Advances in molecular techniques have allowed the efficient recovery of immunoglobulin genes from human B cells in a variety of conditions including influenza vaccination (34
), influenza virus infection (34
), and acute (25
) and chronic (37
) HIV-1 infection. Sorting methods using fluorescently labeled antigen hooks have been used to isolate broadly neutralizing antibodies (43
); however, the use of fluorescently labeled Env proteins to study the vaccine-induced repertoire resulting from candidate HIV-1 vaccination has not been reported. Using Env-specific sorting, we were able to interrogate HIV-1 vaccine-induced antibody maturation (). The activities of the antibodies recovered in this study recapitulated both the neutralization activity and ADCC activity observed in the serum of vaccine recipients although we cannot exclude the possibility that this vaccine induced rare antibodies with greater breadth or activity. In-depth characterization of binding activity, structure, and mutational pathways of antibodies isolated from recipients of candidate HIV-1 vaccines, like that reported here, will permit iterative rational design of future vaccine candidates.
Polyreactivity is a trait of the normal human B cell repertoire, with ~40% of immature peripheral B cells and ~20% of memory B cells showing polyreactivity using the same methods used in this study (48
). Natural autoreactive antibodies have been associated with protection from infectious diseases (5
). Many broadly neutralizing antibodies against HIV-1 isolated to date have evidence of polyreactivity; long, hydrophobic CDR H3 regions; and/or high numbers of somatic mutations (55
). These characteristics suggest that HIV-1 Env may preferentially trigger the normal human polyreactive B cell pool. Thus, it was interesting that this vaccine elicited HIV-1-reactive antibodies that also had evidence of polyreactivity ( and ). If polyreactive B cell precursors are required as the starting points for the development of broad activity, then the ability of this vaccine to elicit polyreactive antibodies suggests that at least one hurdle for vaccine development can at least be partially cleared. The Env-reactive antibodies recovered from this study had slightly longer CDR H3 regions than the antibodies that did not bind Env ( and ); this difference may have resulted from the smaller sample size of the Env-reactive MAbs. However, we did not find exceptionally long CDR H3 regions (), a high degree of somatic mutation (), or broadly neutralizing activity (), suggesting that additional or new vaccine designs will be required to elicit antibodies with those characteristics. It was recently suggested that polyreactive antibodies would be more effective at neutralizing HIV-1 due to polyreactivity-mediated heterotypic binding to virions (37
). Whether the induction of polyreactive antibodies by vaccination, such as observed in the present study, will augment the risk of autoimmune disease is not corroborated by other observations reporting vaccine-induced polyreactive and autoreactive antibodies in 10 to 17% of those receiving hepatitis A (18
), hepatitis B (6
), and influenza vaccines (19
), while no long-term adverse effects were reported (6
). Using techniques similar to those employed in this study, we found that ~20% of isolated rMAbs from subjects immunized with trivalent inactivated influenza vaccine were polyreactive (B. F. Haynes, unpublished data). Vaccinations have been rarely reported to reactivate autoimmune disease (35
); however, most evidence supports the safety of killed or recombinant vaccines in autoimmune disease patients (1
). In cases where it is observed, autoantibody increases following vaccination have been reported to have no clinical consequences (1
Given the high mutation frequency of many broadly neutralizing antibodies (43
), a fundamental question for HIV-1 vaccine development is whether repeated immunization recruits naïve B cells in successive waves or if previously mutated memory B cells are driven to mutate further. The results of this study are consistent with both mechanisms; we found evidence of both unmutated B cells stimulated at late immunization time points (; see also Fig. S3 in the supplemental material) and recall of previously circulating B cells ( and ; see Fig. S1). Whether a vaccine-adjuvant combination can be created that can preferentially drive memory B cells to higher levels of mutation and whether such a strategy will result in the development of breadth of activity remain to be determined.
Using a combination of computational and molecular techniques, we created inferred unmutated ancestor antibodies to further characterize the effect of mutation of HIV-1 specificity. Two antibodies, 3489 and 3491, mediated pseudovirus neutralization; neither UA was able to neutralize, suggesting that neutralization was an activity acquired during somatic hypermutation. Interestingly, 3491_UA bound to a linear peptide in the gp120 C2 region while the recovered rMAb 3491 did not, suggesting that this antibody specificity became more conformationally dependent during affinity maturation. Antibody 3491 was moderately mutated (5.2% HC mutation frequency), and both 3491 and its UA reacted with HEp-2 cells ( and ). This suggested that polyreactivity and Env-epitope reactivity were able to coexist early during antigen-driven somatic hypermutation although loss and regain of these specificities cannot be excluded.
In summary, we have demonstrated that four immunizations with gp120 Env in human volunteers induced both sequential recruitment of unmutated B cells and stimulation of previously recruited B cells. The degree of somatic mutation induced by gp120 Env after four immunizations did not reach the level of mutation reported for broadly neutralizing anti-HIV-1 antibodies. Repetitive boosting with modified Env immunogens may induce higher degrees of somatic mutation in vaccine-elicited antibodies. The RV144 trial (42
) showed nondurable efficacy in the absence of broadly neutralizing antibodies, suggesting that anti-HIV-1 antibodies with narrow neutralization profiles or other activities such as ADCC may be protective in vaccine recipients. Thus, study of the affinity maturation of vaccine-induced antibodies with a variety of anti-HIV-1 activities can guide vaccine development. Future designs that can elicit antibodies that inhibit virus replication through ADCC or other mechanisms (50
) may block infection if the antibodies are present at the time of HIV-1 exposure.