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Failure of immunization with the HIV-1 envelope to induce broadly neutralizing antibodies against conserved epitopes is a major barrier to producing a preventive HIV-1 vaccine. Broadly neutralizing monoclonal antibodies (BnAbs) from those subjects who do produce them after years of chronic HIV-1 infection have one or more unusual characteristics, including polyreactivity for host antigens, extensive somatic hypermutation and long, variable heavy-chain third complementarity-determining regions, factors that may limit their expression by host immunoregulatory mechanisms. The isolation of BnAbs from HIV-1–infected subjects and the use of computationally derived clonal lineages as templates provide a new path for HIV-1 vaccine immunogen design. This approach, which should be applicable to many infectious agents, holds promise for the construction of vaccines that can drive B cells along rare but desirable maturation pathways.
Traditional strategies for vaccine development have relied on killed, attenuated or subunit preparations as homologous ‘prime-boosts’, followed by tests for safety and efficacy1,2. Vaccines developed in this way are used worldwide for both bacterial and viral infectious diseases1–4. Some key viral targets have resisted these classic vaccine development schemes, among them HIV-1, influenza virus and hepatitis C virus (HCV)5–10. Each of these viruses presents the major challenge of antigenic variation, either requiring frequent redevelopment of vaccines (influenza) or inhibiting vaccine development altogether (HIV-1 and HCV). We can therefore take HIV-1 as a paradigm of those viral diseases for which inducing BnAbs is especially difficult.
Most current vaccine strategies (empirical vaccinology1–3, genomics-based ‘reverse vaccinology’11 and structure-based reverse vaccinology12,13) rely on the host to produce a protective response, provided that the appropriate antigen is in the vaccine (Table 1). For many viral vaccines currently in use, the induction of BnAbs is a primary correlate of protection3,4. New strategies have therefore focused on immunogens bearing epitopes that are bound with high affinities by antibodies produced by memory B cells. This approach assumes that the antigens recognized by memory B cells in a vaccine boost are the same as those recognized by naive B cells during the priming immunization. However, in a majority of vaccinated individuals, this and other strategies have not led to an induction of antibodies that neutralize a wide range of strains of HIV-1 or influenza (Table 1). This failure may stem in part from characteristics of the chosen immunogens (for example, glycan masking of HIV-1 envelope protein epitopes9) and limited accessibility of conserved viral epitopes5 (for example, the ‘stem’ and sialic acid–binding epitopes on influenza hemagglutinin (HA)). Work by two of us (B.F.H. and G.K.) and collaborators14 indicates that mimicry of host antigens by some of these conserved epitopes may be another complication of such vaccines, leading to the suppression of potentially useful antibody responses (B.F.H., G.K. et al.14), and lack of a heavy-chain variable region (VH) allelic variant may also limit the breadth or effectiveness of the antibodies induced by the vaccine15 (Box 1).
Several factors can act to prevent the production of BnAbs against pathogens; these are listed below. The B-cell–lineage design approach is likely to help overcome the problems posed by tolerance control and the diversion of B-cell responses, as well as the requirements for extensive somatic hypermutations and specific germline allelic variants.
Making vaccines for infectious agents with transient, cryptic or host-mimicking epitopes requires a detailed understanding of antibody affinity maturation. If we understood the patterns of clonal maturation and selection that lead to the development of rare, broadly protective antibodies16–25, we might be able to design immunogens that increase the likelihood of maturation along these desired but disfavored pathways. Recent data from mouse studies show that the survival and persistence of B cells in the germinal center reaction depends on a high affinity of the B-cell receptors for the antigen26–29 (see Box 2 for a glossary of the terms used). Moreover, for some responses to viral antigens, the antigen that stimulates the memory B cells during affinity maturation and the antigen that initially activates the naive B cells may not be identical15,19–22,30. Thus, to optimize the induction of such a protective antibody response, it may be necessary to use one antigen as the vaccine prime (to trigger naive B cells) and others as boosts to drive the clonal evolution and affinity maturation15,19–22,30,31.
