Antibody B cell responses in HIV-1 infection.

TREIMM-1124; No. of Pages 13

Feature Review

Antibody B cell responses in HIV-1 infection Hugo Mouquet1,2 1 2

Laboratory of Humoral Response to Pathogens, Department of Immunology, Institut Pasteur, Paris, France CNRS-URA 1961, Paris, France

In rare cases, B cells can supply HIV-1-infected individuals with unconventional antibodies equipped to neutralize the wide diversity of viral variants. Innovations in single-cell cloning, high-throughput sequencing, and structural biology methods have enabled the capture and thorough characterization of these exceptionally potent broadly neutralizing antibodies (bNAbs). Here, I review the recent findings in humoral responses to HIV1, focusing on the interplay between naturally occurring bNAbs and the virus both at systemic and mucosal levels. In this context, I discuss how an improved understanding of bNAb generation may provide invaluable insight into the fundamental mechanisms governing adaptive B cell responses to viruses, and how this knowledge is currently contributing to the development of vaccine and therapeutic strategies against HIV-1. Antibodies against HIV-1 More than a century ago, Von Behring and Kitasato provided the first evidence on the role of the humoral response in host protection against infectious agents using serum therapy experiments against diphtheria and tetanus toxins [1]. ‘Briefly expressed, serum therapy works through anti-bodies’ explained Emil Von Behring (http://www.nobelprize.org). We now know that antibodies are valuable immunotherapeutics for a wide range of infectious diseases [2,3], that they can confer mother-to-child protective immunity [4], and that they present the best-known correlates of protection for many licensed vaccines [5]. Indeed, in addition to polyreactive (see Glossary) natural antibodies that act as the first line of defense against invading pathogens, humoral memory is composed of high-affinity antibodies that mediate long-lived immunity against infectious agents, for example, by providing protection against reinfection [5]. Thereby, the induction of high-affinity antibodies that inactivate or neutralize the culprit pathogen is a necessary component for most effective vaccines. Soon after the discovery of HIV-1 as the etiological agent causing acquired immunodeficiency syndrome (AIDS) [6], the existence of antibodies in patients’ sera capable of neutralizing HIV-1 strains in vitro was demonstrated [7,8]. To mediate viral neutralization, antibodies must recognize functional sites on the HIV-1 envelope spike, Corresponding author: Mouquet, H. ([email protected]). Keywords: HIV-1; antibodies; B cells; adaptive immunity; viruses. 1471-4906/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.it.2014.08.007

gp160, which is a hetero-trimer composed of gp120 and gp41 protein subunits [9]. Even so, targeted epitopes on functional gp160 sites must be sufficiently conserved across most HIV-1 quasi-species to enable viral neutralization by one single cross-reacting neutralizing antibody. These conserved functional epitopes are called sites of vulnerability of the HIV-1 envelope protein. Since then, major efforts have been made to develop an efficient HIV-1 vaccine capable of inducing protective immunity, particularly by eliciting cross-neutralizing antibodies. So far, however, all attempts have failed despite the encouraging clinical vaccine trial carried out in Thailand known as the RV144 trial. This trial evaluated on 16 000 Thai subjects a vaccine regimen consisting of a nonreplicating avian pox Glossary Germinal centers: germinal centers are specialized microenvironments within secondary lymphoid organs that support the maturation, proliferation, and differentiation of B lymphocytes during T cell dependent immune responses. These microanatomical structures are divided into light and dark zones; in the dark zone, B cells expand and diversify their antigen receptors through somatic hypermutation (that potentially increases antibody affinity) and class-switching (that alters antibody effector functions), whereas in the light zone B cells are selected for binding to their cognate antigen. HIV-1 broadly neutralizing antibodies (bNAbs): HIV-1 bNAbs are antibodies capable of neutralizing in vitro numerous HIV-1 viral strains from diverse subgroups (or clades). bNAbs can be classified into two groups according to the time they have been isolated and their neutralizing activity. First-generation bNAbs (b12, 2G12, 4E10, and 2F5) were isolated in the 1990s, and exhibit limited neutralization potency and/or breadth. On the contrary, next-generation bNAbs identified from 2009 by single-cell cloning strategies have more potent and broad neutralization activity. Latent HIV-1 reservoirs: the latent reservoir of HIV-1 consists of a pool of resting memory CD4+ T cells that contain integrated DNA of replicationcompetent HIV-1 proviruses. HIV-1 reservoirs are long-lived, and mediate the persistence of infection by being immunologically silent and insensitive to antiretroviral therapy. Polyreactivity: antibody polyreactivity is the ability of an antibody, secreted or expressed at the surface of B cells as a BCR, to bind to various structurally unrelated ligands generally, with a low affinity. Sites of vulnerability: sites of vulnerability of HIV-1 are the epitopes targeted by bNAbs, which are well-conserved regions of the viral envelope glycoprotein among divergent HIV-1 quasi-species. Antibodies recognizing these sites either interfere with the receptor/co-receptor binding to HIV-1 gp120 or with viral fusion. T cell dependent and independent B cell responses: during humoral immune responses to protein antigens, a T cell dependent activation of B lymphocytes is required, and necessitates the cooperation of B cells with helper T cells for the production of high-affinity antibodies. Upon exposure to non-protein molecules such as polysaccharides and lipids, B cells can respond autonomously by producing antibodies without presenting antigen to CD4+ T cells (defined as T cell independent B cell response). Virological synapse: the virological synapse is a specialized site of cell-to-cell contact between cells infected with lymphotropic viruses (e.g., HIV-1 and human T cell leukemia virus type 1; HTLV-1) and uninfected T cells. Virological synapses play a crucial role in the pathogenesis of these viruses by facilitating the transfer of viral material from infected to uninfected T cells.

