Shelley Stewart

Programming the magnitude and persistence of antibody responses with innate immunity

Many successful vaccines induce persistent antibody responses that can last a lifetime. The mechanisms by which they do so remain unclear, but emerging evidence indicates that they activate dendritic cells via Toll-like receptors (TLRs). For example, the yellow fever vaccine YF-17D, one of the most successful empiric vaccines ever developed, activates dendritic cells via multiple TLRs to stimulate proinflammatory cytokines. Triggering specific combinations of TLRs in dendritic cells can induce synergistic production of cytokines, which results in enhanced T-cell responses, but its impact on antibody responses remain unknown. Learning the critical parameters of innate immunity that program such antibody responses remains a major challenge in vaccinology. Here we demonstrate that immunization of mice with synthetic nanoparticles containing antigens plus ligands that signal through TLR4 and TLR7 induces synergistic increases in antigen-specific, neutralizing antibodies compared to immunization with nanoparticles containing antigens plus a single TLR ligand. Consistent with this there was enhanced persistence of germinal centres and of plasma-cell responses, which persisted in the lymph nodes for >1.5 years. Surprisingly, there was no enhancement of the early short-lived plasma-cell response relative to that observed with single TLR ligands. Molecular profiling of activated B cells, isolated 7 days after immunization, indicated that there was early programming towards B-cell memory. Antibody responses were dependent on direct triggering of both TLRs on B cells and dendritic cells, as well as on T-cell help. Immunization protected completely against lethal avian and swine influenza virus strains in mice, and induced robust immunity against pandemic H1N1 influenza in rhesus macaques.

Antigenicity and Immunogenicity of RV144 Vaccine AIDSVAX Clade E Envelope Immunogen Is Enhanced by a gp120 N-Terminal Deletion

ABSTRACT An immune correlates analysis of the RV144 HIV-1 vaccine trial revealed that antibody responses to the gp120 V1/V2 region correlated inversely with infection risk. The RV144 protein immunogens (A244-rp120 and MN-rgp120) were modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of a herpes simplex virus (HSV) gD protein-derived tag (gD). We investigated the effects of these modifications on gp120 expression, antigenicity, and immunogenicity by comparing unmodified A244 gp120 with both Δ11 deletion and gD tag and with Δ11 only. Analysis of A244 gp120, with or without Δ11 or gD, demonstrated that the Δ11 deletion, without the addition of gD, was sufficient for enhanced antigenicity to gp120 C1 region, conformational V2, and V1/V2 gp120 conformational epitopes. RV144 vaccinee serum IgGs bound more avidly to A244 gp120 Δ11 than to the unmodified gp120, and their binding was blocked by C1, V2, and V1/V2 antibodies. Rhesus macaques immunized with the three different forms of A244 gp120 proteins gave similar levels of gp120 antibody titers, although higher antibody titers developed earlier in A244 Δ11 gp120-immunized animals. Conformational V1/V2 monoclonal antibodies (MAbs) gave significantly higher levels of blocking of plasma IgG from A244 Δ11 gp120-immunized animals than IgG from animals immunized with unmodified A244 gp120, thus indicating a qualitative difference in the V1/V2 antibodies induced by A244 Δ11 gp120. These results demonstrate that deletion of N-terminal residues in the RV144 A244 gp120 immunogen improves both envelope antigenicity and immunogenicity.

Stable Docking of Neutralizing Human Immunodeficiency Virus Type 1 gp41 Membrane-Proximal External Region Monoclonal Antibodies 2F5 and 4E10 Is Dependent on the Membrane Immersion Depth of Their Epitope Regions

