Christopher Sundling

Soluble HIV-1 Env trimers in adjuvant elicit potent and diverse functional B cell responses in primates

Broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoproteins (Envs) have proven difficult to elicit by immunization. Therefore, to identify effective Env neutralization targets, efforts are underway to define the specificities of bNAbs in chronically infected individuals. For a prophylactic vaccine, it is equally important to define the immunogenic properties of the heavily glycosylated Env in healthy primates devoid of confounding HIV-induced pathogenic factors. We used rhesus macaques to investigate the magnitude and kinetics of B cell responses stimulated by Env trimers in adjuvant. Robust Env-specific memory B cell responses and high titers of circulating antibodies developed after trimer inoculation. Subsequent immunizations resulted in significant expansion of Env-specific IgG-producing plasma cell populations and circulating Abs that displayed increasing avidity and neutralization capacity. The neutralizing activity elicited with the regimen used was, in most aspects, superior to that elicited by a regimen based on monomeric Env immunization in humans. Despite the potency and breadth of the trimer-elicited response, protection against heterologous rectal simian-HIV (SHIV) challenge was modest, illustrating the challenge of eliciting sufficient titers of cross-reactive protective NAbs in mucosal sites. These data provide important information for the design and evaluation of vaccines aimed at stimulating protective HIV-1 immune responses in humans.

Human B Cell Responses to TLR Ligands Are Differentially Modulated by Myeloid and Plasmacytoid Dendritic Cells

Selected TLR ligands are under evaluation as vaccine adjuvants and are known to activate dendritic cells (DCs) and B cells to affect vaccine-induced Ab responses. However, the relative contribution of the two main human DC subsets, myeloid (MDCs) and plasmacytoid (PDCs), in supporting B cell responses to TLR ligands remains poorly understood. We found that PDCs but not MDCs markedly enhanced B cell proliferation in response to TLR7/8-L, an imidazoquinoline derivative, and to a lesser extent to TLR9 ligands (CpG ODN classes A, B, and C). PDCs strongly enhanced TLR7/8-L-induced proliferation of both memory and naive B cells but were only able to support memory cells to differentiate to CD27high plasmablasts. In response to TLR7/8 stimulation, PDCs mediated the up-regulation of transcription factors B lymphocyte-induced maturation protein 1 and X-box binding protein 1 and enhanced differentiation of B cells into IgM-, IgG-, and IgA-producing cells. Type I IFN produced to high levels by PDCs was the principal mediator of the effects on TLR7/8 stimulation. Although MDCs expressed higher levels of the known B cell growth factors IL-6, IL-10, and B cell-activating factor in response to TLR7/8 stimulation, they were unable to enhance B cell responses in this system. These data help decipher the different roles of PDCs and MDCs for modulating human B cell responses and can contribute to selection of specific TLR ligands as vaccine adjuvants.

High-Resolution Definition of Vaccine-Elicited B Cell Responses Against the HIV Primary Receptor Binding Site

