Immunologic characteristics of HIV‐infected individuals who make broadly neutralizing antibodies

Summary Induction of broadly neutralizing antibodies (bnAbs) capable of inhibiting infection with diverse variants of human immunodeficiency virus type 1 (HIV‐1) is a key, as‐yet‐unachieved goal of prophylactic HIV‐1 vaccine strategies. However, some HIV‐infected individuals develop bnAbs after approximately 2‐4 years of infection, enabling analysis of features of these antibodies and the immunological environment that enables their induction. Distinct subsets of CD4+ T cells play opposing roles in the regulation of humoral responses: T follicular helper (Tfh) cells support germinal center formation and provide help for affinity maturation and the development of memory B cells and plasma cells, while regulatory CD4+ (Treg) cells including T follicular regulatory (Tfr) cells inhibit the germinal center reaction to limit autoantibody production. BnAbs exhibit high somatic mutation frequencies, long third heavy‐chain complementarity determining regions, and/or autoreactivity, suggesting that bnAb generation is likely to be highly dependent on the activity of CD4+ Tfh cells, and may be constrained by host tolerance controls. This review discusses what is known about the immunological environment during HIV‐1 infection, in particular alterations in CD4+ Tfh, Treg, and Tfr populations and autoantibody generation, and how this is related to bnAb development, and considers the implications for HIV‐1 vaccine design.

HIV-1, 19,20 while the role of other antibody-mediated functions in exerting immune pressure is unclear, primarily because antibodies that mediate ADCC and other activities often also neutralize. 21 Regardless, multiple studies have demonstrated that virus neutralization is a driver of both virus and antibody diversity, [22][23][24][25][26] although neutralization of autologous viruses does not always cause the extinction of susceptible virus populations in an infected individual. 25 Because of their ability to neutralize many different circulating strains of HIV-1, broadly neutralizing antibodies (bnAbs) are attractive targets for vaccine development. 27 Passive infusion studies in animals have shown that bnAbs can prevent infection by intravenous 28 and mucosal [28][29][30][31][32][33] challenge, suggesting that a vaccine that elicits robust and durable levels of bnAbs could be protective. Vaccine development strategies that leverage our understanding of antibody-virus coevolution 34 and that use knowledge of antibody-antigen structure relationships 27 are currently being tested in animal models 35,36 and human studies are planned. However, to date, no vaccine has reliably elicited bnAbs, and one possibility is that in addition to optimizing antigen structure, it may be necessary to recreate the immunological environment in which bnAbs develop. Understanding the conditions in which bnAbs have developed is a critical first step toward recreating those conditions.

| The antibody response in HIV-1 infection
During acute HIV-1 infection, there is a vigorous immune response that is unable to contain virus replication or the establishment of latency. 37 In many cases, a single transmitted/founder virus establishes infection and then evolves within the host resulting in a diverse virus population. 38 The earliest changes to the virus population are driven by the CD8 + T-cell response that is induced as viremia increases 39 and places strong selection pressure on the virus, resulting in complete turnover of the virus pool within the first few weeks of infection. 40 The development of antibody responses follows a pattern, with antiviral antibodies being detected first as immune complexes 41 followed by free antibody directed at the HIV-1 envelope (Env) glycoprotein 41 (gp41) subunit, 41,42 and then by the development of Env glycoprotein 120 (gp120)-binding antibodies. 41 These early antibody responses do not neutralize or place selective pressure on virus evolution 41 ; antibodies capable of neutralizing autologous viruses are not detectable until weeks to months after infection is established 19,20 and have little to no activity against heterologous HIV-1 strains. 43 The initial gp41directed antibody response is polyreactive and the antibodies are highly mutated, 42 and evidence indicates that at least some early responding B cells are primed prior to infection by non-HIV-1 antigens such as proteins contained in intestinal microbiota. 44 Acutely HIV-1-infected individuals do not make bnAbs, 41,43 but during chronic HIV-1 infection, neutralization breadth develops to a greater or lesser degree in each person. [45][46][47] Breadth of plasma neutralizing activity develops incrementally, 48 and evidence suggests that protracted exposure to HIV-1 Env is required. [22][23][24][47][48][49][50] Neutralization breadth is not an all-or-nothing phenomenon-in one study, about half of a cohort of 205 chronically infected persons were capable of neutralizing about half of a panel of 219 hard-to-neutralize (tier 2) HIV-1 isolates. 45 When bnAbs arise, neutralization breadth is thought to be mediated by 1-2 specificities per individual, 51,52 although serum mapping studies [53][54][55][56] and algorithms designed to deconvolute neutralization data 57 suggest that in some individuals breadth may be mediated by three or more specificities of antibodies.
In addition, the acquisition of additional neutralization breadth can continue during ongoing HIV-1 infection. For example, in one infected individual (CH505), a CD4-binding site-directed bnAb developed 24 that was aided in its evolution by a cooperating antibody lineage also directed at the CD4 binding site but that did not initially have neutralization breadth. 23 During ongoing infection, this cooperating antibody lineage further evolved, developing bnAb activity. 22 The latter example suggests that the development of neutralization breadth is a dynamic process and that conditions favorable to the evolution of bnAbs may persist during chronic infection in some individuals.

