Hybridoma generation, antibody reactivity, animal studies
We isolated a panel of five human monoclonal antibodies named 4A10, 2O10, 4K8, 6D9, and 2K11 from a single blood sample of a human subject. All antibodies showed HAI activity against both 1918 and 2009 H1N1 pandemic viruses, the related swine influenza viruses from 1930 and 1976, and the H1N1 virus from 1977, but not against strains from the 1940s or H1N1 strains after 1977 (). Ab 6D9 tested negative for functional activity against representative H2N2, H3N2, or H5N1 viruses by HAI (data not shown). Generally, Ab4K8 was the most potent antibody, with an HAI activity <0.01 μg/mL against pandemic H1N1 viruses. We selected this Ab for testing in a lethal mouse model of 1918 influenza virus infection (, ). Ab4K8 protected 6 out of 6 animals from death at both the highest and the intermediate dose levels and 2 out of 6 animals at the lowest dose(). Ab4K8 reduced lung virus titers as compared to the human IgG control by 3.1 log10 PFU/mLat the 200 μg dose, 2.6 log10 PFU/mLat the 20 μg dose, and still 1.3 log10 PFU/mLat the 2 μg dose().
Specific HAI activity of human antibodies against influenza viruses or VLPs
FIGURE 1 Therapeutic efficacy of Ab 4K8 against disease caused by the 1918 A (H1N1) virus in mice. Mice were inoculated on day 0 and treated on day 1 with the indicated antibody and dose. In each group, six mice were monitored every other day for survival (A) (more ...)
Table II Therapeutic efficacy of Ab 4K8 against virus replication in mice inoculated with 1918 influenza A virus. Four mice were inoculated intranasally with 5× LD50 and administered 4K8 antibody or human IgG i.p. 24 h later. Mice were euthanized on day (more ...)
Generation and validation of escape mutations in epitopes recognized by these five antibodies
Ab 4K8 selected for a K166R mutation in the A/USSR/97/1977 context and a K166E mutation in the A/California/04/2009 H1N1 context. To confirm that these mutations alone were sufficient to confer escape, we produced 1918 virus-like particles (VLPs) with mutant HA. The entire panel of antibodies was unable to inhibit hemagglutination of VLPs with K166E, K166N, or K166Q mutations (). Abs 4A10 and 6D9 showed modest HAI activity against VLPs with the K166R mutation, but not 2O10, 4K8, or 2K11(). These data suggested that all five Abs recognized a common epitope, possibly with minor differences in the mode of binding between those five antibodies.
Sequence analysis of the antibody variable gene sequences in the hybridomas
Next, we determined whether shared germline genes were the basis for the recognition of this epitope. Nucleotide sequence determination and sequence analysis of variable gene sequences (, Fig. S1
) using the international ImMunoGeneTics information system (IMGT) (24
) revealed that all of the five antibodies shared usage of the VH
3-7*01 gene and the JH
6*02 gene; furthermore, 4A10, 2O10, and 2K11 used the same VL
1-40*01 gene. Also, 4K8 and 6D9 used both VK
3-20*01 and JK
2. The D genes of these five antibodies were predicted to be of different origins except for 4K8 and 6D9, which shared the D6-13*01 gene (). These data suggested that 4K8 and 6D9 might be derived from the same B cell ancestor clone. Careful review of the junctional sequences showed that indeed 4K8 and 6D9 were clonally related, while 4A10, 2O10, and 2K11 were derived independently of each other and of the 4K8/6D9 clone (). Despite four different clonal origins, the CDR H3s of these antibodies were remarkably similar (); for instance the CDR H3 length of 18 amino acids was identical across this panel. The amino acids in positions 93–95, 96, 100, 100A, 100B, 100D, 100F, and 100H-103 were fully conserved. Interestingly, somatic mutations were shared between the antibodies, for example the tyrosine to histidine mutation in position 100E of 2K11 and 4A10 or the glycineto alaninemutation in position 100G of 2O10 and 4K8 (). Also, several common mutated amino acid residues were found despite differing sequences in the inferred germlineorigin sequence: the glycine in position 96 was encoded entirely by the N1 segment (2K11, 2O10), by the D segment (4A10), or by both (4K8/6D9). The S97 was encoded by the D segment alone (4A10, 4K8/6D9) or by both N1 and D1 (2K11). An aspartic acid was found in position 100 of 2K11 and 2O10 because of their germline; clones 4A10 and 4K8/6D9 acquired this aspartic acid through a somatic mutation, suggesting that this panel converged towards a consensus sequence (). Position 100A was predicted by IMGT to be encoded by the N2 segment alone (2K11, 4K8/6D9), by the D chain and the N2 segment (2O10), or by the D and the J chain (4A10; ). No matter the origin, a threonine was found in all five CDR H3s in this position (). Residues from 100B onward were encoded by the JH
6 gene in all clones; four to six successive tyrosine residues are typically encoded by that germline gene segment(25
), although somatic mutations were found in positions 100C (2K11) and 100E (4A10, 2K11).
Genetic features of H1N1-specific human monoclonal antibodies
FIGURE 2 Comparison of antibody gene junctional sequences reveals four independent clones. The IgH gene segment junctions of the five VH3-7/JH6 antibodies 4A10, 2O10, 4K8, 6D9, and 2K11 are shown in amino acid and DNA sequence. Mutated amino acids and nucleotides (more ...)