Autologous neutralizing antibodies: antibodies that are produced early after the transmission of disease that selectively neutralize the transmitted/founder virus.
B-cell anergy: a type of B cell tolerance that renders antigen-binding B cells unresponsive to their antigen ligands.
B-cell tolerance: a physiologic process that purges or inactivates B cells that are substantially reactive to self-antigens. Most autoreactive B cells are either removed by apoptosis or receptor editing or are rendered unresponsive (that is, anergic).
Broadly neutralizing antibodies (BnAbs): antibodies that neutralize diverse strains of a particular infectious agent.
Committed B cell: a lymphocyte progenitor that has undergone irreversible differentiation to enter the B cell lineage.
Germinal center: antigen-driven histologic structures in immune tissues comprising populations of B and T lymphocytes and follicular dendritic cells. Germinal center B cells present antigens to follicular helper T cells to receive activation signals necessary for immunoglobulin class-switching and somatic hypermutations as well as the generation of memory B cell compartments.
Heavy-chain second and third complementary-determining regions (HCDR1, HCDR2 and HCDR3): three loops from each of the two immunoglobulin polypeptide chains contribute to its antigen-binding surface. The third of these complementarity-determining regions on the heavy chain is particularly variable (because the VH, D and JH segments can all contribute) and often makes a particularly crucial contribution to antigen recognition.
Immunoglobulin class switching: the process by which an antigen drives the switching of an immunoglobulin made by a developing memory B cell from IgM to IgG, IgA or IgE. This process requires the expression of the enzyme AID but is independent of somatic hypermutation. Not all memory B cells undergo class switching, however, and some memory B cells retain surface IgM.
Intermediate ancestor antibodies: antibodies made by intermediates in the clonal lineage that are generated during the affinity maturation of a naive B cell in a germinal center.
Polyreactivity: the property of an antibody binding to multiple and distinct antigens with substantial affinity; a common characteristic of virus-specific antibodies that also bind either host self-antigens or other nonviral antigens.
Somatic hypermutation: a process in germinal centers mediated by the enzyme AID that leads to affinity maturation of the antibody-antigen binding. In HIV-1, broadly neutralizing antibodies typically have more somatic mutations (~15%) than non-neutralizing antibodies isolated from subjects with HIV-1 infection (~7%) (A. Trama, H.-X. Liao and B.F.H., unpublished data).
Simian human immunodeficiency virus (SHIV): a chimeric virus of simian immunodeficiency virus and HIV-1 used in challenge experiments of rhesus macaques vaccinated with HIV-1 envelope.
Unmutated ancestor antibodies: antibodies that represent the BCRs of naive B cells that give rise to clonal lineages of mutated B cells. Unmutated ancestor antibodies can be isolated from transitional or mature naive B cell populations or inferred from analyses of mutated memory B-cell clonal lineages.
VH restriction: recurrent usage of the same VH gene segment in antibody responses from many individuals to the same epitope.
We discuss here a proposed approach to vaccine design based on insights from basic B-cell biology, structural biology and new methods for inferring unmutated ancestor antibodies as estimates of naive B-cell receptors and their clonal progeny. The majority of this discussion will center on HIV-1, for which the preliminary data are available regarding this approach.
Newly generated human B cells are frequently (70–75%) autoreactive and are subject to elimination or inactivation by several physiologic processes32–34. However, not all self-reactive B cells are purged during these processes, and some (20–25%) of the mature, naive B cells circulating in the blood express autoreactive antigen B-cell receptors (BCRs)33–35 (Box 3 and Fig. 1).