| Clinical characteristics of individuals making bnAbs
Longitudinal studies of HIV-1 infection have allowed researchers to map the development of bnAbs. [22][23][24]26,48 During acute/early HIV-1 infection, high viral load and an early decline in circulating CD4 + T cells were associated with the development of neutralization breadth, 48,50 whereas individuals who had low or undetectable viral loads, such as long-term non-progressors, were found to have less neutralization breadth. 49 Time since infection also correlated with the development of breadth, 56 although whether this was due to the need for protracted Env exposure, persistent immune perturbation caused by HIV-1 infection, or both is not clear.
The development of neutralization breadth does not appear to impact the progression of HIV-1 disease 48,67 ; for example, many individuals in these longitudinal cohorts went on antiretroviral therapy as part of standard of care during the course of study, 48,50 including those with high degrees of breadth. 48 Examination of viruses and antibodies taken from the same HIV-1-infected individuals has consistently shown that circulating bnAbs cause extinction of susceptible virus populations despite ongoing viremia, [23][24][25] although viruses susceptible to less potent neutralizing antibodies continue to circulate. 25 Whether individuals who have developed bnAbs exhibit a reduced susceptibility to HIV-1 super-infection has not been addressed, although as bnAbs constitute only part of the overall HIV-specific antibody response they may not reach sufficient titers to do so. The lack of clinical impact of bnAbs is consistent with early bnAb infusion studies in animal models 68 and humans 69 that did not show a lasting impact on HIV-1 viral loads, although newer, more potent bnAbs appear to have a more persistent impact on viral load [70][71][72] and subsequent antibody development. 73 At this time, ongoing clinical trials are being conducted to determine if bnAb infusion can augment other anti-HIV-1 therapies.
The duration and magnitude of Env exposure that is permissive for bnAb development is not completely known. Longitudinal studies suggest that bnAbs do not develop until at least 2 years after HIV-1 infection, but a study of two HIV-1-infected individuals who made Env gp41 membrane proximal external region (MPER) bnAbs showed that activity developed at about 1 year of infection while they were not on antiretroviral therapy. 26 A second study compared individuals taking and not taking antiretroviral therapy and determined that the frequency of individuals making bnAbs was similar between the groups, 74 although not all individuals taking antiretrovirals were completely suppressed. Thus, while prolonged Env exposure appears to be highly correlated with the development of neutralization breadth, there does appear to be a pathway to bnAb development in some individuals that lack prolonged or high-level Env exposure.
Common demographic characteristics do not appear to influence bnAb development. In a large longitudinal study of African HIV-1infected individuals, there was no correlation between neutralization breadth and geographical origin, age, or gender, and no correlation with reported mode of transmission or risk factor. 50 In this same study, there was a correlation with subtype C infection and with the presence of human leukocyte antigen (HLA)-A*03, 50 although these two correlations were not observed in a different study. 47 In this second study, exome sequencing performed on matched sets of individuals with and without bnAbs did not reveal any specific gene that correlated with breadth, although some candidate variants were found. 47 To date, the data suggest that multiple factors contribute to the development of breadth in HIV-1-infected individuals.