We reviewed the variable gene encoded N-terminal part of heavy variable chains for common somatic mutations: 4A10 and 2K11 shared a threonine to serine mutation in position H28. All five antibodies shared a threonine instead of the germline serine in position H35 (). Abs 2O10 and 4K8 shared a lysineto asparaginemutation in position H52. Abs 2O10 and 6D9 mutated towards a threonine from an asparagine in position H76 (). Abs 2O10, 4K8, and 2K11 were found to have a valine instead of an alanine in position H84 (). An aspartic acid took the place of a glutamic acid in position H85 of Abs 4A10 and 4K8. Finally, both Abs 4A10 and 2K11 have a histidineto tyrosinemutation in position L34 of the λ chains in common (Fig. S1
FIGURE 3 Phylogram and sequence alignment to the VH3-7*01 germline sequence of the heavy variable chain genes of Abs 4A10 (cyan), 2O10 (orange), 4K8/6D9 (medium blue), and 2K11 (green) from hybridoma technology and pyrosequencing. Five-letter alphanumeric labels (more ...)
Deep sequencing of the VH3 gene encoded repertoire of this donor
The response of this donor towards 2009 H1N1 was dominated by antibodies encoded by the VH
6 gene segments. We hypothesized that this limited panel of five clones might represent only a small portion of the circulating antigenic site Sa-specific repertoire in this individual. To test this idea, we extracted mRNA from total PBMCs from this donor six months after hybridoma generation and isolated antibody genes using an RT-PCR amplification specific for VH
3. We used pyrosequencing to delineate the entire VH
3 repertoire. We identified sister clones of the above antibodies based on shared V/J usage and identical VDJ junctions to more fully define the genetic diversity within these clones in circulating cells. We obtained 26.2 mega bases of data including 80,687 sequences that passed filter reads. The sequences had an average length of 325 base pairs after removal of primer sequences. Analysis with IMGT identified 60,484 of those sequences as productive; 60,447 belonged to the VH
3 germline. VH
3-23 and VH
3-11 each accounted for 17% of productive VH
3 sequences (the first IMGT assignment was used in case of ambiguities), VH
3-30 for 13%, and VH
3-7 for 9% (Fig. S2
). A total of 1,917 VH
6 IgH were identified, of which 203 shared a CDR H3 length of 18 amino acids with the five neutralizing influenza antibodies we had isolated. Of these 203 sequences, 138 were non-redundant on the protein level belonging to a total of 69clones based on review of the VDJ junctions. Eight of those sequences belonged to the 4A10 lineage (five to clone 2O10, two to clone 4K8/6D9, and 23 to clone 2K11) meaning that every single clone was still found in the peripheral blood of the individual six months after the initial blood draw for hybridoma generation. Since residues DTY at positions 100-100Bwere completely conserved across all antibodies, we screened the remaining clones for the presence of this motif, but none of them shared this DTY motif in CDR H3, suggesting that we likely had identified most of the clones of the VH
6 gene segment-encoded Abs that recognized the influenza HA Sa site.
Intraclonal sequence divergence of the 2K11 clone
While we identified sequences highly related to those of Abs 4A10, 2O10, or 4K8/6D9, none of the sequences from the high throughput analysis was completely identical to those in the original clones. Remarkably, we were able to recover a sequence that was virtually identical to the 2K11 hybridoma cell IgH sequence in the high throughput sequence BQ2Y7. This occurrence may have been linked to the fact that sequences recovered that were related to Ab 2K11 represented by far the biggest clonal family identified and thus provided a more comprehensive snapshot of the evolution of this antibody. Within the panel related to Ab2K11, the AE441 and AIF9Z sequences embodied essentially a germline state with just a single non-silent somatic mutation in the variable gene encoded amino acid sequence, the valine in position 84. On the other hand, the Ab2K11 sequence itself and related sequences were highly mutated, particularly in the CDR H1, with up to five changes in amino acid sequence. While both germline states and highly mutated states were present simultaneously in the peripheral blood, sequences representing many of the intermediate predicted steps were not detected. For example, it was not clear in what order the mutations within CDR H1 of the hybridoma 2K11 occurred except that S31N probably occurred first because it was present in A32OY by itself. Also the S30I mutation probably occurred last within CDR H1 because AUOJR contained all of the other mutations except the aforementioned one. Since the other three clones had fewer representatives than the 2K11 clone, the order of mutations would be more difficult to establish for those clones. Nevertheless, sequence divergence from the germline sequence was readily apparent. Notably, insertion/deletion events seemed to play a minor role in this antibody panel with only sequence BQVKK within the 2K11 clone bearing evidence of a three base pair deletion leading to the loss of a single amino acid residue within CDR H1.
Interclonal sequence convergence
We defined convergence as the same altered amino acid introduced by somatic mutation present in two or more independent clones. Despite sequence divergence within the individual clones, sequence comparison revealed many further examples of interclonal sequence convergence not evident in the hybridoma sequences such as valine in H23 (members of clones 4A10/2K11) or threonine in the same position (2O10, 4K8/6D9, 2K11), leucine in H29 (4A10/2K11), asparagine in H31 (4A10/2K11), glutamine in H46 (4A10/2K11), asparagine in H58 (2O10/4K8/6D9), histidine in H59 (4A10, 2K11), and several others (). Overall, there were 20 positions within the VH
protein sequence with evidence of convergence. Only eight of those 20 positions were found within CDR H1, H2, or the V-GENE encoded portion of H3, so 12 convergence positions were located in the framework regions. Remarkably, a sub clone each within two different clones converged towards a set of similar somatic mutations within the CDR H1 (amino acids SLIN in positions H28-31 in the 2K11 clones and amino acids SLKN in the same positions of the 4A10 lineage). An analysis of silent versus non-silent mutations by framework and CDR is presented in Supplemental Table 1