation of both injectable inactivated vaccine and the nasal spray live attenuated vaccine for the prevention of influenza epidemics and pandemics.

Figure 1

Growth profiles of the X-31 and rgX-31 (A) and X-31ca and rgX-31ca (B) in embryonated chicken eggs. Viruses were grown in embryonated chicken eggs. Eggs were infected with 0.1 PFU/ml of the X-31, rgX-31, X-31ca and rgX-31ca viruses, respectively, and incubated at 25, 30, 33 and 37℃ for 3 days. Virus titers were determined by plaque assay on MDCK cells.

Figure 2

Growth profiles of the X-31, rX-31, X-31ca and rX-31ca viruses in MDCK cells. MDCK cells were infected with 0.01 m.o.i. of the X-31, rgX-31, X-31ca and rgX-31ca viruses and incubated at 25℃ (A), 33℃ (B), 37℃ (C) and 39℃ (D) for 3 days. Supernatants were taken every 24 h for determination of viral titers by plaque assay.

Figure 3

Genotyping of transfectant viruses by multiplex RT-PCR. PCR products of X-31ca reassortants, rgXca-H9N2 (between X-31ca and A/Chicken/Kores/MS96/96) and rgXca-H5N1 (between X-31ca and A/Indonesia/2005) were fractionated by 1% agarose gel and stained with ethidium bromide. Lane 1 and 4: specific for X-31caPB2 (1,002 bp): NP (853 bp): PB1 (447 bp); Lane2 and 5: specific for X-31ca-PA (730 bp): NS (521 bp): M (326 bp); Lane3: specific for haemagglutinin (981 bp) and neuraminidase (552 bp) of H9N2; Lane6: specific for haemagglutinin (510 bp) and neuraminidase (1,286 bp) of H5N1.