| The association between autoimmunity and HIV-1 Infection
Early during the HIV-1 epidemic, clinicians noted that some HIV-1infected individuals would go on to develop autoimmune phenomena, 75,76 usually in the setting of uncontrolled infection. In addition, investigators noted that there was an underreporting of coincident HIV-1 infection and systemic lupus erythematosus (SLE), 77-80 recognizing that lack of reporting does not mean a lack of coincident disease.
It was suggested that SLE and other autoimmune diseases might provide protection against infection, 75 but to date no study has isolated the protective factor. One candidate antibody class, antiphospholipid antibodies that can be found in autoimmune disease, was shown to block infection in vitro but testing of patient samples gave inconclusive results. 15  However, many isolated bnAbs have been shown to have autoreactivity when tested in assays used for the diagnosis of autoimmune disease 22,24,81,82 or when tested on arrays of human proteins. 83,84 While there are bnAbs that do not react with human antigens, 83 most do. For some bnAbs where maturation has been studied from the initial B-cell rearrangement to breadth, development of breadth was correlated with acquisition of autoreactivity. 24 In one case, bnAb activity and autoreactivity were directly correlated, when a bnAb that also had reactivity with double stranded DNA was isolated from a person with coincident HIV-1 infection and SLE, 81 indicating that in that person, bnAb activity derived from an autoreactive B-cell pool.
In addition to autoreactivity, bnAbs usually have a high degree of somatic mutation and/or long heavy-chain complementarity determining region 3 (HCDR3) loops, 85 and no reported bnAb lacks all three characteristics. Autoreactivity, long HCDR3 loops, and high levels of somatic hypermutation are also associated with antibodies made by B cells that are deleted by central and peripheral tolerance controls. 86,87 Furthermore, studies of antibody knock-in mice (engineered to have Thus, while it may not be necessary for a HIV-1 neutralizing antibody to exhibit characteristics of autoantibodies to have breadth, the two appear to be highly correlated. Because of this apparent correlation, we recently completed a study examining the relationship between autoantibodies and bnAb activity. 47 In two different cohorts, we found that HIV-1-infected persons making bnAbs were more likely to have serum autoantibodies as well as perturbations of their circulating T-cell populations, thus suggesting relaxed tolerance controls.
T-cell populations involved in regulation of B-cell responses and their relationship to bnAb induction are discussed in the following sections.

| T follicular helper cells
T follicular helper (Tfh) cells are a subset of CD4 + T cells specialized for provision of help to B cells. Help is provided in a cognate fashion, i.e. antigen bound to B-cell surface Ig is internalized and presented with MHC class II, and Tfh cells with specificity for the peptides presented interact with and provide help to the B cell. 94 The first cognate interactions between B cells and CD4 + T cells occur at the interface between the T-and B-cell zones in lymphoid tissues. 95

| T follicular helper cell differentiation
Tfh cell differentiation is a complex multi-step process that is influenced by numerous heterogeneous signals (reviewed in 112). The first step occurs when naive CD4 + T cells are primed by dendritic cells (DCs) in LNs: an initial Tfh-lineage fate decision is made during the first few rounds of cell division. 113 Factors that influence CD4 + cell Tfh-lineage differentiation include the cytokine environment, ICOS ligation, and signaling via the T-cell receptor (TCR). In mice, IL-6 plays an important role in promoting Tfh cell generation and induces upregulation of the transcription factor B-cell lymphoma 6 (Bcl6), which is a central regulator of Tfh differentiation. [114][115][116] However, although there is evidence that IL-6 can also promote Tfh differentiation in humans, 117 it is a not a good inducer of Bcl6 expression in human CD4 + T cells. 118,119 By contrast, human (but not murine) Tfh cell differentiation is potently induced by activin A 120 and can also be driven by transforming growth factor β (TGFβ), particularly in combination with IL-12 or IL-23. 121 Tfh differentiation is negatively regulated by other cytokines, including IL-2 122,123 and IL-7. 124,125 Tfh cell differentiation also requires ligation of ICOS by its ligand ICOSL. 126 Studies in mice have shown that CD8α(−) DCs at the interface of the follicle and T-cell zone play a prominent role in Tfh cell induction by upregulating expression of ICOSL and also OX40L. 127 The

G-protein-coupled receptor EBI-2 positions T cells in this location,
where Tfh cell differentiation is further enhanced by DC-mediated production of soluble and membrane-bound CD25 (the IL-2 receptor α chain), which binds to and quenches IL-2. 128 Signals received via the TCR during DC-mediated antigen presentation also influence CD4 + differentiation into early Tfh cells, although there is not a simple relationship between TCR signal strength and Tfh cell differentiation. 129 Instead, the duration of signaling appears more important, with Tfh cell induction being associated with prolonged antigen presentation by DC. 130 As CD4 + T cells differentiate into pre-Tfh cells they downregulate expression of C-C chemokine receptor type 7 (CCR7) and P-selectin can move from one GC to another or exit the follicle altogether. 152,153 As Bcl6 expression requires ongoing induction for its maintenance, its expression is downregulated when GC Tfh cells leave the follicle, and they gradually transition to a resting memory state. 152