Human B cells develop from hematopoietic progenitors that express the V(D)J recombinase, [RAG2], recombination activating gene 1 (RAG1) and RAG2 and rearrange the immunoglobulin heavy locus (IGH) gene loci120–123. In pre-B I cells, functional IgM heavy chain (µH) polypeptides formed by these rearrangements associate with surrogate light chains124–126 and Ig-α–Ig-β heterodimers to form pre-BCRs127 that are necessary for cell survival and proliferation120,128,129. These cells exit the cell cycle as pre-B II cells121, initiate rearrangements in the κ or λ light-chain loci35,130 and assemble a mature BCR131,132 that binds antigen120,133 (Fig. 1). The generation of BCRs by genomic rearrangement of V, D and J gene segments and the combinatorial association of IGH, with κ or λ light chains ensures a diverse primary repertoire of BCRs but frequently produces self-reactive B cells32–34.
Most immature B cells in the bone marrow are autoreactive and are normally eliminated or inactivated by immunological tolerance33,34. The remaining B cells mature through the transitional 1 (T1) and T2 stages, which are characterized by changes in membrane IgM and IgD expression and the loss or diminution of markers associated with developmental immaturity134. In the periphery, newly formed (T2) B cells are subject to a second round of immune tolerization before entering the mature B-cell pools33,34.
At least three mechanisms of immunological tolerance deplete the immature and maturing B-cell pools of self-reactivity: apoptosis40,41, cellular inactivation by anergy135,136 and replacement of autoreactive BCRs by secondary V(D)J rearrangement32,137–139. The majority of lymphocytes committed to the B-cell lineage do not reach maturity because they express dysfunctional µH polypeptides and cannot form a pre-BCR140,141 or because they carry self-reactive BCRs33.
Autoreactive BCR numbers decline with increasing B-cell maturity40,137, even in cells drawn from peripheral sites (Fig. 1)142–145. Tolerance mechanisms, especially apoptotic deletion144–146, operate during the transitional stages of B-cell development, and the number of self-reactive cells decreases substantially after entry into the mature pools33.
Despite having multiple tolerance pathways and checkpoints, not all autoreactive B cells are lost during development34,39. In mice, mature follicular B cells are substantially purged of autoreactivity, but the marginal zone and B1 B-cell compartments are enriched for self-reactive cells147. In humans, 20–25% of mature, naive B cells circulating in the blood continue to express autoreactive BCRs33–35.
In germinal centers, antigen-reactive mature B cells are driven to proliferate and express high amounts of AID, an enzyme required for immunoglobulin class-switch recombination and V(D)J hypermutation148. Autoreactivity in the germinal center B cell compartment increases as a result of the incorporation of mutations that alter antibody specificity33,34,43–45 (Fig. 1), although this autoreactivity is decreased in the human bone marrow plasma-cell pool35.
In germinal centers, clonally related B cells rapidly divide, and their clonal evolution is a Darwinian process comprising two mechanisms: hypermutation and affinity-dependent selection29,46,149. Selection clearly is not random, but hypermutation is also nonrandom and is influenced substantially by local DNA sequence150 because of the template specificity of AID148. Furthermore, codon biases conserved in VH and variable light (VL) gene segments increase the likelihood of mutations in the regions that specify antigen-binding domains151. Therefore, even before selection, some evolutionary trajectories for germinal center B cells are favored over others. The continued survival and proliferation of germinal center B cells is strongly correlated with BCR affinity and seems to be determined by the capacity of each B cell to collect and present antigen29,46 to local chemokine (C-X-C motif) receptor 5 (CXCR5)+CD4+ TFH cells152.
Unlike AID-driven hypermutation, in which molecular biases remain constant, clonal selection in the germinal center depends on BCR fitness (affinity and specificity) and changes over the course of clonal maturation. Individual germinal centers are therefore microcosms of Darwinian selection, and each germinal center is essentially an independent ‘experiment’ in clonal evolution with regard to the founding B- and T-cell populations and the order and distribution of the introduced mutations.
The concept of selection imposed by tolerance implies that the full potential of the primary, or germline, BCR repertoire is unavailable to vaccine immunogens: only those naive mature B cells that have been vetted by tolerance are available to respond. For microbial pathogens and vaccine antigens that mimic self-antigen determinants, the pool of mature B cells that can respond may therefore be small or absent.