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Feature Review clade E gp120 vector (ALVAC)-prime and a clades B/E gp120 protein (AIDSVAX)-boost, and showed a modest protection rate of about 31% [10]. Remarkably, antibodies raised against the variable loop 2 of gp120 in vaccinees were found to be the main positive predictive factor for protection [11]. In spite of this, major obstacles still prevent vaccines from achieving sterilizing immunity against the diversity of worldwide circulating HIV-1 strains. Similarly, significant barriers disable the immune system to destroy the virus in infected patients such as certain molecular peculiarities of its envelope protein that prevent an effective antibody recognition and neutralization [12], the generation of viral escape mutants and the persistence of longlived reservoirs of HIV-1 [13]. Nonetheless, rare seropositive individuals are able to produce potent bNAbs capable of neutralizing most of the known HIV-1 stains [14]. In the past 5 years, scrutiny of the human antibody response using methods that directly clone antibodies from single antigen-specific B cells isolated from these patients, has led to important insights as to how the humoral system operates to naturally combat infection. Recent advances in the arena of HIV-1-specific antibodies, notably the discovery and characterization of extremely potent bNAbs have also added new momentum to the quest for effective therapeutic approaches to prevent HIV-1 infection [14]. Here, I discuss the current understanding of how the humoral arm of the immune system naturally tries to fight HIV-1 infection in light of recent progress on the molecular dissection of bNAbs, and their interplay with HIV-1. In an effort to ‘phenocopy’ bNAb-based humoral responses with an adequate vaccine, there has been a major push worldwide to better understand the diverse immunological aspects of bNAb biology, including their gene/structural features, the identification of their epitopes on the viral spike, their antiviral properties, and the development/ maturation of bNAb-expressing B cell lineages during infection. These discoveries are reviewed herein, and in this context, I also discuss the design of antibody-based therapies for AIDS treatment and vaccines against HIV-1. Systemic and mucosal antibody responses to HIV-1 The initial antibody response to HIV-1 can be detected as immune complexes as early as 1 week post-infection [15]. In the following days, circulating anti-gp41 antibodies are produced, followed by production of anti-gp120 antibodies a few weeks later, but none of these antibodies are capable of neutralizing the infecting viral strain (autologous virus) [15]. The first autologous neutralizing antibodies appear several months post-infection and although they are unable to neutralize heterologous viruses (diversified viruses isolated from other seropositive individuals) [16–20], they exert a selective pressure on the virus that subsequently, rapidly evolves by generating escape mutants [19–21]. The development of crossreactive neutralizing antibodies capable of neutralizing dozens of heterologous viruses generally takes 2–4 years after seroconversion [22], but in rare cases could arise earlier [23]. Approximately 20% of HIV-1-infected individuals develop high levels of crossreactive neutralizing antibodies that target different regions of HIV-1 envelope protein [17,24–28]. Among them, rare individuals called ‘Elite 2

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neutralizers’ (representing 1% of seropositive individuals) develop bNAbs [29], which by definition are active against hundreds of different cell-free viral quasi-species from various HIV-1 subtypes. Unexpectedly, the development of bNAbs is not an inherent trait of adults, and infected infants can also mount a bNAb-like immune response against HIV-1, 20 months post-infection [30]. Serological profiling and mapping experiments in elite neutralizers revealed that serum neutralization frequently originates from a limited set of specificities [31–33]. Specifically, gp160-specific bNAbs produced are mostly IgG1, and most of them target known sites of vulnerability [22,24,27,32,33]. Serological neutralizing breadth and potency in HIV-infected patients can be accounted for by combinations of: (i) a multitude of additive/synergetic neutralizing antibodies targeting various epitopes [34]; (ii) clonal variants of a same specificity but with slightly different neutralization spectra [33,35,36]; and (iii) few potent bNAbs recognizing different epitopes and exhibiting complementary neutralizing activity [37,38]. Cell-to-cell transmission of HIV-1 is thought to be one of the major mechanisms contributing to viral spread in vivo [39]. Therefore, it would particularly be interesting to examine whether neutralizing serum antibodies from Elite neutralizers are also active against HIV-1 cell-to-cell spread in vitro. Due to viral escape, bNAbs do not suppress viral replication in infected persons [19,20], but they could contribute to sustain low-level viral loads in some individuals [40]. In this respect, and similar to observations made in infected rhesus monkeys [41,42], B cell depletion induced by rituximab immunotherapy in a HIV-1 seropositive individual with lymphoma led to a drop of neutralizing antibody titers and to a subsequent rebound of plasma viremia [43]. Moreover, HIV-specific IgG2 antibodies were shown to be positively associated with the lack of or slow progression towards AIDS and thus, could participate in keeping the virus in check in certain individuals [44,45]. Finally, HIV-1 antibodies may play a beneficial role by decreasing the risk of a subsequent infection (superinfection), and of vertical transmission in infected pregnant women [179]. Neutralizing antibodies act through their antigen-binding sites by interfering with receptor/co-receptor attachment or viral fusion with the target cells. Moreover, the Fc region of antibodies equips them with different effector functions permitting elimination of infected cells by innate immune cells engaged via the Fc receptors. Interestingly, antibodies responsible for several Fc-mediated mechanisms such as antibody-dependent cell-mediated cytotoxity (ADCC) and antibody-dependent cell-mediated viral inhibition (ADCVI) could reduce the risk of mother-to-child transmission of HIV-1 [46]. Furthermore, these antibodies have been associated with protection in individuals naturally controlling the infection (long-term non-progressors) [47,48] and in RV144 vaccinees [11]. These Fc-mediated properties are thought to be attributable to non- or weakly neutralizing antibodies, but whether serum crossreactive antibodies or bNAbs can also utilize these cellular effector functions remains only partially explored [49,50]. Antibodies have a well-documented role in protection of mucosal tissues against infection by bacteria, viruses, and


Feature Review


protozoa, as well as protection from damage by pathogenassociated toxins; IgA in particular is noteworthy given its prevalence in mucosal tissues [51]. Although mucosal IgAs may constitute an important component for preventing HIV entry at mucosal sites, little is known about the mucosal antibody response to HIV-1 and whether mucosal IgAs could efficiently protect against the infection. At mucosal surfaces, anti-gp41 IgA molecules are transiently produced at a high level during the first 2 weeks of infection, likely by short-lived plasma cells in a T-independent manner [52]. Due to the massive and rapid depletion of gut CD4+ T cells and various types of damage to gut-associated lymphoid tissues during the early stage of infection, that is, germinal center loss [53,54], the T cell dependent induction of long-lived plasma cells producing high-affinity antiviral IgA antibodies is probably impaired [55]. Nevertheless, HIV-infected individuals but also HIV-exposed individuals that are persistently seronegative can develop mucosal HIV-specific IgAs capable of blocking viral transcytosis (translocation throughout epithelial cells) [56–58]. In addition, some mucosa-derived IgA monoclonal antibodies to gp160 have been shown to neutralize HIV-1, by inhibiting its transcytosis and its intracellular replication in epithelial cells [59,60]. Lastly, Bomsel et al. showed that gp41vaccinated macaques were protected against repeated vaginal challenges of high simian–human immunodeficiency virus (SHIV) doses and that all of them developed not only gp41-specific vaginal IgGs with neutralizing and/ or ADCC activities, but also vaginal IgAs that block HIV-1 transcytosis [61]. Bearing in mind that AIDS is primarily a sexually transmitted disease, and that too few studies have been undertaken to investigate mucosal immunity against HIV-1, unraveling the mucosal antibody response in infected individuals is crucial in trying to design strategies to block mucosal transmission of the virus. Thus, HIV-1 infection induces systemic and mucosal B cell responses composed of antibodies with neutralizing and/or antiviral effector properties. However, these conventional antibodies have a limited neutralization efficacy against diversifying HIV-1 strains, and bNAbs more rarely and slowly develop in infected individuals. Indeed, due to both the various strategies used by the virus to escape immune pressure and to the detrimental effects of the infection on the adaptive B cell immunity, the antibody response to HIV-1 is inherently weakened.