| Circulating Tfh populations
Human peripheral blood memory Tfh cells are heterogeneous in phenotype (reviewed in 161). They are found within the CXCR5 + subset of circulating CD4 + T cells, 99 vitro, whereas the CXCR3 + CXCR5 + CD4 + population produces little IL-21 and has a very poor B-cell helper capacity 99,158,160,163 ; furthermore the blood CXCR3 − PD1 + CXCR5 + CD4 + subset has a transcriptional profile most similar to that of GC Tfh cells. 160 Nonetheless, although the CXCR3 − PD1 + subset of resting memory CXCR5 + CD4 + cells have been deemed to constitute resting memory Tfh cells, 160 this does not provide an exclusive or comprehensive definition.
Notably, CXCR3 + CXR5 + memory Tfh cells are generated under some conditions, e.g. the seasonal influenza vaccine elicits ICOS + PD-1 + CXCR3 + CXCR5 + Tfh cells, and the circulating frequency of this population in the periphery correlates with the quantity and avidity of the influenza-specific antibody response. 158,164  168,173,189-191 although Tfh cells are eventually depleted as progression to acquired immunodeficiency syndrome (AIDS) occurs. 192 The expansion in Tfh cells is associated with an increase in GC B cells and plasma cells and elevated IgG production, suggesting that Tfh cells contribute to the dysregulation of B cells and antibody production that occurs during HIV-1 infection. 191 The magnitude and duration of antigenic stimulation are known to be important determinants of the GC Tfh cell response, 193 and although GC Tfh cell frequencies do not generally show a direct correlation with viral load, the increase in GC Tfh cells during chronic SIV/HIV infection is dependent on ongoing antigenic stimulation, as GC Tfh cell frequencies decline when viral replication is contained by antiretroviral therapy. 168,189,191 Sustained LCMV replication in mice is also associated with expansion of GC Tfh cells, and here IL-6 production has been shown to play a key role in driving Tfh cell differentiation during chronic infection. 194,195 GC Tfh cell expansion in chronic HIV-1 infection is also associated with elevated IL-6 production, 189  and IL-21 production in vitro, contributing to their poor helper capacity. 190 The functional capacity of circulating Tfh cells was also found to be impaired: the proportion of cells producing cytokines including IL-21 was reduced, and they again exhibited a reduced B-cell helper capacity in vitro. 163 These defects in Tfh cell function appear to develop very rapidly, during the acute phase of HIV-1 infection. 190,197 Analysis of both GC Tfh cells in SIV-infected macaques 198   First, does the epitope specificity of Tfh cells impact on bnAb induction? As Tfh cells mediate cognate interactions with B cells, Tfh cell epitopes must be physically linked to bnAb epitopes, although they need not necessarily be in Env, e.g. in macaques primed with groupspecific antigen (Gag) plus polymerase (Pol) (Gag-Pol) immunogens and boosted with virus-like particles containing Gag-Pol and Env, Gag-Pol-specific CD4 + T cells were found to enhance Env-specific antibody production. 202 However, as antigens can undergo degradation in vivo, it may be advantageous for Tfh cell and bnAb epitopes to be in close proximity. Second, is Tfh cell avidity important? A recent study of the influenza-virus-specific CD4 + T-cell response indicated that T cells responding to different epitopes exhibited distinct tendencies to develop into Tfh cells, with those exhibiting a higher functional avidity being more likely to become Tfh cells 203 ; but whether Tfh cells of higher avidity also mediate superior help for B cells is not clear.

