Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses

Abstract

Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome, which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG(+) memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains.

Conflict of interest statement

Conflict of interest statement: A.G.G., O.A.O., P.W.H., P.-Y.C.-H., J.M., W.C., Y.K., and M.M. hold stock options in Theraclone Sciences, Inc. M.H. and Y.K. have received consulting fees from Theraclone Sciences for performing the work described in this article.

Figures

Fig. 1.

Anti-M2e mAbs TCN-032 and TCN-031 bind virus particles and virus-infected cells but not M2e-derived synthetic peptide. (A) Purified influenza virus (A/Puerto Rico/8/34) was coated at 10 μg/mL on ELISA wells and binding of anti-M2e mAbs TCN-031, TCN-032, ch14C2, and the HCMV mAbs 2N9 was evaluated using HRP-labeled goat anti-human Fc. (B) The 23mer synthetic peptide of M2 derived from A/Fort Worth/1/50 was coated at 1 μg/mL on ELISA wells and binding of mAbs TCN-031, TCN-032, ch14C2, and 2N9 were evaluated as in A. (C) MDCK cells were infected with A/Puerto Rico/8/34 (PR8) and subsequently stained with mAbs TCN-031, TCN-032, ch14C2 and the HCMV mAb 5J12. Binding of antibodies was detected using Alexafluor 647-conjugated goat anti-Human IgG H&L antibody and quantified by flow cytometry. (D) HEK 293 cells stably transfected with the M2 ectodomain of A/Fort Worth/1/50 (D20) were stained with transient transfection supernatant containing mAbs TCN-031, TCN-032, or the control ch14C2 and analyzed by FMAT for binding to M2 in the presence or absence of 5 μg/mL M2e peptide. Mock-transfected cells are 293 cells stably transfected with vector alone. Results shown for A, B, and C are representative of three experiments, and for D, one experiment.

Fig. 2.

Therapeutic efficacy of anti-M2 mAbs TCN-031 and TCN-032 in mice. Mice (n = 10) were infected by intranasal inoculation with 5 × LD50 A/Vietnam/1203/04 (H5N1) (A and B) or (n = 5) with 5 × LD50 A/Puerto Rico 8/34 (H1N1) (C and D), followed by 3 i.p. injections with mAbs at 24, 72, and 120 h postinfection (a total of three mAb injections per mouse) and weighed daily for 14 d. Percentage-survival is shown in A and C, whereas percent-weight change of mice is shown in B and D. The results shown for the treatment study of mice infected with A/Vietnam/1203/04 (H5N1) are representative of two experiments.

Fig. 3.

Binding of anti-M2e mAbs TCN-031 and TCN-032 to M2 mutants indicates the epitope is located in the highly conserved N terminal of M2e. Mutants with alanine substituted at each position of the M2 ectodomain of A/Fort Worth/1/50 (D20) (A) or 40 wild-type M2 mutants, including A/Vietnam/1203/04 (VN) and A/Hong Kong/483/97 (HK) (B), were transiently transfected into 293 cells. The identity of each wild-type M2 mutant is listed in Table S4. Transfected cells were stained with mAbs TCN-031, TCN-032, or the control ch14C2 and analyzed by FACS for binding to M2 at 24 h posttransfection. The mAbs TCN-031 and TCN-032 do not bind variants with amino acid substitutions at positions 1, 4, or 5 of M2e. (C) The deduced epitope for TCN-031 and TCN-032 occurs in a highly conserved region of M2e and is distinct from that found for ch14C2. Results shown for A and B are representative of three experiments.

Fig. 4.

Anti-M2e mAbs TCN-031 and TCN-032 bind cells that have been infected with H1N1 A/California/4/09. MDCK cells were infected with Influenza A strain H1N1 A/Memphis/14/96, H1N1 A/California/4/09, or mock infected. Twenty-four hours postinfection cells were stained with mAbs TCN-031, TCN-032, or the control ch14C2 and analyzed by FACS for binding to M2. Results shown are for one of three experiments.