A Single Mutation at PB1 Residue 319 Dramatically Increases the Safety of PR8 Live Attenuated Influenza Vaccine in a Murine Model without Compromising Vaccine Efficacy

Abstract

The live attenuated influenza vaccine (LAIV) is preferentially recommended for use in most children yet remains unsafe for the groups most at risk. Here we have improved the safety of a mouse-adapted live attenuated influenza vaccine containing the same attenuating amino acid mutations as in human LAIV by adding an additional mutation at PB1 residue 319. This results in a vaccine with a 20-fold decrease in protective efficacy and a 10,000-fold increase in safety.T he live attenuated influenza vaccine (LAIV) is preferentially recommended for use in most children and has outperformed its inactivated rival in recent clinical trials (1). However, this vaccine is not approved for use in children under 2 years of age or asthmatics due to safety concerns in the initial licensure trials (2-11). In pursuit of a safer vaccine, we previously developed an experimental animal (mouse) model to study the safety and immunogenicity of LAIV by introducing the attenuating mutations of LAIV (PB2 N265S; PB1 K391E, D581G, and A661T) (Fig. 1A) into the genetic background of the mouse-adapted, highly virulent influenza A virus (IAV) strain, A/PR/8/34 (PR8). This resulted in a modified LAIV that retained virulence in C57BL/6 (B6) mice, such that it is possible to identify additional attenuating mutations that further reduce the virulence of this virus (12). Here we extend that work and show that adding a mutation at residue 319 of PB1 conveys a dramatic safety increase with only a modest decrease in immunogenicity.We replaced the natural (and universally conserved) leucine with glutamine at residue 319 of PB1 (Fig. 1A). This amino acid lies underneath the PA linker region and 30 Å from PB1 residue 391 (the nearest amino acid mutation of LAIV) (13). Mutating

A Live Attenuated Influenza Vaccine Elicits Enhanced Heterologous Protection When the Internal Genes of the Vaccine Are Matched to Those of the Challenge Virus

“…Previous studies have shown that the ts mutations from AA/60 attenuate PR8 significantly in mice but that they fail to attenuate Cal/09 in mice and also fail to attenuate various strains of avian influenza viruses (AIVs) in birds (30,38,51). This lack of attenuation in the genetic background of Cal/09 and AIV can be overcome by the addition of other ts or att mutations (e.g., PB1 319Q [52]) or by the insertion of an attenuating PB1 HA epitope tag (53,54). However, it is clear that the AA/60 ts mutations alone do not reproducibly confer a ts or att phenotype across diverse viral genetic backgrounds.…”

“…At the indicated times (12,24,48, and 72 h p.i. ), TCS were collected, and viral titers were determined by tissue culture infectious dose assay (TCID 50 /ml) as described previously (52). Mean values and standard deviations (SD) were calculated using Prism software.…”

A Mutated PB1 Residue 319 Synergizes with the PB2 N265S Mutation of the Live Attenuated Influenza Vaccine to Convey Temperature Sensitivity

“…This SGR virus was found to possess an unexpectedly high degree of temperature sensitivity, which was associated with a novel mutation in PB1 (PB1 319Q). We previously showed that this PB1 mutation confers enhanced temperature sensitivity and attenuation on an LAIV constructed in the genetic background of the IAV strain, A/PR/8/34 (PR8) [55]. Here, we describe the discovery/origin of this mutation and propose a mechanism for its temperature sensitive phenotype.…”

“…LD 50 was calculated by the method of Reed and Muench [58]. The data for PR8 319Q were reproduced from our previous work [55].…”

Study of the biological properties of attenuated variants of the virulent A/WSN/33 strain of influenza virus, obtained by the site-specific mutagenesis of PB2-gene

“…Прямое включение аттенуирующих мутаций в геном актуальных эпидемических штаммов можно рассматривать как новый перспективный подход к получению живых гриппозных вакцин [4,7,16,17,20]. Основным источником аттенуирующих мутаций, согласно данным литературы, рассматривается холодоадаптированный штамм А/Энн Арбор/6/60 (Н2N2) [10].…”