No associations have been reported between HLA class II type and bnAb induction during HIV-1 infection that may support a role for
T-cell responses of particular specificity favoring or disfavoring bnAb induction 47 ; but as many CD4 T-cell epitopes are promiscuously presented by multiple HLA class II alleles, 204 this does not preclude a relationship between epitope recognition and help for bnAb induction.  is spontaneous B-cell activation, GC formation, and autoantibody production, and the animals develop a lupus-like phenotype. 212,213 Likewise increased numbers of circulating CXCR5 + CD4 + T cells, frequencies of which correlate with autoantibody titers, are observed in patients with SLE and other autoimmune diseases including Sjogren's syndrome and myasthenia gravis. 162,214 As discussed above, Tfh cell differentiation is a multi-step process initiated by the signals received by naive CD4 + T cells as they undergo Tfh cell proliferation following antigen recognition on B cells. 109,220,221 In addition to B cells, antigen-specific plasma cells can also present antigen to Tfh cells. However, they do not stimulate Bcl6 expression in Tfh cells and promote their differentiation into non-Tfh cells, providing a negative feedback mechanism for reducing the GC response when large numbers of antigen-specific plasma cells have been generated. 222 Furthermore, as antigenic stimulation is required to sustain Tfh cell responses, the GC response is down-modulated as antigen is cleared 193 ; conversely sustained antigen persistence may promote Tfh expansion in situations of chronic infection or autoimmunity. In addition, GC responses are also regulated by regulatory cell populations that are found within the GC, including CD4 + T follicular regulatory cells (Tfr) 223 and major histocompatibility complex (MHC)-E-restricted regulatory CD8 + T cells. 224  Importantly, although Tfr cells reduce the overall magnitude of the humoral response, the antibody produced is of higher affinity than that generated in the absence of Tfr cells. 240,244 CD4 + Tfr cells develop from thymically derived Tregs that co-opt a Tfh-like differentiation pathway following activation and can also be generated from FoxP3 − precursors. 245 253 However, MHC-E can also present some peptides derived from autoantigens and pathogens, which are recognized by CD8 + T cells. Qa-1 is expressed at high levels on GC CXCR5 + CD4 + cells, while CXCR5 − CD4 + T cells express very low levels of Qa-1; hence, Qa-1-restricted CXCR5 + CD8 + T cells specifically target CXCR5 + Tfh cells. 254 By mediating perforin-dependent lysis of GC Tfh cells, they reduce the GC response and help to prevent autoantibody development. 252,254,255 Although Qa-1-restricted CD8 + Treg cells express CXCR5, they do not express ICOS or PD-1 or markers characteristic of CD4 + Treg cells such as FoxP3. However, they do express ICOSL and CD122 (the IL-2 receptor β chain, which also forms part of the IL-15 receptor). 224 The mechanisms involved in their differentiation are not fully understood, although Helios-dependent STAT5 activation has been shown to be important to enable their survival and prevent terminal differentiation. 256 Whether similar CXCR5 + CD8 + Treg cells contribute to regulation of GC responses in humans remains unclear, although HLA-E-restricted CD8 + Treg cells have been suggested to be involved in the control of type 1 diabetes in humans. 257  HIV-1 infection, 284 although the absolute number of Tfr cells is increased. One report suggests that the Tfr cell frequency in the spleen may be increased during HIV-1 infection, however. 285 Two studies in which the LN Tfr:Tfh cell ratio was analyzed in macaques chronically infected with SIV report conflicting results, with one finding an increase 284 and the other a decrease. 283 In the latter study, the decrease in the Tfr:Tfh cell ratio correlated with an increase in the percentage of GC B cells, suggesting that Tfr cells suppress GC B-cell formation. LN Tfr:Tfh cell ratios have not been investigated during HIV-1 infection, but the ratio of Tfr:Tfh (i.e. CD25 + FoxP3 + :CD25 − FoxP3 − ) cells within the circulating CXCR5 + CD4 + T-cell pool in a cohort of chronically infected individuals was found to be similar to that of uninfected subjects. 47 Whether there are alterations in Tfr cell function during HIV-1/ SIV infections remains to be investigated; however, in experiments in which tonsil cells were infected with HIV-1 in vitro, Tfr cells were found to express increased levels of CTLA-4, lymphocyte activation gene 3 (LAG-3), IL-10, and TGF-β and retained the capacity to impair Tfh cell proliferation and production of IL-21 and IL-4. 284

