Construction And Preliminary Evaluation Of 2.3.4.4 Clade H5 A/B Chimeric Vaccine Candidates

Objective:To construct chimeric A/B influenza virus strains and chimeric A/B attenuated strains containing HA gene of H5 2.3.4.4 lineage H5N6 subtype avian influenza virus and taking B type influenza virus as the framework by using the reverse genetic operation system of B/Yamagata/16/888 plasmid.Taking two rescued strains as vaccine candidates of H5 subtype influenza virus.Two chimeric viruses were given preliminary identification and evaluation of their pathogenicity and immuno-protection,providing new thoughts for the development of novel influenza vaccines and the study of packaging mechanism of influenza B virus.Contents and methods:Rescue and identification of virus.The H5 2.3.4.4 strain HA was modified and the chimeric influenza A/B strain and attenuated influenza A/B strain were rescued by reverse genetic technology.Morphological and sequence identification of the two successful rescued strains were then carried out.Pathogenicity test of chimeric strains in BALB/c mice.Chimeric strain was inoculated with 10⁵EID₅₀/50μl,10⁴EID₅₀/50μl and chimeric attenuated strain was inoculated with 10⁴EID₅₀/50μl,10³EID₅₀/50μl,10²EID₅₀/50μl respectively by nose dropping.Mortality and weight changes of mice were observed and recorded for 14days.Lungs from mice were obtained,crushed and detected for titers using SPF chicken embryos on the 3rd and 6th day post infection.At the same time,the addiction of the virus to mouse tissues was determined.Immunogenicity tests to the rescued strains.Immunogenicity tests wre divided into single immunization group and two immunization groups,all of which were immunized by nasal dropping.In the single immunization experiment,the immune doses of chimeric virus strains were 10⁵EID₅₀/50μl and 10⁴EID₅₀/50μl respectively.And the immune doses of chimeric attenuated virus strains were 10⁴EID₅₀/50μl,10³EID₅₀/50μl and 10²EID₅₀/50μl.In the positive control group,commercialized inactivated H5N1vaccine（RE-8）of H5 2.3.4.4 pedigree was selected.The immunization mode was intramuscular injection.In the negative control group,mice were inoculated with DMEM nasal drops.On day 0 and 21 after immunization,the serum of mice was collected and the titer of hemagglutination inhibition antibody and neutralizing antibody were determined.In the two immunization experiments,mice were immunized for the second time on the 21st day after the first immunization.The dosage and mode of immunization were the same as those of single immunization.On the 0th,21st and 42nd day of immunization,the serum of mice was collected and the titer of hemagglutination inhibition antibody and neutralizing antibody in the serum of mice was determined.Immunoprotection experiments to the rescued strains.The mice in the single immunization group were challenged with 10LD₅₀(3.63log₁₀EID₅₀/50μl)dose of A/cat/Sichuan/SC18/2014（H5N6）wild strain on the 21st day after immunization.Mortality and weight changes of mice were observed and recorded for 14 days.Lungs from mice were obtained and detected for titers using SPF chicken embryos on the 3rd and 6th day after challenge.On the 42nd day after immunization,mice in the two immunization groups were challenged with 100LD₅₀(4.63log₁₀EID₅₀/50μl)dose of A/cat/Sichuan/SC18/2014（H5N6）wild strain.Mortality and weight changes of mice were observed and recorded for 14 days.Lungs from mice were obtained and detected for titers using SPF chicken embryos on the 3rd and 6th day after challenge.Results:Two chimeric influenza A/B strains were successfully rescued.The chimeric influenza A/B strain was named rA/B-H5-NS,and the attenuated chimeric influenza A/B strain was named rA/B-H5-NS110.Sequencing results showed that the sequences of the two rescued viruses were consistent with expectations,and influenza virus-like particles could be seen under electron microscopy.The viral titers of chimeric strain rA/B-H5-NS and chimeric attenuated strain rA/B-H5-NS110 on MDCK cells were 10^6.25TCID₅₀/ml and 10^4.50TCID₅₀/ml,respectively.The viral titers of rA/B-H5-NS and rA/B-H5-NS110 in chicken embryos were 10^7.64EID₅₀/ml and 10^5.25EID₅₀/ml,respectively.The results of pathogenicity test showed that the survival rate of mice was 100%.Viral replication could be detected in the lungs of mice on the 3rd day of infection,and slight replication of chimeric attenuated strains could be detected.No replication of chimeric attenuated strains was detected on the 6th day of infection.The results of immunogenicity experiments showed that the chimeric attenuated strains with different doses could induce the production of hemagglutination inhibition antibodies（HI antibodies）and neutralizing antibodies in mice of single immunization group,and both the highest hemagglutination inhibitory titer and neutralizing antibody titer were 1:640.Chimeric virus strains and RE-8 can also induce HI antibodies and neutralizing antibodies in mice.In the two immunization groups,chimeric attenuated strains at different doses could induce high levels of HI antibodies.The highest hemagglutination inhibition titer was 1:640,and the highest neutralizing antibody titer was 1:5120.Chimeric virus strain and RE-8 can induce high level of HI antibody and neutralizing antibody in mice.The immuno-protection test of mice in single immunization group showed that the survival rate of mice in different doses of chimeric attenuated strain group was 100%,and the weight of mice did not decrease.Only 10²EID₅₀/50μl mice in immunization group detected lung virus replication on the 3rd day after challenge.The survival rate of mice in the chimeric virus group was 100%.There was no significant change in body weight and no pulmonary virus replication was detected.The mortality rate of mice in negative control group was 40%,but the weight of surviving mice decreased to 15%.The clinical symptoms were obvious.At the same time,high levels of lung replication titers were detected on the 3rd and 6th day after challenge.The survival rate of mice in RE-8 group was 100%,the weight of mice increased slightly,and the titer of lung virus decreased on the 6th day after challenge.The results of immuno-protection test of mice in two immunization groups showed that the survival rate of mice in different doses of chimeric attenuated strain group was100%.The weight of mice did not change significantly.No pulmonary virus titer was detected on the 3rd and 6th day after challenge.The survival rate of mice in the chimeric virus strain group was 100%,and the weight of mice did not change that much.No pulmonary virus titer was detected on the 3rd and 6th day after challenge.The mortality rate of mice in negative control group was 100%.All mice died on the 7th day after challenge.High titers of virus replication was detected on the 3rd and 6th day after challenge.The survival rate of mice in RE-8 group was 100%,and the weight of mice did not decrease significantly.Low viral titers could be detected in the lungs of mice on the 3rd and 6th days after exposure.The above experimental results showed that the rescued chimeric attenuated strains could resist the attack of lethal influenza virus and had better immuno protection effect.At the same time,it was found that chimeric virus strains could also induce the production of HI antibodies and have immuno protection effect on mice.Conclusions:Two chimeric influenza A/B strains and chimeric attenuated influenza strains of H5 subtype 2.3.4.4 strain were successfully rescued.Chimeric attenuated strains were less pathogenic to mice as vaccine candidate strains.After immunization,mice serum could produce higher antibodies,which could completely resist the attack of lethal influenza virus and produce immune protection to mice.Chimeric virus strains could also induce mice to produce antibodies and have immuno-protective effects on mice.This study showed that A/B chimeric attenuated strain could be used as vaccine candidates,which laid a foundation for the development of a novel,safe and efficient influenza vaccine.