Vaccine-elicited mAbs bind the HIV-1 Env CD4bs differently from broadly neutralizing infection-induced mAbs. Neutralizing HIV A successful vaccine elicits broadly neutralizing antibodies that bind to viruses and prevent infection, but achieving this feat has been a challenge in HIV vaccine development. A prime target for these antibodies is the HIV envelope glycoprotein Env, which binds to CD4 expressed on T cells, resulting in infection. The CD4 binding site on Env is highly conserved, and broadly reactive neutralizing antibodies have been found that selectively bind to this site in HIV-infected individuals. However, when Env is used as an immunogen, the antibodies elicited have only limited neutralization ability. Sundling et al. use a macaque model of HIV infection to tease apart the differences between the infection- and the vaccine-induced antibodies to the CD4 binding site of Env. One limitation for HIV vaccine studies has been the lack of an animal model that mimics human disease. Sundling et al. found that the immunogenetics of the rhesus macaque immunoglobulin locus are similar to those of humans supporting the use of nonhuman primates for preclinical vaccine studies. The authors then immunized these macaques with Env and compared the elicited antibodies with known infection-induced antibodies targeting the CD4 binding site of HIV. They found that the vaccine-induced antibodies are different from broadly neutralizing infection-induced antibodies in the fine specificities targeted within the CD4 binding site as well as in the degree of somatic hypermutation. However, the vaccine-induced antibodies are not very different from non-broadly neutralizing, infection-induced antibodies. These results provide direction for researchers seeking to develop effective HIV vaccine candidates. The high overall genetic homology between humans and rhesus macaques, coupled with the phenotypic conservation of lymphocyte populations, highlights the potential use of nonhuman primates (NHPs) for the preclinical evaluation of vaccine candidates. For HIV-1, experimental models are needed to identify vaccine regimens capable of eliciting desired immune responses, such as broadly neutralizing antibodies (bNAbs). One important neutralization target on the HIV-1 envelope glycoproteins (Envs) is the conserved primary CD4 receptor binding site (CD4bs). The isolation and characterization of CD4bs-specific neutralizing monoclonal Abs (mAbs) from HIV-1–infected individuals have provided insights into how broadly reactive Abs target this conserved epitope. In contrast, and for reasons that are not understood, current Env immunogens elicit CD4bs-directed Abs with limited neutralization breadth. To facilitate the use of the NHP model to address this and other questions relevant to human humoral immunity, we defined features of the rhesus macaque immunoglobulin (Ig) loci and compared these to the human Ig loci. We then studied Env-immunized rhesus macaques, identified single B cells expressing CD4bs-specific Abs, and sequenced and expressed a panel of functional mAbs. Comparison of vaccine-elicited mAbs with HIV-1 infection–induced mAbs revealed differences in the degree of somatic hypermutation of the Abs as well as in the fine specificities targeted within the CD4bs. These data support the use of the preclinical NHP model to characterize vaccine-induced B cell responses at high resolution.

Vaccine-elicited primate antibodies use a distinct approach to the HIV-1 primary receptor binding site informing vaccine redesign

Significance The development of broadly neutralizing antibodies (bNAbs) to HIV-1 is often thought to be a key component of a successful vaccine. A common target of bNAbs is the conserved CD4 binding site (CD4bs) on the HIV envelope glycoprotein (Env) trimeric spike. Although CD4bs-directed bNAbs have been isolated from infected individuals, elicitation of such bNAbs by Env vaccination has proven difficult. To help understand the limitations of current immunogens, we structurally characterized two vaccine-elicited, CD4bs-directed non-bNAbs from primates. We demonstrate that these vaccine-elicited Abs attempt a vertical approach to the CD4bs, thereby clashing with the variable region of the trimeric spike cap, whereas CD4bs-directed bNAbs adopt angles of approach that avoid such clashes. This analysis can inform future vaccine redesign. HIV-1 neutralization requires Ab accessibility to the functional envelope glycoprotein (Env) spike. We recently reported the isolation of previously unidentified vaccine-elicited, CD4 binding site (CD4bs)-directed mAbs from rhesus macaques immunized with soluble Env trimers, indicating that this region is immunogenic in the context of subunit vaccination. To elucidate the interaction of the trimer-elicited mAbs with gp120 and their insufficient interaction with the HIV-1 primary isolate spike, we crystallized the Fab fragments of two mAbs, GE136 and GE148. Alanine scanning of their complementarity-determining regions, coupled with epitope scanning of their epitopes on gp120, revealed putative contact residues at the Ab/gp120 interface. Docking of the GE136 and GE148 Fabs to gp120, coupled with EM reconstructions of these nonbroadly neutralizing mAbs (non-bNAbs) binding to gp120 monomers and EM modeling to well-ordered trimers, suggested Ab approach to the CD4bs by a vertical angle of access relative to the more lateral mode of interaction used by the CD4bs-directed bNAbs VRC01 and PGV04. Fitting the structures into the available cryo-EM native spike density indicated clashes between these two vaccine-elicited mAbs and the topside variable region spike cap, whereas the bNAbs duck under this quaternary shield to access the CD4bs effectively on primary HIV isolates. These results provide a structural basis for the limited neutralizing breadth observed by current vaccine-induced, CD4bs-directed Abs and highlight the need for better ordered trimer immunogens. The analysis presented here therefore provides valuable information to guide HIV-1 vaccine immunogen redesign.

