RESUMO
Widespread use of a live-attenuated influenza vaccine (LAIV) in the United States (licensed as FluMist) raises the possibility that vaccine viruses will contribute gene segments to the type A influenza virus gene pool. Progeny viruses possessing new genotypes might arise from genetic reassortment between circulating wild-type (wt) and vaccine strains, but it will be difficult to predict whether they will be viable or exhibit novel properties. To begin addressing these uncertainties, reverse-genetics was used to generate 34 reassortant viruses derived from wt influenza virus A/Sydney/5/97 and the corresponding live vaccine strain. The reassortants contained different combinations of vaccine and wt PB2, PB1, PA, NP, M, and NS gene segments whereas all strains encoded wt HA and NA glycoproteins. The phenotypes of the reassortant strains were compared to wt and vaccine viruses by evaluating temperature-sensitive (ts) plaque formation and replication attenuation (att) in ferrets following intranasal inoculation. The results demonstrated that the vaccine virus PB1, PB2, and NP gene segments were dominant when introduced into the wt A/Sydney/5/97 genetic background, producing recombinant viruses that expressed the ts and att phenotypes. A dominant attenuated phenotype also was evident when reassortant strains contained the vaccine M or PA gene segments, even though these polypeptides are not temperature-sensitive. Although the vaccine M and NS gene segments typically are not associated with temperature sensitivity, a number of reassortants containing these vaccine gene segments did exhibit a more restricted ts phenotype. Overall, no reassortant strains were more virulent than wt, and in fact, 33 of the 34 recombinant viruses replicated less efficiently in infected ferrets. These results suggest that genetic reassortment between wt and vaccine strains is unlikely to produce viruses having novel properties that differ substantially from either progenitor, and that the likely outcome of reassortment will be attenuated viruses.
Assuntos
Genes Virais , Vírus da Influenza A/genética , Vacinas contra Influenza/biossíntese , Vírus Reordenados/genética , Recombinação Genética , Vacinas Atenuadas/genética , Proteínas Virais/genética , Animais , Furões , Engenharia Genética , Genótipo , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Fenótipo , RNA Polimerase Dependente de RNA/metabolismo , Vírus Reordenados/fisiologia , Temperatura , Células Tumorais Cultivadas , Vacinas Atenuadas/química , Vacinas Atenuadas/imunologia , Proteínas do Core Viral/química , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Ensaio de Placa Viral , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação ViralRESUMO
The genetic and phenotypic stability of viruses isolated from young children following intranasal administration of the trivalent live-attenuated influenza virus vaccine (LAIV, marketed in the United States as FluMist) was evaluated by determination of genomic sequence and assessment of the cold-adapted (ca), temperature-sensitive (ts) and attenuated (att) phenotypes. The complete genomic sequence was determined for 56 independent isolates obtained from children following vaccination (21 type A/H1N1, 12 A/H3N2, 1 A/H3N1 and 22 type B viruses), 20% of which had no nucleotide misincorporations compared with administered vaccine. The remaining isolates had from one to seven changes per genome. None of the observed misincorporations resulted in predicted amino acid codon substitutions at sites previously shown to contribute to the ca, ts or att phenotypes, and all vaccine-derived isolates retained ca and ts phenotypes consistent with the observation that none of the vaccine recipients displayed distinctive symptoms. The results indicate that LAIV strains undergo very limited genetic change following replication in vaccine recipients and that those changes did not affect vaccine attenuation.
Assuntos
Adaptação Fisiológica , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Animais , Creches , Pré-Escolar , Temperatura Baixa , Feminino , Furões , Humanos , Imunização , Lactente , Recém-Nascido , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza B/genética , Vírus da Influenza B/crescimento & desenvolvimento , Vacinas contra Influenza/efeitos adversos , Vacinas contra Influenza/imunologia , Masculino , Nasofaringe/microbiologiaRESUMO
FluMist is a live-attenuated, trivalent influenza vaccine (LAIV) recently approved for intranasal administration. To demonstrate genetic stability during manufacture of the vaccine viruses in LAIV and a similar vaccine in development (CAIV-T), full genome consensus sequences were determined at multiple manufacturing stages for four influenza type A and five type B strains. The critical cold-adapted (ca), temperature-sensitive (ts) and attenuated (att) mutations were preserved in the virus manufacturing intermediates. Moreover, sequence identity was observed for all vaccine intermediates of the same strain. Minor sequence differences were noted in the shared gene segments of the vaccine viruses and their common progenitor master donor virus (MDV) and several of the hemagglutinin (HA) and neuraminidase (NA) genes contained nucleotide differences when compared to the wild-type parent. Nonetheless, all vaccine viruses retained the ca, ts, and att phenotypes. Thus, genetic and phenotypic stability of the vaccine viruses is maintained during the manufacture of LAIV/CAIV-T vaccines.