memory B lymphocytes (i.e., resembling tissue-resident memory B cells). Importantly, it has recently been shown that gp160-specific B cell clones reside within abnormal B cell subsets, including exhausted tissue-like memory B cells and activated memory B cells, which predominate in HIV-infected individuals and are associated with higher levels of viremia [62]. This constitutes another pathophysiological example of how B-cell immune dysfunctions may contribute to an inadequate humoral response to HIV-1. These alterations arise from systemic bystander effects and possibly from direct interactions of B cells with the viral proteins particularly, gp120 and Nef (reviewed in [63]). Jelicic et al. showed that the direct interaction of HIV-1 gp120 with integrin a4b7 expressed on human B cells in vitro induces repressive signaling events including the production of the immunosuppressive cytokine transforming growth factor (TGF)-b1, induced expression of the inhibitory receptor FcRL4 and decreased expression of CD80 on B cells [64]. This virus-dependent mechanism leads to an inhibition of B cell activation and proliferation as typically observed during the infection, and is potentially a major obstacle hampering the humoral immune response against HIV-1 [64]. Finally, HIV-1 infection negatively impacts on follicular helper T (Tfh) cells, which are key players in establishing humoral responses (see below), and Tfh cell deregulation may undeniably diminish B cell immunity against the virus (reviewed recently in [65]). In addition to these deleterious effects rendering B cell response less effective, and apart from the rapid mutation of variable regions of the HIV-1 envelope protein resulting in an enormous diversity of circulating HIV-1 strains [19,20], the virus – so called Nature’s Master of Disguise [66] – uses a multitude of strategies to avoid or divert antibody B cell recognition (Figure 1). Studies of HIV-specific monoclonal antibodies revealed the peculiar nature of HIV-1 gp160 as the origin of several escape mechanisms including: (i) carbohydrate shielding and shifting [67,68]; (ii) conformation masking [70]; (iii) steric occlusion [71]; (iv) transient epitope exposure [72]; (v) nonfunctional envelope spikes such as gp120–gp41 monomers, gp41 stumps or uncleaved gp160 precursors, which may divert the immune response from functional targets [73,74]; and (vi) low density of functional HIV gp160 on the viral surface [75,76] (Figure 1). Despite such hurdles, bNAbs targeting conserved functional sites on gp120 and gp41 can develop in rare infected individuals.

Multiple viral strategies for disrupting and evading the immune response HIV-1 infection induces major perturbations in B cell development, physiology, and function that likely interfere with the establishment of a normal antiviral humoral response. Deregulation of B cell differentiation and function during HIV-1 infection is now well documented and consists of a variety of abnormalities, which are also associated with pathological changes in lymphoid tissues supporting B cell immune response (follicular hyperplasia and germinal centers alterations) such as: (i) B cell hyperactivity (e.g., hypergammaglobulinemia and polyclonal B cell activation); (ii) increased frequency of peripheral blood immature/transitional B cells; (iii) increased plasmablast differentiation; and (iv) exhaustion of blood tissue-like

Features of HIV-1 neutralizing antibodies In the early 1990s, first-generation HIV-1 bNAbs were isolated using Epstein–Barr virus transformation and phage display methods [77–82]. Their extensive molecular characterization improved our knowledge of the host immune response to HIV-1 by identifying important sites of vulnerability, antibody structure-to-function relations, and numerous viral escape mechanisms as described above [83]. Nevertheless, the uncommon nature of the antibodies (unnatural IgH and IgL pairing for b12, self-reactivity for 2F5/4E10, and an atypical antigen-combining site shaped by swapped VH domains for 2G12), their limited neutralization breadth and/or potency, and the inability of vaccines to induce their development created doubt about the possibility of an antibody-based vaccine. 3


Feature Review

Angenic variability

>

15

nm

Low density surface expression of Env


Nonfunconal Env

Steric occlusion

Glycan shielding

Transient epitope exposure

Conformaons masking TRENDS in Immunology

Figure 1. Envelope-dependent defense mechanisms of HIV-1. The HIV-1 Env or HIV-1 spike is the antigenic target of neutralizing antibodies. As well as from the Env antigenic variability resulting from the low-fidelity reverse transcription of the viral genetic material, several architectural and structural features of the HIV-1 spike limit or block the access of antibodies to conserved neutralizing epitopes: (i) Many hostderived glycans cover the surface of the spike (making up 50% of its mass) to form a ‘glycan armor’ protecting the virus from antibody recognition; (ii) some epitopes are only momentarily exposed to antibodies such as the ‘pre-fusion state’ form of gp41 to which membrane proximal external region-specific broadly neutralizing antibodies bind, and the co-receptor binding site (or CD4-induced site, CD4i) that opens following a conformational change induced by the interaction of gp120 with a CD4 molecule; (iii) similarly, conformational masking of the CD4 binding site (CD4bs) prevents antibody-mediated neutralization; (iv) the large size of immunoglobulins can sterically hinder the antibody binding to some epitopes (such as the CD4i), which can be accessed by Fab fragments alone; (v) most antibodies generated during HIV-1 infection target epitopes on nonfunctional spikes (noncleaved Env precursors, gp120 monomers, and gp41 trunks), which are not expressed by mature functional spikes; and (vi) the small number of gp160 glycoproteins (15 per virion), which are randomly distributed at the HIV surface, likely impair the ability of the antibody to bridge two Env (bivalent antibody binding), therefore reducing avidity effects. Abbreviation: Env, surface envelope glycoprotein.

