Selection of neutralizing antibody escape mutants with type A 

influenza virus HA-specific polyclonal antisera: possible 

significance for antigenic drift

Influenza viruses are highly genetically variable and escape from immunogenic pressure by antigenic changes in their surface proteins, referred to as "antigenic drift" and "antigenic shift." To assess the potential genetic plasticity under strong selection pressure, highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 was passaged 50 times in embryonated chicken eggs in the presence of a neutralizing, polyclonal chicken serum. The resulting mutant acquired major alterations in the neuraminidase (NA)-encoding segment. Extensive deletions and rearrangements were detected, in contrast to only 12 amino acid substitutions within all other segments. Interestingly, this new neuraminidase segment resulted from complex sequence shuffling and insertion of a short fragment originating from the PA segment. Characterization of that novel variant revealed a loss of the neuraminidase protein and enzymatic activity, but its replication efficiency remained comparable to that of the wild type. Using reverse genetics, a recombinant virus consisting of the wild-type backbone and the shortened NA segment could be generated; however, generation of this recombinant virus required the polybasic hemagglutinin cleavage site. Two independent repetitions starting with egg passage 30 in the presence of alternative chicken-derived immune sera selected mutants with similar but different large deletions within the NA segment without any neuraminidase activity, indicating a general mechanism. In chicken, these virus variants were avirulent, even though the HPAIV polybasic hemagglutinin cleavage site was still present. Overall, the variants reported here are the first HPAIV H5N1 strains without a functional neuraminidase shown to grow efficiently without any helper factor. These novel HPAIV variants may facilitate future studies shedding light on the role of neuraminidase in virus replication and pathogenicity.

Section: Discussionmentioning

confidence: 99%

Truncation and Sequence Shuffling of Segment 6 Generate Replication-Competent Neuraminidase-Negative Influenza H5N1 Viruses

Kalthoff

Röhrs

Höper

et al. 2013

“…In different studies, antigenic epitopes in the hemagglutinin (HA) protein were identified by sequencing and structural mapping after generating escape variants in vitro using monoclonal antibodies (21,33,37) or polyclonal (rabbit-or mouse-derived) antiserum (3,23). However, polyclonal antisera from chickens were never used before to generate escape variants, although vaccine escape is a serious problem in influenza virus eradication programs for poultry, especially for HPAIV H5N1 (12).…”

mentioning

confidence: 99%

Highly Pathogenic Avian Influenza Virus Subtype H5N1 Escaping Neutralization: More than HA Variation

Höper

Kalthoff

Hoffmann

2012

Influenza A viruses are one of the major threats in modern health care. Novel viruses arise due to antigenic drift and antigenic shift, leading to escape from the immune system and resulting in a serious problem for disease control. In order to investigate the escape process and to enable predictions of escape, we serially passaged influenza A H5N1 virus in vitro 100 times under immune pressure. The generated escape viruses were characterized phenotypically and in detail by full-genome deep sequencing. Mutations already found in natural isolates were detected, evidencing the in vivo relevance of the in vitro-induced amino acid substitutions. Additionally, several novel alterations were triggered. Altogether, the results imply that our in vitro system is suitable to study influenza A virus evolution and that it might even be possible to predict antigenic changes of influenza A viruses circulating in vaccinated populations.

“…B cells recognize free virions through their external surface molecules, inducing escape mutations in viral surface proteins, such as hemagglutinin and neuraminidase in influenza and GP120 in human immunodeficiency virus (HIV) (8,14,45). CD8…”

mentioning

confidence: 99%

Immune-Induced Evolutionary Selection Focused on a Single Reading Frame in Overlapping Hepatitis B Virus Proteins

Maman

Blancher

Benichou

et al. 2011

Viruses employ various means to evade immune detection. Reduction of CD8؉ T cell epitopes is one of the common strategies used for this purpose. Hepatitis B virus (HBV), a member of the Hepadnaviridae family, has four open reading frames, with about 50% overlap between the genes they encode. We computed the CD8 ؉ T cell epitope density within HBV proteins and the mutations within the epitopes. Our results suggest that HBV accumulates escape mutations that reduce the number of epitopes. These mutations are not equally distributed among genes and reading frames. While the highly expressed core and X proteins are selected to have low epitope density, polymerase, which is expressed at low levels, does not undergo the same selection. In overlapping regions, mutations in one protein-coding sequence also affect the other protein-coding sequence. We show that mutations lead to the removal of epitopes in X and surface proteins even at the expense of the addition of epitopes in polymerase. The total escape mutation rate for overlapping regions is lower than that for nonoverlapping regions. The lower epitope replacement rate for overlapping regions slows the evolutionary escape rate of these regions but leads to the accumulation of mutations more robust in the transfer between hosts, such as mutations preventing proteasomal cleavage into epitopes.