Epitope-Specific Human Influenza Antibody Repertoires Diversify by B Cell Intraclonal Sequence Divergence and Interclonal Convergence

Abstract

We generated from a single blood sample five independent human monoclonal antibodies that recognized the Sa antigenic site on the head of influenza HA and exhibited inhibitory activity against a broad panel of H1N1 strains. All five Abs used the VH3-7 and JH6 gene segments, but at least four independent clones were identified by junctional analysis. High throughput sequence analysis of circulating B cells revealed that each of the independent clones were members of complex phylogenetic lineages that had diversified widely using a pattern of progressive diversification through somatic mutation. Unexpectedly, B cells encoding multiple diverging lineages of these clones, including many containing very few mutations in the antibody genes, persisted in the circulation. Conversely, we noted frequent instances of amino acid sequence convergence in the antigen combining sites exhibited by members of independent clones, suggesting a strong selection for optimal binding sites. We suggest that maintenance in circulation of a wide diversity of somatic variants of dominant clones may facilitate recognition of drift variant virus epitopes that occur in rapidly mutating virus antigens, such as influenza HA. In fact, these Ab clones recognize an epitope that acquired three glycosylation sites mediating escape from previously isolated human antibodies.

Focused antibody response to influenza linked to antigenic drift

“…In addition, within the HA head-specific antibodies, a subgroup of 18 antibodies encoded by V H 3-15 were broadly reactive against former seasonal H1 viruses, whereas the others did not recognize those viruses isolated from at least 1995 to 2007. These patterns and degrees of cross-reactivity between various A viruses and human monoclonal antibodies generated against influenza HA are comparable those found by others (22,23,25,27,28,45,46).…”

“…The dominance of HA head-specific V H 3-7*01/J H 6*02 sequences that we found in the largest V H -related set has been seen in other individual responses following exposure to H1N1pdm09 (28,29). For example, the V H 3-7/J H 6 antibodies rearranged with a different group of 4 D segments (3-16*02, 3-22*01, 6-13*01, and 4-17*01) from those from our donor (3-16*01, 3-3*01 in reading frame 2 and 3, and 3-3*02) (28).…”

“…For example, the V H 3-7/J H 6 antibodies rearranged with a different group of 4 D segments (3-16*02, 3-22*01, 6-13*01, and 4-17*01) from those from our donor (3-16*01, 3-3*01 in reading frame 2 and 3, and 3-3*02) (28). However, all of these D segments are closely related and share an amino acid sequence motif, DTY, at the D-J junction.…”

Genetic measurement of memory B-cell recall using antibody repertoire sequencing

“…This approach has been used to investigate a variety of phenomena, including effects of influenza vaccination, residual disease in leukemia, effects of immune suppression, and differences between memory and naive B-cell compartments (5)(6)(7)(8)(9)(10)(11).…”

Network properties derived from deep sequencing of human B-cell receptor repertoires delineate B-cell populations

“…There are several methods for isolation, amplification, and sequencing of B-cell repertoires. Multiplex PCR amplification, using degenerate PCR primers complementary to germ-line V and J segments have been designed and validated previously (van Dongen et al 2003;Lukowsky et al 2006;Bruggemann et al 2007;Evans et al 2007;van Krieken et al 2007; Vargas et al 2008), used in numerous biological studies (Sanchez et al 2003;Campbell et al 2008;Boyd et al 2009Boyd et al , 2010Krause et al 2011;Jager et al 2012;Lev et al 2012;Maletzki et al 2012), and optimized for clinical use (McClure et al 2006;Harris et al 2012;Sproul and Goodlad 2012), although the potential for biased PCR amplification remains. The 59 rapid amplification of cDNA ends (59 RACE) has also been used (Bertioli 1997;Freeman et al 2009;Varadarajan et al 2011;Warren et al 2011), but can suffer from low efficiency and high levels of nonspecific amplification, contamination by short fragments from RNA degradation, or incomplete cDNA synthesis.…”