In the past 5 years, research into the origin and nature of bNAbs was re-energized by the use of methodologies for producing antigen-specific antibodies from human B cells either by B cell capture [34], originally developed a decade ago as a means to study B cell tolerance and its breakage in autoimmune diseases [69,84], or by screening of cultured B cells [85,86]. The engineering of novel recombinant and soluble HIV-1 envelope proteins used as baits to identify and capture gp160-specific B cells by flow cytometry [87– 89], in combination with a meticulous selection of HIVinfected donors, has enabled single cell antibody cloning techniques to successfully generate hundreds of HIV-specific antibodies and among them, dozens of new extremely potent next-generation bNAbs (Figure 2) [14]. Until recently, mapping and structural definition of the HIV-1 bNAbs epitopes has allowed the identification of several sites of vulnerability on the HIV-1 envelope spike (Figure 2), comprising four major targets: the CD4 binding site (CD4bs), N-glycan-associated epitopes on the V1/V2 loops and the V3 loop, and the membrane proximal external region (MPER) on gp41 (Figure 2) [90]. Using similar technical approaches, the latest research efforts have led to the discovery of two other sites of vulnerability, targeted by 8ANC185 and PGT151-PGT158 bNAbs [91–93]. Both sites involve N-glycan-associated epitopes in the gp120/gp41 bridging regions [91–93] (Figure 2); thus, the 4

glycan-dependent gp120–gp41 interface is a novel ‘supersite’ of vulnerability that should be considered for vaccine immunogens design. Importantly, scrutiny of the humoral response in patients with broad serum neutralizing activity using single B cell capture and cloning approaches first revealed the diversity of the gp160-specific IgG class-switched B cell memory compartment in terms of clonal expansion, targeted epitopes, and neutralization capacity [34,94]. Gene repertoire of anti-HIV-1 memory antibodies showed abnormalities compared to that of healthy controls, in particular a preferential use of heavy-chain variable region 1 (VH1) and immunoglobulin light chain kappa (Igk), and evidence of high rates of somatic hypermutation [34,94]. Increased hypermutation level is also an important gene signature of HIV-1 bNAbs, which carry more than 40% amino acid substitutions in their heavy chains, as well as nucleotide insertions and/or deletions (termed ‘indels’) in heavy and light chain genes that are rare in other antibodies (Figure 3A) [14]. Many bNAbs also express unusually long CDRH3 loops necessary to reach a cryptic region of their epitope, by penetrating the glycan shield or by extracting it from the lipid membrane (Figure 3A) [83,90]. Somatic mutations, spanning both the complementarity determining region (CDR) and the relatively mutation resistant framework regions, increase the affinity of anti-HIV antibodies to their ligand, and are essential for viral neutralization [36,95,96]. Surprisingly, common VH gene segments (VH1–2 and VH1–46) encode next generation anti-CD4bs bNAbs of the VRC01 class originating from different individuals and despite extensive hypermutations (Figure 3B), VRC01-class bNAbs share a large consensus IgH sequence and conserve structural features for contacting the HIV-1 spike [36,97]. Regardless of their neutralization activity, a large fraction of gp160-specific antibodies are polyreactive, reacting with a number of host and other non-HIV antigens [98]. Anti-gp41 bNAbs, 2F5 and 4E10, were the first described to be polyspecific and reactive against several autoantigens, for example, membrane phospholipids and human larynx carcinoma HEp-2 cells [99,180]. In this regard, anti-gp41 antibodies are more frequently poly- and self-reactive than gp120-specific antibodies [94,96,100–102]. Other potent bNAbs were also shown to be weakly poly- or self-reactive such as antibodies to the CD4bs (45-46, 12A12, CH98 and CH103/CH104/CH106) and the glycan-associated V1/2 loops (CH01-CH04) [36,40,86,103]. Nevertheless, the majority of the potent natural HIV-1 bNAbs are not polyreactive and/or autoreactive [98,104]. Antibody polyreactivity could originate either from the preferential selection of mature naı¨ve B cells expressing polyreactive receptors (6% of naı¨ve B cells in healthy humans [69]), which would yield this reactivity unaltered throughout affinity maturation of anti-HIV B cells, or alternatively, from the extensive affinity maturation itself as a byproduct of the accumulated hypermutations, as shown for instance in the CH103 B cell lineage [103,105]. Hence, understanding both the mechanisms linking acquisition of bNAb activity with poly/autoreactivity, and whether polyreactivity is a prerequisite for antibody neutralization are important matters that must be addressed.


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N-glycans V1/V2 loops PG9/PG16, CH01-CH04, CAP256-VRC26 PGT141-PGT145

N156 CD4 binding site

N160 N-glycans V3 loops

b12, VRC01-VRC03/NIH45-46, VRC06b*, VRC23, HJ16, 1NC9, 8ANC134, 12A12, 3BNC60/3BNC117, VRC-PG04, VRC-CH30 - VRC-CH34, CH98, CH103-CH106

N332

PGT121-PGT123/ 10-1074, PGT125-PGT128, PGT135-PGT137, VRC24 CH01-CH04

N301

N276 N262

Unknown 3BNC176

N234

N448

Clustered N-glycans 2G12

N-glycans gp120/gp41 bridging region

N637

8ANC195, PGT151-PGT158

MPER 2F5, 4E10, Z13 m66.6, 10E8 TRENDS in Immunology

Figure 2. Sites of vulnerability on HIV-1 envelope glycoprotein. The HIV-1 envelope crystal structure [171] (PDB ID: 4NCO) was fit onto the cryoelectron microscopy reconstruction of an unliganded membrane-bound HIV-1 Env trimer ([172] (EMDB: 5019 and 5021). Env model that was used was fully glycosylated using predicted glycoforms (except for gp41, gp120 V2 and V4 loops, whose glycans were not modeled because of the disorder of those protein regions in the crystal structure or uncertainty in registry assignment). The figure was generated using UCSF Chimera [173]. N160-dependent V1/V2 loops (orange), N332-dependent V3 loop (purple), CD4 binding-site (pink) and glycans-dependent gp120/gp41 bridging region epitopes (blue and red) are highlighted as defined by PG9 [174,175], PGT128 [176], VRC01 [177], 8ANC195 [93] and PGT151 [91] binding. The MPER of gp41, for which only limited structural information is available in the context of the HIV-1 Env trimer, is also a site of vulnerability and is highlighted in yellow. bNAbs specifically mapped to each site of vulnerability are indicated in boxes. *VRC06b epitope is overlapping both the CD4bs and the CD4i. Abbreviations: bNAbs, broadly neutralizing antibodies; Env, surface envelope glycoprotein; MPER, membrane proximal external region.

