Reverse Genetics Approaches for the Development of Influenza Vaccines

Abstract

Influenza viruses cause annual seasonal epidemics and occasional pandemics of human respiratory disease. Influenza virus infections represent a serious public health and economic problem, which are most effectively prevented through vaccination. However, influenza viruses undergo continual antigenic variation, which requires either the annual reformulation of seasonal influenza vaccines or the rapid generation of vaccines against potential pandemic virus strains. The segmented nature of influenza virus allows for the reassortment between two or more viruses within a co-infected cell, and this characteristic has also been harnessed in the laboratory to generate reassortant viruses for their use as either inactivated or live-attenuated influenza vaccines. With the implementation of plasmid-based reverse genetics techniques, it is now possible to engineer recombinant influenza viruses entirely from full-length complementary DNA copies of the viral genome by transfection of susceptible cells. These reverse genetics systems have provided investigators with novel and powerful approaches to answer important questions about the biology of influenza viruses, including the function of viral proteins, their interaction with cellular host factors and the mechanisms of influenza virus transmission and pathogenesis. In addition, reverse genetics techniques have allowed the generation of recombinant influenza viruses, providing a powerful technology to develop both inactivated and live-attenuated influenza vaccines. In this review, we will summarize the current knowledge of state-of-the-art, plasmid-based, influenza reverse genetics approaches and their implementation to provide rapid, convenient, safe and more effective influenza inactivated or live-attenuated vaccines.

Interplay of PA-X and NS1 Proteins in Replication and Pathogenesis of a Temperature-Sensitive 2009 Pandemic H1N1 Influenza A Virus

“…In order to generate a pH1N1 LAIV virus, we introduced four ts mutations identified in previous studies into the PB2 (N265S) and PB1 (K391E, D581G, and A661T) ORFs of pH1N1 (Fig. 1A) (7,9,10,39). No mutation was introduced into the viral NP, since pH1N1 NP already contains a G at position 34.…”

“…First, because a recombinant pH1N1 harboring inhibitory PA-X and NS1 proteins could lead to a more virulent virus (35)(36)(37)(38), we selected, for safety reasons, to use the ca, ts, and att pH1N1 LAIV virus. Second, we and others have previously shown that LAIVs provide a more sensitive approach to study mutations affecting overall viral replication and pathogenicity (5,7,(9)(10)(11)39). Finally, the results obtained from our studies could be used to identify and develop more efficient LAIV candidates with acceptable safety profiles by modulating the virus' ability to control the host immune responses (7,11).…”

Historical review on thymosin α1 in oncology: preclinical and clinical experiences

“…The current challenge is to develop strategies that allow strong antibody responses to the HA stem region, the less immunogenic region of the HA molecule. Several approaches have already been tested including the use of headless HA molecules, HA molecules with hyperglycosylated heads, or sequential immunization with chimeric HA molecules [28,29]. Both humoral and cellmediated arms of the immune system should be activated to confer broadly protective immunity.…”

In vivo rescue of recombinant Zika virus from an infectious cDNA clone and its implications in vaccine development

“…In 1981, Racaniello and Baltimore provided the basis of this approach with poliovirus 19 . Since then, numerous reverse genetics systems have been developed for positive-and negative-stranded RNA viruses 4,18,[20][21][22][23][24] . Plasmid-based reverse genetic approaches allow the rescue of infectious recombinant viruses by the transfection of one or more plasmids encoding the components necessary for the de novo generation of infectious virus particles into cultured cells.…”

“…Importantly, reverse genetic techniques are powerful platforms to modify the viral genome and they have provided critical insights into replication and pathogenesis of multiple viruses 18 . Importantly, viral reverse genetic approaches have also been used as platforms to develop novel and more effective live-attenuated vaccines (LAVs) against viral infections and have significantly contributed to the development of antiviral treatments 18,20 . However, there are intrinsic limitations in the in vitro recovery of recombinant viruses using reverse genetic approaches, mostly associated with cell culture-adaptive mutations that might restrict and/or change the phenotype of the virus in vivo 25,26 .…”