Plasmablasts induced by pandemic H1N1 infection are highly cross-reactive and have accumulated particularly high levels of variable gene somatic hypermutation. (A and B) Pandemic H1N1 reactive mAbs isolated from infected patients (1000, EM, 70, 1009) were assayed for binding to annual H1N1 influenza strain whole virus. The minimum detectable concentration is defined as two standard deviations above the mean binding of 48 randomly chosen naive B cell antibodies (Fig. S1 A). Bars are color coded to approximate levels of cross-reactivity to the annual vaccine (circulating) strains of recent years. Panels A and B use the same color scheme. Each value is representative of at least two replicate ELISAs repeated until a single consistent minimum concentration was established. The center numeral equals total antibodies. (C) Analysis of the variable gene sequences from plasmablasts of the four pandemic H1N1-infected patients indicated that ∼16.5% of the pandemic H1N1-induced plasmablasts were clonally related (shared identical VH and JH genes and CDR3 junctions). (D) The average number of somatic hypermutations in the pandemic H1N1 patient plasmablast variable region genes compared with primary IgG plasmablast responses to vaccinia (small pox) or the anthrax vaccine, or after at least 4 boosters with the anthrax vaccine. To account for the obvious outlier in the pandemic H1N1 group (patient-EM), median values are indicated by the bar. Student’s t tests excluding the outlier indicated a p-value of <0.04 for the remaining five pandemic H1N1 samples compared with the IgG memory and germinal center (GC) cells or the primary IgG plasmablast responses (0.2 with EM included) and a p-value of <0.0001 against the IgM populations. Notably, besides patient EM, each individual set of VH genes averaged significantly more mutations than the IgG memory and GC or the primary responses (Fig. S3 A). Each point represents one individual donor and is averaged from 25–75 sequences, except for the primary response to anthrax from which only 10 VH genes could be cloned from single cells because of the highly limited response. Mutations accumulated per individual sequence are depicted in Fig. S3. Detailed sequence characteristics are provided in Tables S1–S3. The naive, IgG and IgM GC and memory populations are derived from historical data (Zheng et al., 2004, Zheng et al., 2005; Koelsch et al., 2007; Wrammert et al., 2008).

The neutralizing antibodies bind to three nonoverlapping epitopes in either the stalk or the globular head of the HA molecule. (A) Competition ELISA assays were used to determine the similarity in specificity between the various neutralizing antibodies. Shown is the percentage of competition of each antibody in an ELISA binding assay against all other neutralizing antibodies. A 10-fold molar excess of unlabeled antibody was used to inhibit a biotinylated antibody. Percent competition is calculated as the reduction in absorbance relative to the level of inhibition of any particular antibody against itself. Colors indicate degree of inhibition of antibody binding, as indicated. Antibody C179 is a commercial antibody that binds to the stalk region of the HA molecule identifying epitope-1. Epitope-2 and -3 are each on the HA-head active site. 1000-2G06 and the nonneutralizing, but HA-binding, antibodies had no competition with any of the other HA-reactive antibodies and are therefore not shown. VH gene usage of the individual antibodies is listed on the right. All assays were performed in duplicate. (B) Plasmids encoding full-length WT H5-TH04 (A/Thailand/2-SP-33/2004 [H5N1]) and its mutants were transiently transfected into 293T cells. 24 h after transfection, cells were harvested for FACS analysis, and binding of indicated antibodies was tested at 10 µg/ml. The cell surface HA expression of each of the mutants were verified with a ferret anti-H5N1 serum (not depicted). Antibody F10 was one of the antibodies used to characterize the HA stalk epitope by x-ray crystallography (Sui et al., 2009) and served as a positive control for the binding pattern expected of HA stalk–reactive antibodies to these HA mutants.

Breadth of in vivo prophylactic efficacy in mice. 6–8-wk-old BALB/c mice were treated with 200 µg (10 mg/kg of body weight) EM-4C04, 70-1F02, or 1009-3B06 human mAb intraperitoneally. Control mice were treated with PBS only, a control mAb or polyclonal human IgG. 12 h later, they were challenged with a 3xLD50 dose of mouse adapted pandemic H1N1, PR/8/34, or FM/1/47 influenza virus. All mice were monitored daily for body weight changes and any signs of morbidity and mortality. Percentage of initial body weight (left) and survival curves (right) are plotted. Infected, untreated mice showed clear signs of sickness ∼4–5 d after infection and perished by day 8–9. Figure shows one representative experiments of at least three independent repeat experiments. Antibody treatment conferred significant protection as determined by comparison of weights in untreated versus prophylaxis, and at the time of treatment versus 12 d after infection (unpaired, two-tailed Student’s t test, P < 0.05). The log-rank test indicated significant survival as well (P < 0.003).