Thus, HIV-1 bNAbs share common molecular features required for overcoming the various gp160-dependent mechanisms protecting the sites of vulnerability on the virion. Two main conserved signatures stand out: intensive somatic gene modifications (high rates of hypermutation and gene indels) and polyreactivity; both of which may constitute roadblocks for the immune system to generate bNAbs. Development of next generation HIV-1 bNAbs Genetic and biological host factors that determine or influence the generation of HIV-1 bNAbs are not fully understood, and are an area of active investigation. From an immunological standpoint, genetic predisposition to the natural control of HIV-1 (HIV-1 controller phenotype) is principally linked to certain HLA class I alleles predicted to enable efficient effector CD8+ T cell responses [106]. Whether specific genetic traits are associated with the development of bNAbs in Elite neutralizers remains an

open question. Conversely, it has been proposed that certain characteristics of the immune system could inherently limit the development of HIV-1 bNAbs explaining per se the rarity of such a phenomenon. At the gene level, the scarcity of some specific V(D)J rearrangements encoding germline precursors of HIV-1 bNAbs in the human immunoglobulin gene repertoire, such as those creating exceptionally long CDRH3s, could represent a restricting factor [107]. By contrast, the great number of DNA modifications: somatic hypermutations, and indels that accumulates in bNAbs-encoding genes through multiple rounds of affinity maturation is undoubtedly a major difficulty to overcome. While the affinity maturation process leading to the generation and selection of bNAb-expressing B cells is not well understood yet, chronic stimulation by extended antigen exposure appears to be mandatory, and this is rather associated with the progression of the disease than with its control (as in HIV-1 controllers) [26,108]. However, it is important to note that viral replication and evolution also 5


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HIV-1 bNAbs

(A)

Increased VL mutaons

(B)

Long CDRH3

CD4bs bNAbs

Inseron N58 R71

W50

Deleon

VH

VL

VH

VL

VL

VH 1-2 GL

Short CDRL3

Increased VH mutaons

VH TRENDS in Immunology

Figure 3. Immunoglobulin gene and structural features of bNAbs. Crystal structure of 10-1074 Fab [35] (PDB ID: 4FQC) is used to display some of the molecular features of HIV-1 bNAbs (A). Most bNAbs exhibit unusual numbers of somatic hypermutations in heavy and light chain-coding genes not only in the complementary determining region but also in the more mutation-resistant framework regions [95]. Hypermutation-induced amino acid substitutions (IgH, red spheres; IgL blue spheres), frequently accompanied with nucleotide insertions and/or deletions, allow adequate shaping of the antigen-binding site to fit complex neutralizing epitopes expressed on gp120 and gp41 glycoprotein subunits. On average, the CDRH3 length of IgG memory B cell antibodies from uninfected healthy individuals is about 14 amino acids. In order to access hidden portions of their epitopes, many bNAbs have extra-long CDRH3 loops (generally beyond 20 residues, and up to 35 amino acids for CAP256-VRC26), which appear to be the product of germline VDJ rearrangements. Crystal structure of 3BNC60 [36] (PDB ID: 3RPI) is used to display common molecular features of CD4bs bNAbs of the VRC01 class (B). Many CD4bs bNAbs, isolated from different donors, originate from the VH1-2 gene segment (VH1-2 GL). Despite extensive hypermutations, these antibodies share a large consensus IgH sequence, including three germline residues (blue spheres: W50, N58, and R71), therefore conserving structural features necessary for contacting the HIV spike. In addition, the CDRL3 length of CD4bs bNAbs is usually short (5 residues) to avoid steric clashes with gp120. The figure was generated using MacPyMOL (The PyMOL Molecular Graphics System 2011) [178]. Abbreviations: bNAbs, broadly neutralizing antibodies; VH, heavy chain variable domain; VL, light chain variable domain.

occurs in HIV-1 controllers [109], and that some of these individuals also fit the Elite neutralizer category [34]. In addition to the persistence of stimulating antigen, viral diversification within sites of vulnerability is a crucial event driving the evolution of B cells from the precursors originally recruited against transmitted-founder viruses to the affinity maturated clones expressing bNAbs. This was elegantly shown for anti-CD4bs antibody, CH103 [103], and more recently for anti-V1/V2 loops antibody clonal lineage, CAP256-VRC26, which was initiated by the interaction of its B cell precursor with a superinfecting virus [110]. Neutralizing antibodies and HIV-1 are thus coevolving in a recurring ordered manner; first, the virus evades antibody recognition by mutating the targeted epitope, and then, the immune system fights back by extending the maturation of the B cell lineage to give birth to new clonal antibody variants with increased neutralization breadth, which are able to neutralize the escape mutant viruses [68,103,110]. Importantly, this process could be more complex, potentially involving additional B cell lineages that would cooperatively intervene by driving the early steps of the viral evolution necessary to induce the bNAbs lineages, as recently described for CH103 antibody [111]. What favors the priming, maturation, and selection of B cell clones harboring bNAbs specificities in the context of the B cell competition in germinal centers (GCs) is not known, but it certainly implicates an effective access to antigen and to survival signals provided by Tfh cells [112]. Indeed, by interacting with GC B cells through specific surface receptors/ligands [such as MHC class II-peptideTCR, CD40–CD40L, inducible co-stimulatory molecule (ICOS)–ICOSL, and signaling lymphocytic activation molecule (SLAM) receptors], and by stimulating them with interleukin (IL)-21, Tfh cells play a crucial role in humoral responses by orchestrating B cell maturation and 6