This censoring of the primary BCR repertoire by tolerance sets up a roadblock in the development of effective HIV-1 vaccines, as the success of naive B cells in humoral responses is largely determined by BCR affinity26–29. If immunological tolerance reduces the BCR affinity and the number of naive B cells that can recognize HIV-1–neutralizing epitopes, the humoral responses to those determinants will be suppressed. Indeed, HIV-1 infection and experimental HIV-1 vaccines are extremely inefficient in selecting B cells that go on to secrete high-affinity, broadly neutralizing HIV-1 antibodies36–39.
The predicted effects of immune tolerance on the production of HIV-1 BnAbs have been illustrated in 2F5 immunoglobulin, variable (V), diversity (D), joining (J) knock-in (2F5 VDJ-KI) mice that contain the human VDJ gene rearrangement of the 2F5 BnAb38,39. In 2F5 VDJ-KI mice, early B-cell development is normal, but the generation of immature B cells is severely impaired in a manner that is diagnostic of apoptotic tolerization of autoreactive B cells40,41. Subsequent studies have shown that the 2F5 monoclonal antibody (mAb) avidly binds both mouse and human kynureninase, an enzyme of tryptophan metabolism, at an α-helical motif that matches exactly the 2F5 HIV-1 envelope protein (Env) glycoprotein subunit 41 (gp41) membrane-proximal external region (MPER) epitope ELDKWA42 (G. Yang, B.F.H. and G.K., unpublished data).
Although immunologic tolerance eliminates most autoreactivity33,34, antigen-driven, somatic hypermutation in mature, germinal center B cells can generate de novo self-reactivity, and these B-cell mutants can become memory B cells43–45. Hypermutation of immunoglobulin genes is driven by activation-induced cytidine deaminase (AID). Natural selection of mutant germinal center B cells not only drives affinity maturation for exogenous immunogens26,46–48 but also creates newly autoreactive B cells that are only weakly regulated by T cells29,49–52.
Accumulation of mutations in the germinal center eventually compromises antigen binding and cell survival29,46,53 (Box 3). Indeed, the frequency of V(D)J mutations approaches a ceiling above which further mutation can only lower BCR affinity and decrease cell fitness52–54. The mean frequency of human immunoglobulin mutations in secondary immune responses is approximately 6%30,55,56, and the substantially higher frequencies (10–15%) of V(D)J mutations present in genes encoding HIV-1 BnAbs14,36 suggest atypical pathways of clonal evolution and/or selection. In contrast to clonal debilitation by a high mutational burden52–54, HIV-1 BnAbs seem to require extraordinarily high frequencies of V(D)J misincorporation14,36. A plausible explanation for this unusual characteristic is the serial induction and selection of V(D)J hypermutation by distinct antigens. This explanation also suggests pathways for generating BnAb responses that are normally proscribed by the effects of tolerance.
The low efficiency with which infection and immunization elicit BnAbs and the unusually high frequency of immunoglobulin mutations present in most BnAb gene rearrangements imply that BnAb B cells are the products of disfavored and tortuous pathways of clonal evolution as a result of their long, variable heavy-chain third complementarity-determining regions (HCDR3s) or polyreactivity and their need for extensive somatic hypermutation. Because BCR affinity is the crucial determinant of the fitness of germinal center B cells, it should be possible to select immunogens that direct germinal center B-cell evolution along normally disfavored pathways and promote the maturation of typically subdominant or disfavored B-cell clonal lineages. Any method for directed somatic evolution must take into account the complex and inter-related processes of immunoglobulin hypermutation, affinity-driven selection and cognate interaction with T follicular helper (TFH) cells. These hurdles are real but not insurmountable. Indeed, the BnAb responses elicited by HIV-1 infection may be an example of fortuitous sequential immunizations that favor BnAb development from nonreactive, naive B cells57,58.
