Journal: The Journal of Experimental Medicine

Article Title: Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection

doi: 10.1084/jem.20101352

Figure Lengend Snippet: Summary of clinical data for patients with acute pandemic H1N1 virus infections

Article Snippet: A/Brevig Mission/1/1918 (H1N1) was purchased from Sino Biological.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection

doi: 10.1084/jem.20101352

Figure Lengend Snippet: 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 hi CD38 hi cells (dot plots are gated on CD3 − CD20 lo/− lymphocytes). The plasmablasts are defined herein as CD3 − CD20 lo/− CD19 + CD38 hi CD27 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.

Article Snippet: A/Brevig Mission/1/1918 (H1N1) was purchased from Sino Biological.

Techniques: Infection, Enzyme-linked Immunospot, Recombinant, Flow Cytometry, Isolation, FACS, Clone Assay, Binding Assay, Purification, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection

doi: 10.1084/jem.20101352

Figure Lengend Snippet: 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 ( , ; ; ).

Article Snippet: A/Brevig Mission/1/1918 (H1N1) was purchased from Sino Biological.

Techniques: Infection, Isolation, Binding Assay, Concentration Assay, Clone Assay, Sequencing, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection

doi: 10.1084/jem.20101352

Figure Lengend Snippet: 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 ) against recombinant HA (original curves are in 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 ) 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 . (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.

Article Snippet: A/Brevig Mission/1/1918 (H1N1) was purchased from Sino Biological.

Techniques: Infection, In Vitro, Functional Assay, Recombinant, Concentration Assay, Neutralization, Enzyme-linked Immunosorbent Assay, Binding Assay, Competitive ELISA, Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection

doi: 10.1084/jem.20101352

Figure Lengend Snippet: 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.

Article Snippet: A/Brevig Mission/1/1918 (H1N1) was purchased from Sino Biological.

Techniques: In Vivo, Infection, Injection, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection

doi: 10.1084/jem.20101352

Figure Lengend Snippet: 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).

Article Snippet: A/Brevig Mission/1/1918 (H1N1) was purchased from Sino Biological.

Techniques: In Vivo, Infection, Two Tailed Test