Measurement of neutralizing antibody responses against H5N1 clades in immunized mice and ferrets using pseudotypes expressing influenza hemagglutinin and neuraminidase.
Neutralizing antibody is associated with the prevention and clearance of influenza virus infection. Microneutralization (MN) and hemagglutination inhibition (HI) assays are currently used to evaluate neutralizing antibody responses against human and avian influenza viruses, including H5N1. The MN assay is somewhat labor intensive, while HI is a surrogate for neutralization. Moreover, use of replication competent viruses in these assays requires biosafety level 3 (BSL-3) containment. Therefore, a neutralization assay that does not require BSL-3 facilities would be advantageous. Toward this goal, we generated a panel of pseudotypes expressing influenza hemagglutinin (HA) and neuraminidase (NA) and developed a pseudotype-based neutralization (PN) assay. Here we demonstrate that HA/NA pseudotypes mimic release and entry of influenza virus and that the PN assay exhibits good specificity and reveals quantitative difference in neutralizing antibody titers against different H5N1 clades and subclades. Using immune ferret sera, we demonstrated excellent correlation between the PN, MN, and HI assays. Thus, we conclude that the PN assay is a sensitive and quantifiable method to measure neutralizing antibodies against diverse clades and subclades of H5N1 influenza virus.
Tsai
Cheguo
C
The Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
Caillet
Catherine
C
Hu
Hongxing
H
Zhou
Fan
F
Ding
Heng
H
Zhang
Guoliang
G
Zhou
Boping
B
Wang
Shixia
S
Lu
Shan
S
Buchy
Philippe
P
Deubel
Vincent
V
Vogel
Frederick R
FR
Zhou
Paul
P
D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood* / Q000276:immunology; D000914:Antibodies, Viral / Q000097:blood* / Q000276:immunology; D002460:Cell Line; D004285:Dogs; D005260:Female; D005289:Ferrets; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology; D006389:Hemagglutinins, Viral / Q000235:genetics / Q000276:immunology; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000276:immunology* / Q000502:physiology; D007252:Influenza Vaccines / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009439:Neuraminidase / Q000235:genetics / Q000276:immunology; D009500:Neutralization Tests / Q000379:methods*; D009976:Orthomyxoviridae Infections / Q000276:immunology* / Q000517:prevention & control; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology; D053586:Virus Internalization; D057074:Virus Release
D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D007252:Influenza Vaccines; D014764:Viral Proteins; C487630:NA protein, influenza A virus; D009439:Neuraminidase