The initial antibody response to HIV-1 after transmission is to non-neutralizing epitopes on gp41 (refs. 30,59). The first antibody response that can neutralize the transmitted or founder virus in vitro appears only ~12–16 weeks after transmission. This antibody is to gp120 and is of extremely limited breadth60,61.
Antibodies to the HIV-1 envelope that neutralize a broad range of HIV-1 isolates have not yet been induced in high titers by vaccination and are present only in a minority of subjects with chronic HIV-1 infection36 (Fig. 2 and Table 2). Moreover, only ~20% of these subjects eventually make plasma BnAb and then only after 4 or more years of infection57. It is probable that an individual will need BnAbs of more than one specificity for protection24,62,63; therefore, B-cell lineage vaccine design will probably require multiple lineages of B cells driven to make multiple specificities of BnAbs.
Passive infusion of human broadly neutralizing mAbs can protect against challenge with simian HIVs (SHIVs) at concentrations of antibodies thought to be achievable by immunization64–67. Passive protection studies of BnAb administration in rhesus macaques suggest that a plasma concentration 100 times the in vitro 50% inhibitory concentration is needed to protect from SHIV acquisition68. Thus, a major goal of HIV-1 vaccine development is to find strategies for inducing antibodies that have sufficient HIV-1 neutralization breadth to be globally effective.
Recent advances in isolating human mAbs using single-cell sorting of plasmablasts/plasma cells30,55, antigen-specific memory B cells decorated with labeled antigen24,69,70 and clonal cultures of memory B cells31,68,71 have led to the isolation of mAbs that recognize new targets for HIV-1 vaccine development (Fig. 2 and Table 2). Those BnAbs that are made in the setting of chronic HIV-1 infection have one or more of the following unusual traits: restricted VH usage, long HCDR3s, a high number of somatic mutations or antibody polyreactivity for self- or other non–HIV-1 antigens14,36. Several HIV-1 antibodies have been reverted experimentally to their unmutated ancestral state and were found to bind weakly or undetectably to native HIV-1 Env15,19,21,22. These observations suggest a strategy in which different or non-native immunogens are used to prime the Env response followed by the use of other immunogens to boost it15,19–21,23,30,31. Thus, the B-cell–lineage vaccine design strategy discussed below is an effort to drive rare or complex B-cell maturation pathways.
We anticipate three general steps for any lineage-based approach to vaccine design (Fig. 3). First, the identification of a set of clonally related memory B cells using single-cell technology to obtain the native immunoglobulin heavy (VDJ) and light (VJ) gene pairs. Second, use of the computational methods described below to infer the unmutated ancestral BCR (that is, the presumptive receptor of the targeted naive B cell), along with probable intermediate ancestor BCRs at key clonal lineage branch points. Finally, the design of immunogens with an enhanced affinity for unmutated and intermediate ancestor BCRs using the unmutated and intermediate ancestor paratopes as structural templates (Fig. 3). Thus, in contrast to the usual vaccine immunogens that prime and boost with a common immunogen, a vaccination protocol based on B-cell lineage may prime with one immunogen, boost with another and potentially boost further with a sequence of several different immunogens15,19–23,30,31 (Fig. 3). For example, whereas a gp140 Env antigen did not bind the inferred unmutated ancestor of a human BnAb, it was capable of binding if Env was deglycosylated21. Immunization of rhesus macaques showed that the deglycosylated Env that was bound by the unmutated ancestor antibody was superior to the native Env as an immunogen21.
It is noteworthy that variability of the antibody repertoire among individuals poses a potential problem for this strategy: a clonal lineage isolated from one subject may not be relevant for inducing a similar antibody in another subject. Even so, recent observations of limited VH gene segment usage suggest that for some viral-neutralizing epitopes, the relevant immunoglobulin repertoire is restricted to a very small number of VH families, and that the maturation pathways may be similar among individuals23,70,72 (Box 1). Examples of the convergent evolution of human antibodies in different individuals come from analyses of influenza and HIV-1 VH1-69 antibodies, in which similar VH1-69 neutralizing antibodies can be isolated from different subjects73–78. Another example comes from the structures of V1/V2 loop conformational (quaternary) antibodies in which the antibodies have very similar HCDR3 structures but arise from different VH families31,68,79,80. Recently, use of 454 deep-sequencing technology has shown convergent evolution and restricted VH gene segment usage in the maturation of BnAbs23,30,72. Determining how distinct the affinity maturation pathways are for each specificity of HIV-1 BnAbs will require experimental testing.
