Application of Reverse Genetics for Producing Attenuated Vaccine Strains against Highly Pathogenic Avian Influenza Viruses

Abstract

In this study, reverse genetics was applied to produce vaccine candidate strains against highly pathogenic avian influenza viruses (HPAIVs) of the H5N1 subtype. The H5 subtype vaccine strains were generated by a reverse genetics method in a biosafety level 2 facility. The strain contained the HA gene from the H5N1 subtype HPAIV attenuated by genetic modification at the cleavage site, the NA gene derived from the H5N1 subtype HPAI or the H5N3 subtype of avian influenza virus and internal genes from A/Puerto Rico/8/34. Vaccination with an inactivated recombinant virus with oil-emulsion completely protected chickens from a homologous viral challenge with a 640 HAU or 3,200 HAU/vaccination dose. Vaccination with a higher dose of antigen, 3,200 HAU, was effective at increasing survival and efficiently reduced viral shedding even when challenged by a virus of a different HA clade. The feasibility of differentiation of infected from vaccinated animals (DIVA) was demonstrated against a challenge with H5N1 HPAIVs when the recombinant H5N3 subtype viruses were used as the antigens of the vaccine. Our study demonstrated that the use of reverse genetics would be an option to promptly produce an inactivated vaccine with better matching of antigenicity to a circulating strain.

Immunization with the H5N1 Recombinant Vaccine Candidate Induces High Protection in Chickens against Vietnamese Highly Pathogenic Avian Influenza Virus Strains

“…For example, Kandeil [20] immunized one-week-old chickens with 256 HAU of recombinant H5N1 virus. On the other hand, under a higher dose of immunization (640-3200 HAU), the recombinant Miyazaki H5N1 strain (clade 2.2) induced a higher HI titre (12.31-23.78, log 2 ) [22] but could not reach 100% of positive sera, as in our study (12/12, HI ≥ 4 log 2 ). Besides, the cross reactivity of the antibody against the heterologous HA (clade 2.3.2.1c, Akita strain) was observed in only one of eight vaccinated chickens with substantially lower HI titre (5.45, log 2 ).…”

“…In principal, the "6 + 2" reverse genetics allows the generation of a virus strain from a plasmid set containing all eight segments of the influenza A genome [6]. However, it has been demonstrated that some recombinant strains did not grow in eggs to quantities sufficient for immunization tests and further vaccine production; therefore, these strains could not be used as vaccine candidate strains [20,22,23]. In our study, a recombinant strain accommodating HA and NA from ST-2009 HPAI H5N1 (clade 1.1) was also produced on the PR8 background but was unable to replicate in eggs (negative in the HA assay).…”

Evaluation of the Protective Efficacy of Poly I:C as an Adjuvant for H9N2 Subtype Avian Influenza Inactivated Vaccine and Its Mechanism of Action in Ducks

“…However, H9N2 can survive for long periods as a persistent infection and be excreted at high concentrations in feces [ 4 , 5 ]. Vaccination has been proven effective in protecting chickens against AIV and is being used in several countries to control the disease [ 6 ]. However, H9N2 underwent significant antigenic drift in China and escaped from protection of the present vaccine [ 7 ].…”

Вакцины Против Гриппа Лошадей

“…Вводя мутации в гены внутренних белков вируса, а также удаляя факторы вирулентности высокопатогенных вирусов гриппа, можно получать аттенуированные штаммы (45,47). M. Quinlivan с соавт.…”

“…Jung et al, 2010; E. Hoffmann et al, 2010; Y. Uchida et al, 2014). Reverse genetics allows generation of artificial recombinant influenza viruses and provides the possibility to rapidly and easily modify the antigenic characteristics of the vaccine strain by genetic manipulation.…”