differentiation in GCs [112]. Moreover, the fact that activated Tfh cells can move freely between different GCs and join ongoing GC reactions to provide help to B cells, may ensure an efficient humoral response against pathogens diversifying their antigenic epitopes such as HIV-1 [113]. Recent works truly provide compelling evidence supporting the role of Tfh cells in the maturation and selection of bNAb-expressing B cells [65,114]. In this regard, Crotty and colleagues identified in humans a population of circulating memory Tfh cells (with a CD4+PD1+CXCR3 CXCR5+ phenotype), robustly supplying help to B cells, and which positively correlates with the presence of bNAbs in elite neutralizers [115]. Finally, the recent study by Gitlin et al. shed light on how B cell clonal selection is regulated in the GC. The authors showed that high-affinity GC B cells are selected to proliferate the greatest number of times in the GC dark zone, with each round of cell division leading to greater diversification via somatic mutation [116]. Hence, this feed-forward loop may provide a mechanistic explanation for the preferential selection and maturation of high affinity HIV-1 bNAb-expressing B cell clones that exhibit high levels of hypermutation. Human HIV-1 bNAbs usually belong to highly expanded clonal lineages (or clonotypes), and represent the most terminally differentiated members, suggesting that their elicitation during the infection is a progressive and slow process [117]. Recent advances in the development of highthroughput DNA sequencing technologies now permit investigations on antibody gene repertoires (also called antibodyomes) at an unprecedented level [118]. Next-generation high-throughput sequencing of Ig genes (Ig-seq), along with computational and structural biology tools, constitutes a promising approach for a better understanding of the humoral response to HIV-1. For instance, studies using 454-pyrosequencing and structural biology methods


Feature Review have provided precious information about the affinity maturation pathways giving rise to VRC01-like bNAbs [97,119]. Similar Ig-seq approaches are now being used to reconstitute the ‘family trees’ of other bNAbs such as the PGTs (PGT121-PGT123 and PGT135-137) and anti-gp41 10E8 [120–122]. Importantly, by facilitating the mapping of HIV-1 bNAb clonotypes, the Ig-seq approaches may be a crucial step in establishing vaccine schemes that could instruct B cells to follow the tortuous but inescapable maturation pathways (a strategy called ‘B-cell lineage immunogen design’) [123]. The priming of suitable B cell progenitors and their clonal progeny is a pivotal event in this scenario. Hence, efforts are currently being made to rationally design HIV-1 envelope-derived immunogens that could efficiently activate bNAb B cell precursors, especially for attractive candidates such as VRC01-like antibodies that derive from germline-encoded B cell receptors (BCRs) with the same VH gene family (VH1) [124,125]. It is still unclear whether during infection, the envelope expressed by transmitted/founder viruses correspond to the stimulating antigens of bNAb B cell precursors [103], or if as suggested by some authors, B cells selected against unrelated antigens are subsequently crossreacting with envelope proteins leading to their recruitment in the immune response to HIV-1 [123]. Such a scenario may occur at mucosal sites where B cells primed by commensal bacteria could then crossreact with HIV-1 as a result of a molecular mimetism between microbial antigens and gp41 protein [126]. Polyreactivity is a frequent property of gp160-specific antibodies [98], and since most polyreactive B cells are removed from the B cell repertoire during development [69,127], it has been proposed that physiological tolerance might prevent the genesis of HIV-1 bNAbs [128,129]. However, so far this concept has only been validated for 2F5 and 4E10 [130–132], and it remains to be verified if such a rule could also apply to the other polyreactive bNAbs. On the contrary, it is essential to highlight that somatic hypermutations can create polyreactive BCRs, which are commonly expressed by several B cell populations in healthy humans such as IgG+ memory B lymphocytes [98,133]. Furthermore, although the magnitude and spectrum of polyreactivity for HIV-1 bNAbs still need to be accurately defined, only a minority of them appears to be as highly poly- and self-reactive as the archetypes 2F5 and 4E10, which when knocked in on mouse B cells in their affinity maturated form failed to pass central and peripheral tolerance checkpoints [104,130–132]. In this model, it has been suggested that the molecular-mimicry-based crossreactivity of 2F5 and 4E10 with autoantigens (kynureninase enzyme and RNA splice factor 3B, respectively) is responsible for the tolerization of 2F5- and 4E10-expressing B cells [134]. Interestingly, this developmental blockade could be bypassed using an appropriate immunogen/adjuvants combination, allowing the production of 2F5 antibodies through the activation/expansion of residual anergic 2F5-expressing B cells [135]. Are B cell precursors expressing germline BCRs of potent next-generation bNAbs also counterselected in such models? Can HIV-1 infection interfere with normal tolerance mechanisms allowing the selection of ordinarily ‘banned’ clones? More answers are required to


adjudicate on this particular matter. Of note, if immune tolerance mechanisms truly interfere with bNAb development, one can hypothesize that tolerance breakage occurring during certain autoimmune diseases may facilitate the acquisition of self-reactivity associated with bNAb activity. In support of this, a recent study has shown that an HIV-1-infected individual who subsequently developed a systemic lupus erythematous autoimmune disease could develop polyreactive anti-CD4bs bNAbs exhibiting crossreactivity to human autoantigens including double-stranded DNA [40]. Last but not least, polyreactive binding exhibited by HIV-1 antibodies could be functionally important to increase their avidity against virions displaying low-density spikes at their surfaces [96,136]. Therefore, moderate polyreactivity might be beneficial for HIV-1 antibodies to combat the infection without representing a substantial obstacle to the development of most HIV-1 bNAbs. Taken together, the data indicates that multiple host and viral obstacles hamper the generation of HIV-1 bNAbs, potentially accounting for the rarity of this event in infected humans. B cell deregulation and viral defense mechanisms are major impediments that weaken the humoral immune response to HIV-1. Difficulties for the natural development of bNAbs also lie in their unusual molecular characteristics, which are ‘forged’ for counteracting the protecting mechanisms used by HIV-1 to escape from antibody pressure. Without being counterselected due to potentially harmful polyreactivity levels, highly mutated anti-HIV-1 antibodies need to be positively selected during the immune response especially against escape variants, and this requires the pivotal help of Tfh cells, which are themselves profoundly altered by the infection. Considering that in the course of the infection, the virus is escaping and diversifying at a high rate, many successive B cell checkpoints must to be overcome in order for bNAbs to develop. Antiviral activity of broadly HIV-1 neutralizing antibodies and therapeutic potential The antiviral activity of HIV-1 antibodies is typically measured in vitro using cell-free pseudovirus particles and reporter cell lines, such as the HeLa-derived TzMbl cell [137]. In these assays, which measure the inhibition of free virus binding to cellular receptors and/or of viral fusion (Figure 4), first-generation bNAbs can achieve neutralization breadth but only several orders of magnitude less than the recently isolated bNAbs, which can neutralize up to 95% of HIV-1 strains at low concentration [14]. Over the course of the epidemic, circulating HIV-1 strains progressively became more resistant to neutralization by antigp120 but not anti-gp41 antibodies; nevertheless, contemporary HIV-1 variants are still sensitive to neutralization by most first- and next-generation anti-gp160 bNAbs [35,138–140]. HIV-1 antibodies can also access viruses present at the virological synapse [141], but only a subset of potent HIV-1 bNAbs are capable of inhibiting most of the steps implicated in the lymphocyte-to-lymphocyte spread of HIV-1 including the formation of virological synapses and the transfer of viral material from infected cells to the targets 7