B-cell–lineage vaccine design requires the inference of unmutated ancestor antibodies and their intermediates from the V(D)J sequences of clonally related, mutated antibodies, as depicted in the clonal lineages in Figure 3 and Box 4, Figure 4. Functional antibody genes are assembled from a fixed set of gene segments. In humans, the numbers of VH, DH and JH gene segments per haploid genome are approximately 38–46, 23 and 6, respectively, with some variation among individuals. In addition to this combinatorial diversity, there is diversity in the locations of the recombination sites for each junction. Together there are on the order of 109 different V, D and J gene segment combinations. Although this number seems large, it is a tiny fraction of the 4350 possible nucleotide sequences of comparable length (350 bases). This enormous reduction in the space of possible ancestors makes quantitative inference plausible15,23,30,31.
The known unrearranged germline sequences of the V, D and J gene segments and the rules for their recombination provide very restrictive prior distributions on possible ancestors. From the likelihood of the sequence data, given the ancestor and the substitution model, as well as the prior distribution on the ancestors, one can apply Bayes’ rule to compute the probability of any hypothesized ancestor. Bayes’ rule describes the relationship between the conditional probabilities relating data and hypotheses. If we can compute the probability of the data given each hypothesis (the likelihood), Bayes’ rule tells us how to compute the probability of any given hypothesis given the data.
The posterior probability at each position in the unmutated ancestor can be computed from the posteriors over the gene segments and over other parameters of the rearrangement. The complete probability function provides a measure of the certainty of the inference at each position in addition to the most likely nucleotide state itself. This additional information may be crucial to ensuring the relevance of the antigen binding assays performed on the synthesized unmutated ancestor. Some of the intermediate forms of the antibody genes through which a given member of the clone passed can be similarly inferred, though not all of them can be (Fig. 3). The more members of the antibody clone that are able to be isolated, the higher the resolution with which one can reconstruct the clonal intermediates30.
The inference of the posterior probabilities of the unmutated common ancestor and intermediates proceeds in several steps, outlined as follows and in Figure 4. First, one starts with an initial estimate of the clonal lineage tree, which will be iteratively updated. Each node of the tree is an antibody intermediate, and the root of the tree is the unmutated common ancestor antibody. The likelihood function is a function over the nucleotides at each position of the undetermined sequences at the nodes of the tree. Second, the likelihood is computed backwards from the observed sequences (O1 and O2) at the tips of the tree to each node in sequence through the substitution model back to the unmutated ancestor. Third, the likelihood at the unmutated ancestor is used to align the germline gene segments to compute the prior probability on nucleotides in the unmutated ancestor. Normalizing the product of the likelihood and the prior probability gives the posterior probability of the unmutated ancestor. The posterior probabilities are then propagated back up the tree through all the intermediates, again using the substitution matrix and Bayes’ rule. One may re-estimate the clonal tree and repeat the steps above until the tree stabilizes and the posterior probabilities converge.
The starting point for any likelihood-based phylogenetic analysis is a model for the introduction of changes along the branches. To infer the unmutated ancestral V(D)J gene arrangements of a clonal lineage (Fig. 3), one needs a model for somatic mutation describing the probability that a given nucleotide that initially has state n1 will, after the passage of t units of evolutionary time, have state n2. This substitution model would allow for the computation of the likelihood of the observed data, given any hypothesized ancestor, from which, as described in Box 4 and ref. 81, the posterior probability for any such ancestor can be computed.