Generation of human mAbs against pandemic H1N1 influenza virus from infected patients. (A and B) Magnitude of the plasmablast response observed in peripheral blood of six pandemic H1N1-infected patients and 22 healthy (noninfected/nonvaccinated) donors by ELISPOT analysis. (A) Representative ELISPOT. Numbers of plasmablasts secreting antibody reactive to pandemic H1N1 is compared with the total number of IgG-secreting cells from each PBMC sample (numerals). All ELISPOT assays were performed in duplicate. (B) Summary of all the donors analyzed; each dot represents one patient or control. (C and D) Specificity of the sorted plasmablasts measured by ELISPOT analysis. Representative ELISPOT showing plasmablasts producing antibodies reactive with total IgG or pandemic H1N1 whole virus, annual influenza vaccine (2009/2010 TIV vaccine), or recombinant HA from pandemic H1N1, the previous year’s annual vaccine H1N1 strain (A/Brisbane/59/2007), or the previous years H3N2 strain (A/Brisbane/10/2007). (D) Summary of the frequency of whole IgG secreting cells specific to pandemic H1N1 whole virus, recombinant HA from pandemic H1N1, and recombinant HA from the previous year’s vaccine. Donors EM1 and SF1000 were not analyzed in this fashion, as the antigens were not available for live-cell analyses at that time point in the pandemic. (E) Sorting of plasmablast cells from pandemic H1N1 influenza–infected patients to generate mAbs. Flow cytometry plots show the percentage of CD27^hiCD38^hi cells (dot plots are gated on CD3⁻CD20^lo/− lymphocytes). The plasmablasts are defined herein as CD3⁻CD20^lo/−CD19⁺CD38^hiCD27^hi cells. (right) An example of post-sort purity of ungated cells (verified for each sample). Single plasmablasts were isolated from the sorted fraction by cell sorting, and variable antibody genes were cloned from individual cells (see Materials and methods). (F and G) Scatchard plots of binding of the isolated mAbs to pandemic H1N1 whole-purified virus (F) and pandemic H1N1 recombinant HA (G) as measured by ELISA. Antibodies were scored positive (frequency above plots) if they bound at least two standard deviations greater than the mean absorbance of naive B cell antibodies at 10 µg/ml (detailed in Fig. S1 A). Antibodies were tested at 10 µg/ml and threefold serial dilutions until a nonbinding concentration was determined. Each antibody was tested in at least two (and typically more) replicates for specificity and affinity estimations. Note that only 14 of 15 HA-binding antibodies have curves in G because one of the HA-reactive antibodies only binds HA on whole virions, not on the recombinant protein.

HA-specific antibodies induced by pandemic H1N1 infection bind cross-reactive neutralizing epitopes. (A) In vitro functional analysis of 15 antibodies from indicated patients that bound pandemic H1N1 influenza recombinant HA protein. The left panel shows HAI minimum effective antibody concentration, the middle panel shows PRNT50 plaque reduction neutralization minimum effective antibody concentration, and the right panel shows ELISA binding summarized as minimum positive concentration (as defined for Fig. 2) against recombinant HA (original curves are in Fig. 1 F and Fig. S2 A). The antibodies are grouped based on whether they show HAI and/or neutralizing (neut) function. Antibody 1009-3B06 was only tested for binding to whole virus, as this antibody did not bind to rHA due to binding of a quaternary or conformationally sensitive epitope that is not present in the recombinant protein. HAI and neutralization assays were performed in duplicate and repeated at least three times. ELISA curves are provided in Fig. S2 A. (B) ELISA binding as shown by minimum positive concentration (defined for Fig. 2) of neutralizing mAbs to rHA or whole virions from pandemic H1N1 or other influenza strains (ELISA binding curves are provided in Fig. S2 A). Three binding patterns (epitopes 1 and 2, and 3) were observed that coincided with specificity comparisons by competitive ELISA, as illustrated in Fig. 4 A. (C) Three representative neutralizing antibodies (EM-4C04, 70-1F02, and 1009-3B06) were used for HAI and microneutralization (MN) activity against pandemic H1N1 and several other annual or laboratory H1N1 influenza strains. Experiments were performed in duplicates and repeated at least three times. Minimum effective concentration is shown for both assays.

In vivo prophylactic and therapeutic efficacy of human mAbs against pandemic H1N1 influenza virus. 6–8-wk-old BALB/c mice were infected with a 3xLD50 dose of highly pathogenic, mouse-adapted 2009 pandemic H1N1 influenza (A/California/04/09). 24, 48, and 60 h after infection, 200 µg (10 mg/kg of body weight) of EM-4C04, 70-F02, or 1009-3B06 human mAb were injected intraperitoneally. All mice were monitored daily for body weight changes and any signs of morbidity and mortality. Percentage of initial body weight is plotted, and the number of surviving mice is shown in the lower right of each plot. Infected, untreated mice showed clear signs of sickness around day 4–5 after infection and perished by day 8–9. Prophylactic treatment is shown on the left for comparison. Antibody treatment conferred significant protection as determined by comparison of weights in untreated versus prophylaxis and at the time of treatment versus 12 d after infection (unpaired, two-tailed Student’s t test, P < 0.05). The log-rank test indicated significant survival as well (P < 0.001). Figure shows one representative experiments of at least three independent repeat experiments.