Human Immunodeficiency Virus Type 1 Env Trimer Immunization of Macaques and Impact of Priming with Viral Vector or Stabilized Core Protein

ABSTRACT Currently there is limited information about the quality of immune responses elicited by candidate human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-based immunogens in primates. Here we describe a comprehensive analysis of neutralizing antibody and T-cell responses obtained in cynomolgus macaques by three selected immunization regimens. We used the previously described YU2-based gp140 protein trimers administered in an adjuvant, preceded by two distinct priming strategies: either alphavirus replicon particles expressing matched gp140 trimers or gp120 core proteins stabilized in the CD4-bound conformation. The rationale for priming with replicon particles was to evaluate the impact of the expression platform on trimer immunogenicity. The stable core proteins were chosen in an attempt to expand selectively lymphocytes recognizing common determinants between the core and trimers to broaden the immune response. The results presented here demonstrate that the platform by which Env trimers were delivered in the priming (either protein or replicon vector) had little impact on the overall immune response. In contrast, priming with stable core proteins followed by a trimer boost strikingly focused the T-cell response on the core sequences of HIV-1 Env. The specificity of the T-cell response was distinctly different from that of the responses obtained in animals immunized with trimers alone and was shown to be mediated by CD4+ T cells. However, this regimen showed limited or no improvement in the neutralizing antibody responses, suggesting that further immunogen design efforts are required to successfully focus the B-cell response on conserved neutralizing determinants of HIV-1 Env.

Influence of Novel CD4 Binding-Defective HIV-1 Envelope Glycoprotein Immunogens on Neutralizing Antibody and T-Cell Responses in Nonhuman Primates

ABSTRACT The high-affinity in vivo interaction between soluble HIV-1 envelope glycoprotein (Env) immunogens and primate CD4 results in conformational changes that alter the immunogenicity of the gp120 subunit. Because the conserved binding site on gp120 that directly interacts with CD4 is a major vaccine target, we sought to better understand the impact of in vivo Env-CD4 interactions during vaccination. Rhesus macaques were immunized with soluble wild-type (WT) Env trimers, and two trimer immunogens rendered CD4 binding defective through distinct mechanisms. In one variant, we introduced a mutation that directly disrupts CD4 binding (368D/R). In the second variant, we introduced three mutations (423I/M, 425N/K, and 431G/E) that disrupt CD4 binding indirectly by altering a gp120 subdomain known as the bridging sheet, which is required for locking Env into a stable interaction with CD4. Following immunization, Env-specific binding antibody titers and frequencies of Env-specific memory B cells were comparable between the groups. However, the quality of neutralizing antibody responses induced by the variants was distinctly different. Antibodies against the coreceptor binding site were elicited by WT trimers but not the CD4 binding-defective trimers, while antibodies against the CD4 binding site were elicited by the WT and the 423I/M, 425N/K, and 431G/E trimers but not the 368D/R trimers. Furthermore, the CD4 binding-defective trimer variants stimulated less potent neutralizing antibody activity against neutralization-sensitive viruses than WT trimers. Overall, our studies do not reveal any potential negative effects imparted by the in vivo interaction between WT Env and primate CD4 on the generation of functional T cells and antibodies in response to soluble Env vaccination.

CTA1-DD adjuvant promotes strong immunity against human immunodeficiency virus type 1 envelope glycoproteins following mucosal immunization

Strategies to induce potent and broad antibody responses against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) at both systemic and mucosal sites represent a central goal for HIV-1 vaccine development. Here, we show that the non-toxic CTA1-DD adjuvant promoted mucosal and systemic humoral and cell-mediated immune responses following intranasal (i.n.) immunizations with trimeric or monomeric forms of HIV-1 Env in mice and in non-human primates. Env-specific IgG subclasses in the serum of immunized mice reflected a balanced Th1/Th2 type of response. Strikingly, i.n. immunizations with Env and the CTA1-DD adjuvant induced substantial levels of mucosal anti-Env IgA in bronchial alveolar lavage and also detectable levels in vaginal secretions. By contrast, parenteral immunizations of Env formulated in Ribi did not stimulate mucosal IgA responses, while the two adjuvants induced a similar distribution of Env-specific IgG-subclasses in serum. A single parenteral boost with Env in Ribi adjuvant into mice previously primed i.n. with Env and CTA1-DD, augmented the serum anti-Env IgG levels to similar magnitudes as those observed after three intraperitoneal immunizations with Env in Ribi. The augmenting potency of CTA1-DD was similar to that of LTK63 or CpG oligodeoxynucleotides (ODN). However, in contrast to CpG ODN, the effect of CTA1-DD and LTK63 appeared to be independent of MyD88 and toll-like receptor signalling. This is the first demonstration that CTA1-DD augments specific immune responses also in non-human primates, suggesting that this adjuvant could be explored further as a clinically safe mucosal vaccine adjuvant for humoral and cell-mediated immunity against HIV-1 Env.

