Development of a Mouse-Adapted Live Attenuated Influenza Virus That PermitsIn VivoAnalysis of Enhancements to the Safety of Live Attenuated Influenza Virus Vaccine

Abstract

The live attenuated influenza virus vaccine (LAIV) is preferentially recommended for use in persons 2 through 49 years of age but has not been approved for children under 2 or asthmatics due to safety concerns. Therefore, increasing safety is desirable. Here we describe a murine LAIV with reduced pathogenicity that retains lethality at high doses and further demonstrate that we can enhance safety in vivo through mutations within NS1. This model may permit preliminary safety analysis of improved LAIVs. Influenza A virus is a respiratory pathogen that infects through the upper airway and leads to pathology via replication in the lower airway (1). The temperature gradient between these two areas in people enabled the development of the cold-adapted, live attenuated influenza virus vaccine (LAIV [FluMist]) that replicates in the cooler upper respiratory tract to trigger a protective immune response but cannot damage the lower respiratory tract due to the elevated temperatures restricting replication (2). This temperature-sensitive (ts) attenuated (att) phenotype is imparted by five mutations within the viral replicative machinery: namely, PB2 N265S, PB1 K391E, D581G, and A661T, and NP D34G (3, 4). Although this vaccine has an overall acceptable safety profile, it is not approved for use in children under 2 years of age due to concerns about elevated hospitalizations due to wheezing (5, 6). For this reason, it is also not approved for use in asthmatics. Therefore, development of vaccines with increased safety over LAIV is desirable. Currently, no mouse model exists for the adequate assessment of the safety of the LAIV. In experimental animal studies with LAIV, elevated doses of LAIV do not elicit pathology, rendering determination of safety impossible (7-10). Here, we describe a model with which we can assess alterations in vaccine safety.The parental strain for our vaccine is a well-characterized, murine-lethal strain of influenza A virus (A/Puerto Rico/8/34 H1N1, PR8). We introduced four ts att mutations from LAIV into PR8 (NP D34G is natively present) via site-directed mutagenesis (Agilent) and rescued this virus using plasmid-based reverse genetics techniques (11). This ts att virus (referred to henceforth as "PR8 LAIV") has been previously characterized in cell culture, but its phenotype in mice was not demonstrated (8). PR8 wild-type (WT) virus has a 50% lethal dose (LD 50 ) in C57BL/6 (B6) mice of 10 to 25 PFU (12, 13). Thus, we sought to ascertain the LD 50 of PR8 LAIV (Fig. 1). Groups of mice (n ϭ 5) were intranasally

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

“…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.…”

“…1). PR8 LAIV shows impaired growth at 39°C in all cell types tested (12). In contrast, PR8 LAIV 319Q shows dramatically reduced replication at 37°C, and no virus was detected at 39°C in A549 and MDCK cells.…”

“…Five-to 7-week-old female B6 mice (Jackson Laboratory) were infected intranasally after light anesthetization with increasing doses of PR8 319Q and PR8 LAIV 319Q. Data from Cox et al on the safety of LAIV in C57BL/6 mice is replicated for comparison (12). Addition of PB1 319Q to LAIV increased safety by 10,000-fold (from 10 2 focus-forming units [FFU] to 10 6 FFU), as determined by comparing maximum safe doses in mice (i.e., the maximum dose at which no weight loss was observed), as PR8 LAIV has a maximum safe dose of 100 FFU ( Fig.…”

Rearrangement of Influenza Virus Spliced Segments for the Development of Live-Attenuated Vaccines

“…Importantly, the modified Ms or Ms/NSs influenza virus was highly attenuated in a mouse model of infection and able to confer, upon a single intranasal immunization, full protection against a lethal challenge with wild-type (WT) PR8 virus. Notably, mice immunized with the modified Ms or Ms/NSs virus were better protected against subsequent WT PR8 virus lethal challenge than mice immunized with our previously described PR8 ts LAIV (14). In addition, the ability to encode NS1 and NEP (NS segment) and M1 and M2 (M segment) proteins as separate, nonoverlapping transcripts represents an excellent option to independently study mutations in these viral proteins without affecting the viral products encoded by other segments of the viral genome.…”

“…However, LAIV is recommended only for immunocompetent 2-to 49-year-old persons (13). Moreover, the attenuated phenotype of the virus used in LAIV is conferred by just five point mutations, located in PB2 (N265S), PB1 (K391E, E581G, A661T), and NP (34G) (14)(15)(16), that make the virus temperature sensitive (ts). The concern is that reversion of any or a combination of the five mutations could result in a replication-competent and potentially pathogenic virus.…”