ABSTRACT The binding of neutralizing antibodies 2F5 and 4E10 to human immunodeficiency virus type 1 (HIV-1) gp41 involves both the viral membrane and gp41 membrane proximal external region (MPER) epitopes. In this study, we have used several biophysical tools to examine the secondary structure, orientation, and depth of immersion of gp41 MPER peptides in liposomes and to determine how the orientation of the MPER with lipids affects the binding kinetics of monoclonal antibodies (MAbs) 2F5 and 4E10. The binding of 2F5 and 4E10 both to their respective nominal epitopes and to a biepitope (includes 2F5 and 4E10 epitopes) MPER peptide-liposome conjugate was best described by a two-step encounter-docking model. Analysis of the binding kinetics and the effect of temperature on the binding stability of 2F5 and 4E10 to MPER peptide-liposome conjugates revealed that the docking of 4E10 was relatively slower and thermodynamically less favorable. The results of fluorescence-quenching and fluorescence resonance energy transfer experiments showed that the 2F5 epitope was more solvent exposed, whereas the 4E10 epitope was immersed in the polar-apolar interfacial region of the lipid bilayer. A circular dichroism spectroscopic study demonstrated that the nominal epitope and biepitope MPER peptides adopted ordered structures with differing helical contents when anchored to liposomes. Furthermore, anchoring of MPER peptides to the membrane via a hydrophobic anchor sequence was required for efficient MAb docking. These results support the model that the ability of 2F5 and 4E10 to bind to membrane lipid is required for stable docking to membrane-embedded MPER residues. These data have important implications for the design and use of peptide-liposome conjugates as immunogens for the induction of MPER-neutralizing antibodies.

Recognition of synthetic glycopeptides by HIV-1 broadly neutralizing antibodies and their unmutated ancestors

Significance A current key goal of HIV-1 vaccine development is to learn how to induce antibodies that will neutralize many diverse HIV-1 strains. Current HIV-1 vaccines elicit strain-specific neutralizing antibodies, whereas broadly neutralizing antibodies (BnAbs) are not induced and only arise in select HIV-1 chronically infected individuals. One strategy for induction of favored antibody responses is to design and produce homogeneous immunogens with selective expression of BnAb but not dominant epitopes. In this study, we describe the binding properties of chemically synthesized variable loop 1/2 (V1V2) glycopeptides that bind both to mature HIV-1 envelope BnAbs and the receptors of their naïve B cells. These results demonstrate that such synthetic glycopeptides can be immunogens that selectively target BnAb naïve B cells. Current HIV-1 vaccines elicit strain-specific neutralizing antibodies. Broadly neutralizing antibodies (BnAbs) are not induced by current vaccines, but are found in plasma in ∼20% of HIV-1–infected individuals after several years of infection. One strategy for induction of unfavored antibody responses is to produce homogeneous immunogens that selectively express BnAb epitopes but minimally express dominant strain-specific epitopes. Here we report that synthetic, homogeneously glycosylated peptides that bind avidly to variable loop 1/2 (V1V2) BnAbs PG9 and CH01 bind minimally to strain-specific neutralizing V2 antibodies that are targeted to the same envelope polypeptide site. Both oligomannose derivatization and conformational stabilization by disulfide-linked dimer formation of synthetic V1V2 peptides were required for strong binding of V1V2 BnAbs. An HIV-1 vaccine should target BnAb unmutated common ancestor (UCA) B-cell receptors of naïve B cells, but to date no HIV-1 envelope constructs have been found that bind to the UCA of V1V2 BnAb PG9. We demonstrate herein that V1V2 glycopeptide dimers bearing Man5GlcNAc2 glycan units bind with apparent nanomolar affinities to UCAs of V1V2 BnAbs PG9 and CH01 and with micromolar affinity to the UCA of a V2 strain-specific antibody. The higher-affinity binding of these V1V2 glycopeptides to BnAbs and their UCAs renders these glycopeptide constructs particularly attractive immunogens for targeting subdominant HIV-1 envelope V1V2-neutralizing antibody-producing B cells.

Induction of antibodies in rhesus macaques that recognize a fusion-intermediate conformation of HIV-1 gp41.