The goal of the B-cell–lineage vaccine design strategy described here is to derive proteins (or peptides) with an enhanced affinity for the unmutated and intermediate ancestor antibodies of a BnAb clonal lineage compared to existing antigens. The method of choice for finding such proteins will clearly depend on the extent of the structural information available (Tables 3 and and4).4). Ideally, one might have the crystal structures for the complex of the mature antibody Fab with antigen, the structures of the unmutated ancestor and of one or more intermediate ancestors and, perhaps, a structure of an unmutated ancestor–antigen or intermediate ancestor–antigen complex. It is possible that the native antigen on a virion will not bind tightly enough to the unmutated ancestor to enable a determination of the structure of that complex. In the absence of any direct structural information, cases in which the antibody footprint has been mapped by one or more indirect methods can also be considered (for example, Env mutational analysis).
Computational methods for ligand design are becoming more robust and, for certain immunogen-design applications, will probably be valuable82. We anticipate, however, that for the epitopes presented by HIV-1 Env, the available structural information may be too restricted to rely primarily on computational approaches. The interface between an antibody and a tightly bound antigen is generally between 750 Å2 and 1,000 Å2, and on the surface of gp120, for example, such an interface might include several loops from different segments of gp120. Even if both the structure of the mature antibody–Env complex and that of the unmutated ancestor antibody were known, the computational design of a modified Env with an enhanced affinity for the unmutated ancestor would be challenging. Selection approaches should, at least in the near term, be more satisfactory and reliable.
For continuous epitopes, phage display is a well-developed selection method for finding high-affinity peptides83,84. The best-studied continuous epitopes on HIV-1 Env are those for the antibodies that are directed against the MPER of gp41: 2F5 and 4E10. Efforts to obtain high titers of neutralizing antibodies by immunization with peptides or other MPER immunogens bearing the sequence of these epitopes have generally been unsuccessful, presumably in part because a peptide, even if cyclized, only rarely adopts the conformation required for recognition in the context of gp41. In a computational effort to design suitable immunogens, the 2F5 epitope was grafted onto computationally selected protein scaffolds that presented the peptide epitope in the conformation seen in its complex with the 2F5 antibody85. These immunogens indeed elicited antibodies that recognized the epitope in its presented conformation but did not neutralize viral infectivity85. The MPER epitopes are exposed only on the fusion-intermediate conformation of gp41 (ref. 86). To have neutralizing activity, these antibodies must have a membrane-targeting segment at the tip of their HCDR3 in addition to a high-affinity site for the peptide epitope87. In this manner, a liposome containing the 2F5 gp41-neutralizing epitope induces rhesus macaque antibodies to the epitope—again in the absence of neutralizing activity—indicating a lack of induction of polyreactive (lipid-binding) gp41 BnAbs14 and showing the necessity of potent adjuvants to overcome peripheral tolerance controls.
One can map differences between the antibody 2F5 and its most probable unmutated ancestor onto the 2F5 Fab peptide–epitope complex. The side chains on the peptide that contact the antibody are all within a ten-residue stretch, and several of these (an AspLysTrp sequence, in particular) must clearly be an anchor segment, even for a complex with the unmutated ancestor antibody. Randomization of no more than five positions in the peptide would cover contacts with all the residues in the unmutated ancestor antibody that are different from their counterparts in the mature antibody. Phage display libraries can accommodate this extent of sequence variation (about 3 × 106 members), and therefore a direct lineage-based, experimental approach to finding potential immunogens is possible through selection of peptides that bind unmutated or intermediate ancestor antibodies from such libraries.
For discontinuous epitopes on gp120 that are antigenic on cell-surface–expressed, trimeric Env, one can devise a selection scheme for variant Envs based on the same kind of single-cell sorting and subsequent sequencing that is used to derive the antibodies. Cells would be transfected with a library of Env-encoding vectors selectively randomized at a few positions, and the tag used for the sorting would be a fluorescently labeled version of the unmutated ancestor antibody. A procedure would then be required to select only those cells expressing an Env variant with a high affinity for the antibody.
