Search

The ectodomain of matrix protein 2 (M2e) of influenza A viruses is a universal influenza A vaccine candidate. Here, we report potential evasion strategies of influenza A viruses under in vivo passive anti-M2e IgG immune selection pressure in severe combined immune-deficient (SCID) mice. A/Puerto Rico/8/34-infected SCID mice were treated with the M2e-specific mouse IgG monoclonal antibodies (MAbs) MAb 65 (IgG2a) or MAb 37 (IgG1), which recognize amino acids 5 to 15 in M2e, or with MAb 148 (IgG1), which binds to the invariant N terminus of M2e. Treatment of challenged SCID mice with any of these MAbs significantly prolonged survival compared to isotype control IgG treatment. Furthermore, M2e-specific IgG2a protected significantly better than IgG1, and even resulted in virus clearance in some of the SCID mice. Deep sequencing analysis of viral RNA isolated at different time points after treatment revealed that the sequence variation in M2e was limited to P10H/L and/or I11T in anti-M2e MAb-treated mice. Remarkably, in half of the samples isolated from moribund MAb 37-treated mice and in all MAb 148-treated mice, virus was isolated with a wild-type M2 sequence but with nonsynonymous mutations in the polymerases and/or the hemagglutinin genes. Some of these mutations were associated with delayed M2 and other viral gene expression and with increased resistance to anti-M2e MAb treatment of SCID mice. Treatment with M2e-specific MAbs thus selects for viruses with limited variation in M2e. Importantly, influenza A viruses may also undergo an alternative escape route by acquiring mutations that result in delayed wild-type M2 expression. IMPORTANCE Broadly protective influenza vaccine candidates may have a higher barrier to immune evasion compared to conventional influenza vaccines. We used Illumina MiSeq deep sequence analysis to study the mutational patterns in A/Puerto Rico/8/34 viruses that evolve in chronically infected SCID mice that were treated with different M2e-specific MAbs. We show that under these circumstances, viruses emerged in vivo with mutations in M2e that were limited to positions 10 and 11. Moreover, we discovered an alternative route for anti-M2e antibody immune escape, in which a virus is selected with wild-type M2e but with mutations in other gene segments that result in delayed M2 and other viral protein expression. Delayed expression of the viral antigen that is targeted by a protective antibody thus represents an influenza virus immune escape mechanism that does not involve epitope alterations.

Influenza virus neuraminidase (NA) has been under intense study recently as a vaccine antigen, yet there remain unanswered questions regarding the immune response directed toward NA. Antibodies (Abs) that can inhibit NA activity have been shown to aid in the control of disease caused by influenza virus infection in humans and animal models, yet how and if interactions between the Fc portion of anti-NA Abs and Fcγ receptors (FcγR) contribute to protection has not yet been extensively studied. Herein, we show that poly- and monoclonal anti-NA IgG antibodies with NA inhibitory activity can control A(H1N1)pdm09 infection in the absence of FcγRs, but FcγR interaction aided in viral clearance from the lungs. In contrast, a mouse-human chimeric anti-NA IgG1 that was incapable of mediating NA inhibition (NI) solely relied on FcγR interaction to protect transgenic mice (with a humanized FcγR compartment) against A(H1N1)pdm09 infection. As such, this study suggests that NA-specific antibodies contribute to protection against influenza A virus infection even in the absence of NI activity and supports protection through multiple effector mechanisms. IMPORTANCE There is a pressing need for next-generation influenza vaccine strategies that are better able to manage antigenic drift and the cocirculation of multiple drift variants and that consistently improve vaccine effectiveness. Influenza virus NA is a key target antigen as a component of a next-generation vaccine in the influenza field, with evidence for a role in protective immunity in humans. However, mechanisms of protection provided by antibodies directed to NA remain largely unexplored. Herein, we show that antibody Fc interaction with Fcγ receptors (FcγRs) expressed on effector cells contributes to viral control in a murine model of influenza. Importantly, a chimeric mouse-human IgG1 with no direct antiviral activity was demonstrated to solely rely on FcγRs to protect mice from disease. Therefore, antibodies without NA enzymatic inhibitory activity may also play a role in controlling influenza viruses and should be of consideration when designing NA-based vaccines and assessing immunogenicity., (Copyright © 2019 Job et al.)