A component to the problem of inducing broad neutralizing HIV-1 gp41 membrane proximal external region (MPER) antibodies is the need to focus the antibody response to the transiently exposed MPER pre-hairpin intermediate neutralization epitope. Here we describe a HIV-1 envelope (Env) gp140 oligomer prime followed by MPER peptide-liposomes boost strategy for eliciting serum antibody responses in rhesus macaques that bind to a gp41 fusion intermediate protein. This Env-liposome immunization strategy induced antibodies to the 2F5 neutralizing epitope 664DKW residues, and these antibodies preferentially bound to a gp41 fusion intermediate construct as well as to MPER scaffolds stabilized in the 2F5-bound conformation. However, no serum lipid binding activity was observed nor was serum neutralizing activity for HIV-1 pseudoviruses present. Nonetheless, the Env-liposome prime-boost immunization strategy induced antibodies that recognized a gp41 fusion intermediate protein and was successful in focusing the antibody response to the desired epitope.

Chemical synthesis of highly congested gp120 V1V2 N-glycopeptide antigens for potential HIV-1-directed vaccines.

Critical to the search for an effective HIV-1 vaccine is the development of immunogens capable of inducing broadly neutralizing antibodies (BnAbs). A key first step in this process is to design immunogens that can be recognized by known BnAbs. The monoclonal antibody PG9 is a BnAb that neutralizes diverse strains of HIV-1 by targeting a conserved carbohydrate-protein epitope in the variable 1 and 2 (V1V2) region of the viral envelope. Important for recognition are two closely spaced N-glycans at Asn(160) and Asn(156). Glycopeptides containing this synthetically challenging bis-N-glycosylated motif were prepared by convergent assembly, and were shown to be antigenic for PG9. Synthetic glycopeptides such as these may be useful for the development of HIV-1 vaccines based on the envelope V1V2 BnAb epitope.

Isolation of HIV-1-neutralizing mucosal monoclonal antibodies from human colostrum.

Background Generation of potent anti-HIV antibody responses in mucosal compartments is a potential requirement of a transmission-blocking HIV vaccine. HIV-specific, functional antibody responses are present in breast milk, and these mucosal antibody responses may play a role in protection of the majority of HIV-exposed, breastfeeding infants. Therefore, characterization of HIV-specific antibodies produced by B cells in milk could guide the development of vaccines that elicit protective mucosal antibody responses. Methods We isolated B cells from colostrum of an HIV-infected lactating woman with a detectable neutralization response in milk and recombinantly produced and characterized the resulting HIV-1 Envelope (Env)-specific monoclonal antibodies (mAbs). Results The identified HIV-1 Env-specific colostrum mAbs, CH07 and CH08, represent two of the first mucosally-derived anti-HIV antibodies yet to be reported. Colostrum mAb CH07 is a highly-autoreactive, weakly-neutralizing gp140-specific mAb that binds to linear epitopes in the gp120 C5 region and gp41 fusion domain. In contrast, colostrum mAb CH08 is a nonpolyreactive CD4-inducible (CD4i) gp120-specific mAb with moderate breadth of neutralization. Conclusions These novel HIV-neutralizing mAbs isolated from a mucosal compartment provide insight into the ability of mucosal B cell populations to produce functional anti-HIV antibodies that may contribute to protection against virus acquisition at mucosal surfaces.

Surface plasmon resonance measurements of plasma antibody avidity during primary and secondary responses to anthrax protective antigen

Establishment of humoral immunity against pathogens is dependent on events that occur in the germinal center and the subsequent induction of high-affinity neutralizing antibodies. Quantitative assays that allow monitoring of affinity maturation and duration of antibody responses can provide useful information regarding the efficacy of vaccines and adjuvants. Using an anthrax protective antigen (rPA) and alum model antigen/adjuvant system, we describe a methodology for monitoring antigen-specific serum antibody concentration and avidity by surface plasmon resonance during primary and secondary immune responses. Our analyses showed that following a priming dose in mice, rPA-specific antibody concentration and avidity increases over time and reaches a maximal response in about six weeks, but gradually declines in the absence of antigenic boost. Germinal center reactions were observed early with maximal development achieved during the primary response, which coincided with peak antibody avidity responses to primary immunization. Boosting with antigen resulted in a rapid increase in rPA-specific antibody concentration and five-fold increase in avidity, which was not dependent on sustained GC development. The described methodology couples surface plasmon resonance-based plasma avidity measurements with germinal center analysis and provides a novel way to monitor humoral responses that can play a role in facilitating vaccine and adjuvant development.