The recognition of the HIV-1 envelope by several classes of BnAbs includes glycans presented by conformational protein epitopes. Such antibodies account for ~25% of the broadly neutralizing activity in the plasma of subjects selected for broad activity62,88. By analogy with selection from phage-displayed libraries, synthetic libraries of glycans or peptide–glycan complexes could be screened to select potential immunogens with a high affinity for the unmutated and intermediate ancestor antibodies of clonal lineages89. A large-scale synthesis of the chosen glycoconjugates could then yield the bulk material for immunization trials90,91.
The approaches discussed here should be equally applicable to the design of influenza vaccines. On the influenza virus HA, two conserved epitopes have received recent attention: a patch that covers the fusion peptide on the stem of the elongated HA trimer74,75,78, and the pocket for binding sialic acid, the influenza-virus receptor92. Screens of three phage-displayed libraries of human antibodies from quite different sources yielded similar antibodies directed against the stem epitope, and additional human mAbs of this kind have been identified subsequently by B-cell sorting. Conservation of the stem epitope may be partly a consequence of low exposure resulting from the tight packing of HA on the virion surface and, hence, a low immunogenicity on intact virus particles. An antibody from a vaccinated subject has been characterized that binds the sialic acid–binding pocket, mimics most of the sialic acid contacts and neutralizes a very broad range of H1 seasonal strains of influenza92.
The phenomenon of ‘original antigenic sin’, sometimes seen in influenza vaccination, is the recall of specificities of antibodies to prior infections by a new vaccination93. For HIV-1 and HCV, B-cell–lineage design will be for primary immunizations of individuals with no prior infection, so the original antigenic sin phenomenon is not expected to occur in this context.
Should the B-cell–lineage vaccine design strategy be successful, could it drive the survival of B-cell clones that are sufficiently autoreactive to be pathogenic? It is key in this context to note that polyreactivity is a normal component of the immune response94 and that polyreactive BnAbs are not necessarily expected to be pathogenic when produced. Indeed, polyreactivity of HIV-1 antibodies has been suggested to improve their protective effect95, and, in some cases, polyreactivity is required for antibody neutralization87.
HIV-1 is a paradigm for viruses that express conserved epitopes on their envelope proteins, which, by various mechanisms, are prevented from efficiently inducing antibodies. Among these mechanisms, at least in the case of HIV-1, is the physiological control of immunological tolerance to viral epitopes that structurally mimic self-antigens. It is therefore understandable why conventional immunization strategies for BnAb induction have not as yet succeeded.
With recombinant antibody technology, clonal cultures of memory B cells and 454 deep sequencing, numerous clonal lineages of BnAbs can now be detected and analyzed. We anticipate optimizing immunogens for high-affinity binding to antibodies (BCRs of clonally related B cells) at multiple stages of clonal lineage development by combining the analysis of these lineages with structural analyses of the antibodies and their ligands. The work described here outlines an approach for testing this strategy for inducing B-cell maturation along pathways that would not be taken in response to conventional, single-immunogen vaccines.
The authors acknowledge H.-X. Liao, M. Anthony Moody, S. Munir Alam, L. Verkoczy, M. Bonsignori and G.D. Tomaras at Duke University School of Medicine and D. Dimitrov at the National Cancer Institute, US National Institutes of Health (NIH), USA, for discussions and key collaborations on the work cited in this review; H.-X. Liao for Figure 3; and K. McClammy and S. Devine for expert secretarial assistance. This work was supported by a Vaccine Development Center grant in the Collaboration for AIDS Vaccine Discovery Program from the Bill and Melinda Gates Foundation, by the NIH, National Institute of Allergy and Infectious Diseases (NIAID), Division of AIDS grant for the Center for HIV/AIDS Vaccine Immunology, cooperative agreement U19 AI-067854, and by an NIAID ‘Modeling Immunity for Biodefense’ contract. S.C.H. is an investigator of the Howard Hughes Medical Institute.
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