| Relationship between CD4 + Treg cells/CD4 + Tfr cells and bnAb induction in HIV-1/SIV infection
Regulatory CD4 + T-cell populations, in particular Tfr cells, control the overall magnitude of the GC response and regulate the stringency of B-cell selection within GCs, limiting the production of lower-affinity antibodies and auto-reactive antibodies. They may, therefore, constrain the production of HIV-1 bnAbs, which, as discussed earlier, commonly exhibit extensive somatic hypermutation and show evidence of autoreactivity, suggesting a need for strong GC Tfh cell activity and relaxed selection controls for their generation. One study in SIV-infected macaques found that circulating CD4 + Treg numbers correlated positively with autoantibody titers and the spectrum of autoantigens recognized, which does not support a relationship between relaxation of host tolerance controls and generation of antibodies with greater autoreactivity 286 ; however, LN Tfr cell numbers and the Tfr:Tfh cell ratio were not addressed. Conversely in another SIV study, the LN Tfr cell frequency (% FoxP3 + CD25 + CXCR5 + CD4 + T cells) was found to show an inverse correlation with the frequency of LN Tfh cells and the avidity of antibodies recognizing the SIV gp120 envelope protein in plasma, indicating a role for Tfr cells in constraining the maturation of the envelope-reactive antibody response. 282 We recently analyzed circulating CD4 + Treg and CD4 + Tfr cell populations in subjects chronically infected with HIV-1 who had generated bnAbs and matched individuals who had developed little or no antibody neutralization breadth. 47 The frequency of CD4 + Treg cells within lymphocytes was significantly lower in the bnAb group, although there was no difference between groups in the percentage of CD4 + Treg cells within CD4 + T cells, the circulating frequency of Tfr cells, or the Tfr:Tfh ratio (ratio of CD25 + FoxP3 + :CD25 − FoxP3 − cells within circulating CXCR5 + CD4 + T cells). Interestingly, however, PD-1 was expressed at significantly higher levels on both CD4 + Treg and CD4 + Tfr cells in the subjects who had generated bnAbs. As PD-1 inhibits the function of CD4 + Treg and CD4 + Tfr cells, 239 this suggested that the suppressive capacity of CD4 + Treg/Tfr cells in the bnAbproducing subjects may be impaired, an observation supported by the finding that the PD-1 hi subset of CD4 + Treg cells from some HIVseronegative donors exhibited an impaired capacity to inhibit the proliferation of conventional CD4 + T cells in vitro. 47 As discussed above, autoantibodies were also detected in a higher proportion of the group of subjects who produced bnAbs than those who did not. Together, these findings suggest that bnAb development during HIV-1 infection may be favored by a relaxation in regulatory CD4 + T-cell control of antibody production that enables a strong GC response and permits some degree of autoreactive antibody production. 47

| IMPLICATIONS FOR DESIGN OF BNAB-INDUCING VACCINES
A major focus of efforts to develop bnAb-inducing vaccines has been on antigen design. [287][288][289] Dissection of the pathways by which bnAbs evolve in HIV-1-infected individuals has led to recognition of the need for antigens that are able to trigger B cells expressing the unmutated common ancestors of mature bnAbs, drive the selection of daughter cells expressing antibodies with potential for further maturation along bnAb lineages, and ultimately enable bnAb evolution. 34 However, in addition to design of appropriate vaccine antigens, immunization strategies need to be devised that will elicit an environment support- Vaccine development efforts are, thus, likely to benefit from consideration of these three inter-related factors. First, a strong antigenspecific CD4 + Tfh cell response needs to be elicited. As discussed above, it is currently unclear whether this should ideally target particular CD4 + epitopes within the vaccine antigen, although it can be speculated that targeting of high-affinity epitopes in close proximity to bnAb epitopes may be beneficial. However, the use of immunization regimes (e.g. vectors and adjuvants) that promotes CD4 + T-cell differentiation into Tfh cells will be of great importance. For example, recent studies in murine models demonstrated that adenoviral vectors elicited a strong Tfh cell response to encoded vaccine antigens, 290 and that adenoviral priming enabled induction of strong humoral responses to a subsequent protein boost delivered with a relatively weak adjuvant 291 ; and adenoviral vectors have also been shown to elicit strong Tfh cell responses to encoded HIV antigens in macaques. 292 Second, immunization platforms need to be developed that will enable high level and prolonged antigen availability. This is of importance to sustain the Tfh cell response and the GC reaction and enable B cells to undergo multiple rounds of somatic hypermutation. Repeat immunizations, vectors that drive prolonged antigen expression, and/or platforms enabling slow antigen release over time (e.g. nanoparticle delivery systems 293 ) may all be helpful. Third, it may be necessary to transiently modulate host constraints on autoreactive antibody development at the time of bnAb induction to enable the evolution of bnAbs whose development is constrained by host tolerance controls. For example, this could be achieved by the use of immune modulatory strategies such as transient CTLA-4 blockade, 294 although this type of approach will need to be carefully safety-tested in animal models prior to study in human trials.
In summary, study of the mechanisms that enable bnAb induction in some HIV-infected individuals has given important insight into how bnAb induction may be achieved by vaccination. The benefit to rational vaccine design strategies is such that achievement of bnAb induction by vaccination now seems very likely.
F I G U R E 1 Factors associated with bnAb development in HIV-1-infected individuals. HIV-1-infected individuals who make bnAbs have been found to have perturbations of their immune system. In contrast, demographic characteristics have not been found to correlate with bnAb development

Not correlated with bnAb development
Likelihood of bnab development