Complement-dependent lysis of influenza a virus-infected cells by broadly cross-reactive human monoclonal antibodies

Abstract

We characterized human monoclonal antibodies (MAbs) cloned from influenza virus-infected patients and from influenza vaccine recipients by complement-dependent lysis (CDL) assay. Most MAbs active in CDL were neutralizing, but not all neutralizing MAbs can mediate CDL. Two of the three stalk-specific neutralizing MAbs tested were able to mediate CDL and were more cross-reactive to temporally distant H1N1 strains than the conventional hemagglutination-inhibiting and neutralizing MAbs. One of the stalk-specific MAbs was subtype cross-reactive to H1 and H2 hemagglutinins, suggesting a role for stalk-specific antibodies in protection against influenza illness, especially by a novel viral subtype which can cause pandemics.

Fig. 1.

CDL activities against target cells infected with seasonal H1N1 and the 2009 pandemic H1N1 strains by seven MAbs at lower antibody concentrations. MAbs 1009-3B06, 1009-3F01, TIV-1, 1009-3B05, 1009-3E06, 70-1F02, and TIV-2, which had shown moderate to high levels (14 to ∼44%) of CDL lysis of A/Solomon Islands/3/2006 (H1N1)- and/or A/California/7/2009 (H1N1)-infected A549 target cells (Table 1), were tested at lower antibody concentrations against target cells infected with the same seasonal H1N1 and 2009 pandemic H1N1 strains. All assays were performed in triplicate. Averages and standard deviations for three independent experiments are shown.

Fig. 2.

Cross-reactivities of MAbs against target cells infected with temporally distant seasonal H1N1 strains. Six MAbs were tested at 4 μg/ml (MAbs 1009-3B06 and TIV-2 were tested at 5 μg/ml) against A549 cells infected with recent seasonal strains A/Brisbane/59/2007 (H1N1), A/Solomon Islands/3/2006 (H1N1), and A/New Caledonia/20/1999 (H1N1) and older strains A/USSR/90/1977 (H1N1) and A/Puerto Rico/8/1934 (H1N1) in CDL assays. Low-Tox guinea pig complement was added at a final dilution of 1:20. All assays were performed in triplicate.

Fig. 3.

Subtype cross-reactivity of MAb 70-1F02 against target cells infected with H1N1 and H2N1 strains. (A) CDL assays were used to test the stalk-specific MAbs 1009-3B05 (white), 1009-3E06 (light gray), and 70-1F02 (dark gray) against target cells infected with influenza virus strains A/Solomon Islands/3/2006 (H1N1) and A/Puerto Rico/8/1934 (H1N1) and a reassortant virus, X-27 (H2N1), which has the HA of A/Rockefeller Institute/5/1957 (H2N2) and the NA of A/NWS/1934 (H1N1). All assays were performed in triplicate. (B) Two MAbs, 1009-3B05 and 70-1F02, were compared in CDL assays against target cells infected with the A/Solomon Islands/3/2006 (H1N1) virus or the X-27 (H2N1) reassortant virus. All assays were performed in triplicate. Averages and standard deviations for three independent experiments are shown. The percent specific lysis values for MAb 70-1F02 were significantly higher against the H2N1-infected cells than those for MAb 1009-3B05 at both 4 and 32 μg/ml (P = 0.013 and P = 0.018, respectively, as determined by Student's t test, assuming unequal variances).