Modulation of Nonneutralizing HIV-1 gp41 Responses by an MHC-Restricted TH Epitope Overlapping Those of Membrane Proximal External Region Broadly Neutralizing Antibodies

A goal of HIV-1 vaccine development is to elicit broadly neutralizing Abs (BnAbs), but current immunization strategies fail to induce BnAbs, and for unknown reasons, often induce nonneutralizing Abs instead. To explore potential host genetic contributions controlling Ab responses to the HIV-1 Envelope, we have used congenic strains to identify a critical role for MHC class II restriction in modulating Ab responses to the membrane proximal external region (MPER) of gp41, a key vaccine target. Immunized H-2d–congenic strains had more rapid, sustained, and elevated MPER+ Ab titers than those bearing other haplotypes, regardless of immunogen, adjuvant, or prime or boost regimen used, including formulations designed to provide T cell help. H-2d–restricted MPER+ serum Ab responses depended on CD4 TH interactions with class II (as revealed in immunized intra–H-2d/b congenic or CD154−/− H-2d strains, and by selective abrogation of MPER restimulated, H-2d–restricted primed splenocytes by class II–blocking Abs), and failed to neutralize HIV-1 in the TZM-b/l neutralization assay, coinciding with lack of specificity for an aspartate residue in the neutralization core of BnAb 2F5. Unexpectedly, H-2d–restricted MPER+ responses functionally mapped to a core TH epitope partially overlapping the 2F5/z13/4E10 BnAb epitopes as well as nonneutralizing B cell–Ab binding residues. We propose that class II restriction contributes to the general heterogeneity of nonneutralizing gp41 responses induced by Envelope. Moreover, the proximity of TH and B cell epitopes in this restriction may have to be considered in redesigning minimal MPER immunogens aimed at exclusively binding BnAb epitopes and triggering MPER+ BnAbs.

Vaccine-Induced HIV-1 Envelope gp120 Constant Region 1-Specific Antibodies Expose a CD4-Inducible Epitope and Block the Interaction of HIV-1 gp140 with Galactosylceramide

ABSTRACT Mucosal epithelial cell surface galactosylceramide (Galcer) has been postulated to be a receptor for HIV-1 envelope (Env) interactions with mucosal epithelial cells. Disruption of the HIV-1 Env interaction with such alternate receptors could be one strategy to prevent HIV-1 entry through the mucosal barrier. To study antibody modulation of HIV-1 Env-Galcer interactions, we used Galcer-containing liposomes to assess whether natural- and vaccine-induced monoclonal antibodies can block HIV-1 Env binding to Galcer. HIV-1 Env gp140 proteins bound to Galcer liposomes with Kd s (dissociation constants) in the nanomolar range. Several HIV-1 ALVAC/AIDSVAX vaccinee-derived monoclonal antibodies (MAbs) specific for the gp120 first constant (C1) region blocked Galcer binding of a transmitted/founder HIV-1 Env gp140. Among the C1-specific MAbs that showed Galcer blocking, the antibody-dependent cellular cytotoxicity-mediating CH38 IgG and its natural IgA isotype were the most potent blocking antibodies. C1-specific IgG monoclonal antibodies that blocked Env binding to Galcer induced upregulation of the gp120 CD4-inducible (CD4i) epitope bound by MAb 17B, demonstrating that a conformational change in gp120 may be required for Galcer blocking. However, the MAb 17B itself did not block Env-Galcer binding, suggesting that the C1 antibody-induced gp120 conformational changes resulted in alteration in a Galcer binding site distant from the CD4i 17B MAb binding site. IMPORTANCE Galactosyl ceramide, a glycosphingolipid, has been postulated to be a receptor for the HIV-1 envelope glycoprotein (Env) interaction with mucosal epithelial cells. Here, we have mimicked this interaction by using an artificial membrane containing synthetic Galcer and recombinant HIV-1 Env proteins to identify antibodies that would block the HIV-1 Env-Galcer interaction. Our study revealed that a class of vaccine-induced human antibodies potently blocks HIV-1 Env-Galcer binding by perturbing the HIV-1 Env conformation.