Human and rhesus plasmacytoid dendritic cell and B-cell responses to Toll-like receptor stimulation

Interferon‐α (IFN‐α) produced at high levels by human plasmacytoid dendritic cells (pDCs) can specifically regulate B‐cell activation to Toll‐like receptor (TLR) 7/8 stimulation. To explore the influence of IFN‐α and pDCs on B‐cell functions in vivo, studies in non‐human primates that closely resemble humans in terms of TLR expression on different subsets of immune cells are valuable. Here, we performed a side‐by side comparison of the response pattern between human and rhesus macaque B cells and pDCs in vitro to well‐defined TLR ligands and tested whether IFN‐α enhanced B‐cell function comparably. We found that both human and rhesus B cells proliferated while pDCs from both species produced high levels of IFN‐α in response to ligands targeting TLR7/8 and TLR9. Both human and rhesus B‐cell proliferation to TLR7/8 ligand and CpG class C was significantly increased in the presence of IFN‐α. Although both human and rhesus B cells produced IgM upon stimulation, only human B cells acquired high expression of CD27 associated with plasmablast formation. Instead, rhesus B‐cell differentiation and IgM levels correlated to down‐regulation of CD20. These data suggest that the response pattern of human and rhesus B cells and pDCs to TLR7/8 and TLR9 is similar, although some differences in the cell surface phenotype of the differentiating cells exist. A more thorough understanding of potential similarities and differences between human and rhesus cells and their response to potential vaccine components will provide important information for translating non‐human primate studies into human trials.

Isolation of antibody V(D)J sequences from single cell sorted rhesus macaque B cells

Studies in nonhuman primates offer information of high relevance to clinical medicine due to their close genetic relationship with humans. Here, we established an optimized protocol for the isolation of antibody V(D)J sequences from rhesus macaque B cells. Nested PCR primers were designed to align to sequences flanking the V(D)J coding region to enable amplification of highly mutated antibody sequences. The primers were evaluated using cDNA from bulk PBMCs as well as from single-sorted memory and naïve B cells from several macaques to ascertain effective germline coverage. The nested PCR efficiency reached 60.6% positive wells for heavy chain amplification, 39.2% for kappa chain, and 23.7% for lambda chain sequences. Matching heavy and light chain sequences, indicating antibodies that potentially can be cloned, were obtained in 50% of the positive wells. Using these primers, we found that the efficiency and specificity of V(D)J amplifications were markedly improved compared to when primers designed for human Ab isolation were used. In particular, the amplification of recombined light chain VJ sequences was improved. Thus, we describe the design and testing of a new set of rhesus-specific primers that enable efficient and specific amplification of heavy, kappa and lambda V(D)J genes from single sorted B cells. The use of these primers will facilitate future efforts to clone and express rhesus macaque MAbs for genetic, functional and structural analyses.

Single-Cell and Deep Sequencing of IgG-Switched Macaque B Cells Reveal a Diverse Ig Repertoire following Immunization

The nonhuman primate model is important for preclinical evaluation of prophylactic and therapeutic intervention strategies. The recent description of the rhesus macaque germline Ig loci and establishment of a database of germline gene segments offer improved opportunities to delineate Ig gene usage in the overall B cell repertoire as well as in response to vaccination. We applied 454-pyrosequencing and single-cell RT-PCR of bulk and sorted memory B cells, respectively, to investigate IGHV gene segment expression in rhesus macaques. The two methods gave remarkably concordant results and identified groups of gene segments that are frequently or rarely used. We further examined the VH repertoire of Ag-specific memory B cells induced by immunization with recombinant HIV-1 envelope glycoproteins, an important vaccine component. We demonstrate that HIV-1 envelope glycoprotein immunization activates a highly polyclonal response composed of most of the expressed VH gene segments, illustrating the considerable genetic diversity of responding B cells following vaccination.