Feature Review

Cell-free viral neutralizaon


Inhibion of cell-to -cell viral spread

Fc-dependent anviral acvity

Inhibion of viral transcytosis1 Intracellular viral neutralizaon2 1

2

Infected cell

CD4

CCR5

Infected cell

Target cell

Epithelial cells

Fc receptor Perforin, granzymes

Target cell

Eﬀector cell Plasma cell

TRENDS in Immunology

Figure 4. Antiviral activity of HIV-1 bNAbs. For decades, the major antiviral property considered for anti-gp160 antibodies was HIV neutralization in in vitro assays. In these systems, cell-free viral neutralization relies on the inhibition by antibodies of receptor/co-receptor attachment or viral fusion with the target cells. However, only a subset of potent HIV-1 bNAbs determined by this assay can efficiently block one of the most important modes of viral spread in vivo, which occurs through cell-to-cell transmission. Moreover, as already shown for some anti-gp160 antibodies, bNAbs may facilitate the clearance of infected cells by innate immune cells through Fc-dependent mechanisms such as antibody-dependent cell-mediated cytotoxicity. In this case, the antigen-binding site of the IgG molecule attaches cell-surface Env, and its Fc fragment is bound by Fc receptors expressed on effectors cells such as natural killer cells, which lyse the infected cells via the secretion of proteolytic enzymes, granzymes, and perforin. bNabs could also have a crucial role for the protection against HIV-1 infection at mucosal sites. Anti-HIV antibodies could inhibit the passage of HIV-1 from the lumen to the basolateral pole (HIV-1 transcytosis) (1) but also, neutralize virions inside epithelial cells (2) and in the lamina propria where they are produced by plasma cells. Abbreviations: bNAbs, broadly neutralizing antibodies; CCR5, chemokine CC receptor 5.

(Figure 4) [142]. These antibodies can also block viral transmission from infected lymphocytes to plasmacytoid dendritic cells, and compromise innate sensing of HIV-1 [142]. Similarly, viral transfer from infected immature dendritic cells to T cells can be blocked by HIV-1 bNAbs in an Fc-dependent manner, more efficiently by anti-gp120 than anti-gp41 antibodies, but not by non-neutralizing IgG [144]. Finally, anti-gp120 but not anti-gp41 bNAbs can inhibit the transfer of viruses from infected macrophages to T cells [143]. Overall, antibody specificity and effector function are key components to an efficient block of the HIV-1 cell-to-cell spread. Therefore, since viral spread from infected immune cells (T cells, dendritic cells, and macrophages) to uninfected CD4+ T cells is probably the dominant mode of HIV-1 propagation in vivo, next generation anti-gp120 bNAbs may represent the most desirable candidates for therapeutic interventions. Mucosal transmission of HIV-1 can be blocked in vitro and in humanized mice by HIV-1 bNAbs with modest neutralization capacity especially when expressed as monomeric or dimeric IgA [145–148] (Figure 4). Additionally, when first-generation bNAbs are injected as IgG in nonhuman primates challenged rectally or vaginally with SHIV, they protect against infection [149–154]. Whether bNAbs are effective in these in vivo models due to their ability to directly interfere with viral transcytosis and/or to decrease the infectivity of transcytosed viruses is not known. In this regard, the effect of HIV-1 IgG antibodies on HIV-1 transcytosis through epithelial cells in vitro is still disputed [155,156]. Thus, the inhibitory activity of next-generation bNAbs (as IgG and IgAs) against HIV-1 8

transcytosis remains to be evaluated. Finally, understanding the precise mechanisms allowing the transport of intravenously injected IgG antibodies to mucosal surfaces, and whether it implicates the neonatal Fc receptor (FcRn) [156,157], are key points to be addressed. Numerous studies have established that prophylactic passive transfer of first-generation bNAbs (2G12, b12, 2F5, and 4E10) to monkeys protects them against SHIV infection [49,149–151,154,158–162]. However, antibody immunoprophylaxis in humanized mice and in macaques using next-generation bNAbs has proven to be more efficient, suppressing viremia for a prolonged period of time and requiring much lower amounts of passively transferred antibody than for first-generation bNAbs [163–167]. For instance, passive administration of PGT121 as prophylactic treatment in rhesus macaques vaginally challenged with SHIV induced complete protection at serum concentrations at least two logs lower than the ones preconized for first-generation bNAbs [165]. More importantly, single bNAbs or a cocktail of two used as therapeutics in chronically infected macaques can suppress viral loads to undetectable levels, without the emergence of resistant viral strains [166,167]. Remarkably, antibody treatment also leads to a decrease in cell-associated HIV DNA, and improves HIV-specific T cell immunity [166,167], possibly suggesting a vaccine-like effect of bNAb immunotherapy [160]. It remains to be determined whether passive administration of HIV-1 bNAbs could purge latently infected cells undergoing reactivation. A part of the answer was revealed in a recent study by Halper-Stromberg et al. in which a combination of three potent bNAbs (3BNC117, PG16, and


