As binding to host CD4 receptors is required for infection, diverse coreceptor CCR5-dependent HIV strains maintain the CD4BS in a mostly constant structure. We identified IgA class antibodies specific for the CD4BS 416–433 epitope produced by survivors of prolonged HIV infection that neutralized heterologous viral strains with exceptional potency. A conclusive determination of IgA specificity was possible through use of an electrophilic 416–433 peptide that mimics the CD4 binding function of the native CD4BS expressed by HIV. The corresponding region of full-length gp120 is expressed in a sterically accessible form on the protein surface [17
]. IgA recognition of the CD4BS was confirmed by competitive inhibition of IgA binding to the electrophilic peptide and gp120 by CD4. Furthermore, mutation studies indicated the shared binding specificity of CD4 and the IgAs. Immunoadsorption and epitope-specific chromatography procedures indicated the 416–433 region as the major neutralizing epitope recognized by the IgA. This is corroborated by the observed association between certain epitope mutations and altered neutralizing potency. Consistent with recognition of the conserved CD4BS region, the IgAs neutralized genetically diverse HIV strains. The IgAs contain subsets of antibodies that bind gp120 non-covalently and antibodies that proceed to hydrolyze gp120 after completing the non-covalent binding step [27
]. Our binding assays using the electrophilic 416–433 peptide mimetic detect both types of antibodies. We did not measure the contributions of these activities in virus neutralization. Non-covalent CD4BS binding alone is sufficient to neutralize the virus, and gp120 hydrolysis holds the potential of enhancing the neutralizing potency [26
Adaptive synthesis of anti-CD4BS neutralizing antibodies in the natural course of infection is widely acknowledged as an immunologically disfavored process [13
]. This is consistent with the superantigenic character of the 421–433 region of the CD4BS. The epitope is recognized weakly by the framework regions (FRs) of antibodies produced without HIV infection that bind gp120 reversibly [29
] and catalyze its degradation [26
]. Superantigen binding to the B cell receptor FRs is thought to stimulate the cells non-productively, and premature apoptosis occurs [31
], consistent with infrequent production of neutralizing antibodies to the CD4BS. Serum antibodies to the 421–433 CD4BS epitope are also increased in the autoimmune disease systemic lupus erythematosus [46
], and recombinant antibodies to this epitope from lupus patients neutralize HIV [38
]. HIV infection and lupus are rarely coexistent. We had assumed that the lupus antibodies are produced due to dysfunctional autoimmune reactivity. The studies reported here indicate that HIV infection itself stimulates production of powerful neutralizing antibodies to the CD4BS despite its superantigenic character. IgA concentrations in human blood are about 2 mg/ml [47
]. Depending on the HIV-1 strain, the 50% effective concentrations of IgAs from the long-term survivors in were 75–800,000 fold lower than the physiological IgA concentrations. HIV neutralization by unfractionated plasma from the 3 patients was also evident. We studied only 3 patients with very prolonged infection, and all 3 were positive for the neutralizing IgA. Also, our studies were limited to IgA from patients who contracted HIV by transfusion of contaminated blood products as children. Future research is needed to determine the time course of infection-induced production of neutralizing IgAs, whether the neutralizing response is restricted to IgA class antibodies and whether the response can be induced by sexually transmitted infection occurring after the immune system has reached full maturity. Similarly, our findings do not necessarily imply that anti-CD4BS IgA production impedes HIV disease progression. Detailed longitudinal studies on a larger cohort of patients that take into account other host and viral factors known to influence disease progression are necessary to address these points.
HIV presents few epitopes suitable for vaccine targeting, reflecting an unusual capacity to evade traditional protective immunity mechanisms. The essential role of the 421–433 epitope in CD4BS binding and its sequence conservation qualify the epitope as a vaccine target. However, the superantigenic character of the epitope poses a challenge. The immune response to microbial infection is a stochastic process relying on certain high probability B lymphocyte differentiation events. The process entails antigen binding to the antibody complementarity determining regions (CDRs), driving adaptive selection of somatically diversified B cell receptors with the highest antigen binding affinity. Understanding how B cells of the LTS19–21
patients described here bypass restrictions on production of neutralizing anti-CD4BS antibodies may provide insight to designing a vaccine that induces similar antibodies. The potential bypass mechanisms are: (a)
The B cells may slowly produce antibodies that bind the CD4BS via
their CDRs with no utilization of the pre-existing CD4BS binding site located in the FRs; and (b)
Cellular down-regulation due to CD4BS binding to the antibody FR site may be effectively counteracted by a favorable differentiation signal generated upon simultaneous engagement of another epitope on the same gp120 molecule by the CDRs. The latter bypass mechanism is supported by our recent report of antibodies with binary epitope reactivity directed to the 421–433 CD4BS epitope and another spatially distinct gp120 epitope induced by immunization of mice with an electrophilic mimetic of full-length gp120 [48
]. Importantly, targeting of the 421–433 epitope is the first vaccine approach that induces the synthesis of antibodies with ability to neutralize genetically diverse HIV strains.
The inability to replicate the native structure of the CD4BS has held back development of a candidate vaccine that induces neutralizing antibodies to genetically diverse HIV strains. A conformational epitope recognized by monoclonal antibody b12 directed to the CD4BS was previously proposed as an HIV vaccine target [49
]. Accurate mimicry of conformational epitopes, however, is difficult, and efforts to develop a vaccine candidate mimicking the epitope recognized by antibody b12 have not been fruitful. The linear character of the 421–433 region should facilitate development of immunogens for induction of neutralizing antibody synthesis. Mimicry of the native 421–433 region of HIV by the E-416–433 probe is evident from its recognition by CD4 and neutralizing antibodies from infected subjects. Findings of potent and broad HIV neutralization by human antibodies to the 421–433 region described in the present study and the prospect of inducing accelerated synthesis of similar antibodies using experimental immunogens offer encouragement that an effective HIV vaccine directed to the CD4BS can be developed.