Rapid cloning of high-affinity human monoclonal antibodies against influenza virus

Abstract

Pre-existing neutralizing antibody provides the first line of defence against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14-21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B-cell receptor (BCR) repertoire that in some donors was dominated by only a few B-cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over 50 human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high-affinity mAbs from humans within a month after vaccination. The panel of influenza-virus-specific human mAbs allowed us to address the issue of original antigenic sin (OAS): the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared with the virus strain present in the vaccine. However, we found that most of the influenza-virus-specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal, healthy adults receiving influenza vaccination.

Figure 1. Analysis of the B cell response induced by influenza vaccination

a, PBMCs collected from 10 donors were assayed for influenza specific IgG secreting ASCs by ELISPOT assay at days 0, 7, 14, 28 and 80 days after vaccination. Each sample was measured in duplicate, averaged and plotted as ASC/10⁶ PBMCs over time post-vaccination. b, ASCs were measured in blood by flow cytometric analysis. Shown is the frequency of the ASC gate (CD3^-/CD20^-/low/CD19⁺/CD27^hi/CD38^hi) for a representative donor and a summary for all ten donors normalized to total CD20⁺/CD19⁺ B cell numbers. c, HLA-DR and Intracellular expression of Ki-67 by ASCs compared to naive or memory B cells. d, The majority of ASCs at day 7 after influenza vaccination are influenza specific. Influenza and total IgG-specific ELISPOT assays from several donors were similar. e, Percentage of influenza specific memory cells per total IgG positive memory cells after mitogen stimulation as measured by ELISPOT at days 0, 7, 14, 28 and 80 days post vaccination as previously described.

Figure 2. The ASC response after influenza vaccination is pauci-clonal and highly diversified by somatic hypermutation

a, Comparison of the mean proportion (line) of all clonal variable region sequences from day 7 ASCs of 14 donors (points), including: the bulk RNA of 10⁴ to 10⁵ ASCs from 10 donors and verification by single cell RT-PCR for 4 donors (average 37 sequences per donor). The ASCs were the most clonally-related population (t-test p ≤ 0.0003). Dotted lines indicate donors from which memory and ASCs were analyzed simultaneously. Other B cell populations were from historical data analyzed in a similar fashion from our laboratory- (see Methods, Supplemental data at Nature online) b, Each point is the average frequency of somatic mutations per sequence from each donor (n values within Methods). On average the anti-influenza ASCs had accumulated more mutations than either the IgG (t-test p =0.003) or IgM (p = <0.0001) memory and GC populations. c, Indicated is the proportion of all variable genes from each B cell population with the number of somatic mutations denoted in the legend (n-values are at the center of each pie chart).

Figure 3. High affinity mAbs generated from single influenza specific ASCs

a, Recombinant monoclonal antibodies from day 7 IgG anti-influenza ASCs (Sup. Fig. 1b) bind to a mixture of the three influenza vaccine strain virions with high affinity. In total, 71% of the ASC antibodies bound either native antigens of influenza viruses freshly grown in eggs (62% or 53/86), or to antigens within the vaccine only (9% or 8/86, not shown). Each of the five donors were influenza specific (by donor 34, 13, 11, 15, and 21 antibodies were generated of which 45% to 85% were influenza specific). Individual antibody strain specificities are in Table 1 and Sup. Fig. 2. b, 0/86 naïve B cell antibodies bound influenza. c, Analysis by immunoprecipitation and Western blot (Sup. Fig. 3) identified the specific viral antigens bound. Hemagglutination assays were used to identify those antibodies that were inhibiting (Table 1 and Methods).

Figure 4. Specificity for the newly introduced influenza B strain in the vaccine suggests a minimal impact of OAS

a, Influenza B strains used for the vaccine since 1989. Throughout the Figure, strain names are color coded for the Yamagata lineage (green) and the Victoria lineage (orange/red). b, Phylogenetic tree illustrating the similarity of recent influenza B strains and the years in which each strain was included in the vaccine. The three vaccine strains tested herein (larger font) included B/Malaysia/2506/2004 (2006/7 season) that is most similar to the 2002-2004 strain (B/Hong Kong/33/2001). Conversely, the 2005/6 vaccine strain, B/Shanghai/361/2002, is more divergent. c, All anti-B strain antibodies reacted with equal or greater affinity to the current year’s vaccines when tested by ELISA.