Feature Review 10-1074; bNAbs tri-mix) could obstruct the establishment of the latent reservoir in humanized mice when administered early after infection [50]. For established infections, bNAbs in the presence of a combination of inducers of viral transcription was shown to interfere with the maintenance of the HIV-1 reservoir, as measured by viremia rebound following antibody decay [50]. Strikingly, bNAbs displaying efficient and long lasting therapeutic or prophylactic properties in infected humanized mice and non-human primates are those that effectively interfere with cell-tocell spread of HIV-1 in vitro [142,163]. Similarly, antibodies unable to inhibit intercellular viral spread failed to control viremia in these animal models. Therefore, the ability of potent bNAbs to block cell-contact-mediated viral transmission could possibly explain why these antibodies efficiently protect in vivo from infection. Finally, other mechanisms such as Fc-dependent effector function may play a critical role for in vivo protection as formerly shown for b12 [49,149], and more recently for a potent bNAbs trimix (3BNC117, PG16, and 10-1074) used in infected humanized mice [50]; this possibility must be further explored in the case of potent bNAbs. Antibody-mediated immunotherapy against HIV-1 was tested a decade ago in infected humans using first-generation bNAbs (2G12, 4E10, and 2F5). Antibodies passively administered alone or in combination before antiretroviral therapy (ART) cessation failed to control viral rebound due to the rapid emergence of HIV-1 escape variants [168,169]. Despite the discouraging outcome of early clinical trials, the use of potent next-generation human HIV-1 bNAbs offers new therapeutic opportunities. Indeed, considering the promising results obtained in infected animal models, after treatment with a single antibody or cocktail of antibody molecules with or without ART treatment, next generation bNAbs (i.e., anti-CD4bs VRC01/45-46 and 3BNC117, and anti-glycans/V3 loop PGT121 and 101074) should be evaluated in new clinical trials. The effectiveness of passive immunization with bNAbs could be tested in various clinical circumstances including mother-to-child HIV transmission [170] or in cohorts of seropositive patients not tolerating or resistant to ART [14]. Concluding remarks Humoral immune responses are fundamental to the host protection against pathogens, and rely on the extraordinary diversity of antibody molecules that ensure the recognition of a theoretically infinite number of foreign antigens. Since the beginning of the AIDS epidemic 30 years ago, intensive research efforts have been engaged to dissect the interplay between HIV-1 and neutralizing antibodies. However, only recently did the discovery and characterization at a molecular level of broadly neutralizing HIV-1 antibodies radically modify our conception of antiviral humoral immunity, bringing hopes for the elaboration of an effective vaccine as well as immunotherapeutic strategies. Groundbreaking findings were made possible by significant technical advances and importantly, by interdisciplinary and collaborative research enterprises. Studies have revealed unparalleled insights on the genetic diversity, structural features, and antiviral properties of HIV-1 bNAbs, and we are now better appreciating the


Box 1. Outstanding questions Are B cell defects commonly found during infection also characteristic of Elite neutralizers? Are tolerance checkpoints effective in HIV-1 infection, and do they prevent the development of polyreactive anti-HIV-1 antibodies? Does the polyreactivity of some bNAbs play a role in their neutralization activity? What are the stimulating antigens initiating HIV-1 B cell responses, especially B cell lineages of bNAbs? Does B cell competition in germinal centers interfere with the development of bNAbs clonal lineages? Can crossreactive neutralizing antibodies naturally be produced at mucosal sites, and can they protect against mucosal transmission? Can potent bNAbs isolated at systemic level efficiently protect mucosa from infection? What are the mechanisms involved in mucosal immunity in vivo; does it implicate antibody binding to neonatal Fc receptor (FcRn)? Can new-generation bNAbs exhibit in vitro and in vivo Fcdependent antiviral properties such as ADCC? Can new generation bNAbs affect the HIV-1 latent reservoirs? Can the immunotherapy-mediated protection achieved in monkeys be reproduced in humans? Can the ‘B cell lineage immunogen design’ vaccine strategy lead to bNAbs development in animal models and humans?

processes involved in B cell selection, maturation, and dynamics enabling their elicitation in rare individuals. Many immune evasion mechanisms used by the virus to ‘trick’ antibodies have been elucidated, and research on bNAbs has uncovered several functionally conserved neutralizing sites, identified as the Achilles heels on the virus. Evolutionary pathways of HIV-1-specific B cell clones chasing constantly mutating viral targets are starting to be elucidated, and might guide future vaccine-design strategies. Considering that these bNAbs molecules are extremely potent prophylactic and therapeutic reagents in non-human primates models, they also offer unique opportunities as potential treatment alternatives to antiviral drugs alone in humans, and their efficacy should be evaluated in clinical trials (Box 1). Acknowledgments We are grateful to Jean-Philippe Julien and Ian A. Wilson (The Scripps Research Institute) for providing us the structural model used in Figure 2, and to Caroline Eden (Icahn School of Medicine at Mount Sinai) for helpful comments and manuscript editing. This work was supported by the European Research Council (ERC) – Seventh Framework Program (ERC-2013-StG 337146). H.M. was supported by the G5 Institut Pasteur Program and the Milieu Inte´rieur Program (ANR-10LABX-69-01).

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13

Antibody responses during hepatitis B viral infection.

Antibody responses in HIV infection.

Antibody and B cell responses to Plasmodium sporozoites.

Commentary on: Antibody and B Cell Responses to Plasmodium Sporozoites.

Syk tyrosine kinase is critical for B cell antibody responses and memory B cell survival.

Impaired colonic B-cell responses by gastrointestinal Bacillus anthracis infection.

B cell responses to influenza infection and vaccination.

Antibody responses to Haemophilus influenzae type B vaccines in men with human immunodeficiency virus infection.

Virus-specific antibody secreting cell, memory B-cell, and sero-antibody responses in the human influenza challenge model.

macrophage infection by HIV1.

Single B-cell deconvolution of peanut-specific antibody responses in allergic patients.

Sca-1 Deficient Mice.

Targeted DNA vaccines eliciting crossreactive anti-idiotypic antibody responses against human B cell malignancies in mice.

Antibody-dependent CD56+ T cell responses are functionally impaired in long-term HIV-1 infection.

Identification of a potent synthetic HIV1 immunogen compromising gag-P24 tandem T- and B-cell epitopes.

Enhancing T Cell Immune Responses by B Cell-based Therapeutic Vaccine Against Chronic Virus Infection.

An Atypical Splenic B Cell Progenitor Population Supports Antibody Production during Plasmodium Infection in Mice.

Detection of core antibody in hepatitis B infection.

Immune function in aged mice. IV. Loss of T cell and B cell function in thymus-dependent antibody responses.

NKT Cell Responses to B Cell Lymphoma.

Targeting of Ly9 (CD229) Disrupts Marginal Zone and B1 B Cell Homeostasis and Antibody Responses.

Polyanhydride Nanovaccines Induce Germinal Center B Cell Formation and Sustained Serum Antibody Responses.

Anergy and suppression in B-cell responses.

B-cell responses in autoimmune diseases.