Antibody pressure by a human monoclonal antibody targeting the 2009 pandemic H1N1 virus hemagglutinin drives the emergence of a virus with increased virulence in mice

Abstract

In 2009, a novel H1N1 influenza A virus (2009 pH1N1) emerged and caused a pandemic. A human monoclonal antibody (hMAb; EM4C04), highly specific for the 2009 pH1N1 virus hemagglutinin (HA), was isolated from a severely ill 2009 pH1N1 virus-infected patient. We postulated that under immune pressure with EM4C04, the 2009 pH1N1 virus would undergo antigenic drift and mutate at sites that would identify the antibody binding site. To do so, we infected MDCK cells in the presence of EM4C04 and generated 11 escape mutants, displaying 7 distinct amino acid substitutions in the HA. Six substitutions greatly reduced MAb binding (K123N, D131E, K133T, G134S, K157N, and G158E). Residues 131, 133, and 134 are contiguous with residues 157 and 158 in the globular domain structure and contribute to a novel pH1N1 antibody epitope. One mutation near the receptor binding site, S186P, increased the binding affinity of the HA to the receptor. 186P and 131E are present in the highly virulent 1918 virus HA and were recently identified as virulence determinants in a mouse-passaged pH1N1 virus. We found that pH1N1 escape variants expressing these substitutions enhanced replication and lethality in mice compared to wild-type 2009 pH1N1 virus. The increased virulence of these viruses was associated with an increased affinity for α2,3 sialic acid receptors. Our study demonstrates that antibody pressure by an hMAb targeting a novel epitope in the Sa region of 2009 pH1N1 HA is able to inadvertently drive the development of a more virulent virus with altered receptor binding properties. This broadens our understanding of antigenic drift.

Importance: Influenza viruses accumulate amino acid substitutions to evade the antibody response in a process known as antigenic drift, making it necessary to vaccinate against influenza annually. Mapping human monoclonal antibody (hMAb) epitopes is a necessary step towards understanding antigenic drift in humans. We defined the specificity of an hMAb that specifically targeted the 2009 pH1N1 virus and describe a novel epitope. In addition, we identified a previously unappreciated potential for antibody escape to enhance the pathogenicity of a virus. The escape mutation that we identified with in vitro immune pressure was independently reported by other investigators using in vivo selection in nonimmune mice. Although in vitro generation of escape mutants is unlikely to recapitulate antigenic drift in its entirety, the data demonstrate that pressure by a human monoclonal antibody targeting a novel epitope in the hemagglutinin of the 2009 pandemic H1N1 virus can inadvertently drive the development of escape mutants, of which a subset have increased virulence and altered receptor binding properties.

FIG 1

Binding analysis of escape mutants. (A) A binding ELISA was performed using plates coated with each of the indicated viruses in triplicate. Serial dilutions of EM4C04 were added to the wells, followed by the addition of a secondary anti-human antibody conjugated to HRP. OD values were read at 450 nm. The negative control was wells that had PBS alone added without virus. (B) Hemagglutination inhibition (HAI) assay comparing the HAI titers of CA/09 wt and mutant viruses. The HAI titer represents the inverse of the highest MAb dilution at which hemagglutination was inhibited. (C) Neutralization assay of CA/09 wt and mutant viruses. The neutralization titer represents the inverse of the highest MAb dilution at which an infectivity of 100 TCID₅₀ of the virus was neutralized. HAI and neutralization assays were performed in quadruplicate.

FIG 2

Map of EM4C04 escape mutations on the HA surface. (A and C) Diagram of the 2009 pH1N1 HA protein with both a side view (A) and a top view (C). (A) The locations of the amino acid residues identified as escape mutations that allow escape from EM4C04 neutralization are highlighted in red (residues 131, 133, 134, 157, and 158), blue (123), and purple (186). (B) A zoomed-in view of the locations of the five amino acid residues that likely comprise the EM4C04 target epitope is shown with the residues labeled. A sialic acid molecule is shown (highlighted in green) to indicate the location of the receptor binding site of the HA protein.

FIG 3

Weight loss and lethality in mice infected with the D131E and S186P CA/09 mutants. Weight loss (A) and mortality (B) were monitored for 14 days after intranasal inoculation with 10⁵ TCID₅₀ of biological and recombinant CA/09 wt and mutant viruses in groups of five mice. Mice were euthanized if they lost at least 25% of their original body weight. In panel A, the numbers beside each line represent the number of mice that survived per group of five mice. Numbers in parentheses represent results from multiple groups.