Many successful vaccines induce persistent antibody responses that can last a lifetime. The mechanisms by which they do so remain unclear, but emerging evidence suggests that they activate dendritic cells (DCs) via Toll-like receptors (TLRs)1,2. For example, the yellow fever vaccine YF-17D, one of the most successful empiric vaccines ever developed3, activates DCs via multiple TLRs to stimulate pro-inflammatory cytokines4,5. Triggering specific combinations of TLRs in DCs can induce synergistic production of cytokines6, which results in enhanced T cell responses, but its impact on antibody responses remain unknown. Learning the critical parameters of innate immunity that programs such antibody responses remains a major challenge in vaccinology. Here we demonstrate that immunization of mice with synthetic nanoparticles containing antigens plus Toll-like receptor (TLR) ligands 4 + 7 induces synergistic increases in antigen-specific, neutralizing antibodies compared to immunization with a single TLR ligand. Consistent with this there was enhanced persistence of germinal centers (GCs), 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 early programming towards B cell memory. Antibody responses were dependent on direct triggering of both TLRs on B cells and dendritic cells (DCs), 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.

Summary Induction of HIV-1 broad neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development but has remained challenging partially due to unusual traits of bnAbs, including high somatic hypermutation (SHM) frequencies and in-frame insertions and deletions (indels). Here we examined the propensity and functional requirement for indels within HIV-1 bnAbs. High-throughput sequencing of the immunoglobulin (Ig) VHDJH genes in HIV-1 infected and uninfected individuals revealed that the indel frequency was elevated among HIV-1-infected subjects, with no unique properties attributable to bnAb-producing individuals. This increased indel occurrence depended only on the frequency of SHM point-mutations. Indel-encoded regions were generally proximal to antigen binding sites. Additionally, reconstruction of a HIV-1 CD4-binding site bnAb clonal lineage revealed that a large compound VHDJH indel was required for bnAb activity. Thus, vaccine development should focus on designing regimens targeted at sustained activation of bnAb lineages to achieve the required SHM and indel events.

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.

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.The two broadly neutralizing human monoclonal antibodies (MAbs) 2F5 and 4E10 target conserved core amino acid residues that lie in the membrane proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 (6,9,18,25,29). Structural studies of 2F5 and 4E10 in complex with their nominal epitope peptides led to the proposition that the long hydrophobic heavy chain CDR3 (CDR H3) loop might be involved in binding to the virion membrane due to the lack of direct contact of the tip of the CDR H3 loop with their bound epitopes (6, 25). MAbs 2F5 and 4E10 indeed were found to have enhanced binding to gp41 MPER in the presence of membrane (12,25). Subsequent studies have revealed the lipid reactivity of both the 2F5 and 4E10 MAbs (2, 14, 23, 27), emphasizing the need to understand how MAbs 2F5 and 4E10 recognize their epitopes in the context of a membrane-gp41 MPER interface.It has been hypothesized that the ability of MAbs 2F5 and 4E10 to interact with membrane lipids is required for binding to the membrane-bound gp41 MPER region and subsequent HIV-1 neutralization (2, 14, 15). The binding of both the 2F5 and 4E10 MAbs to their epitope peptides presented on synthetic liposomes was remarkably different from tha...

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.

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 Man 5 GlcNAc 2 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 strainspecific 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.

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 Asn160 and Asn156. 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.

A class of high-affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time-dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]-Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 x 10(6) sites per cell. Approximately 9-14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo-uterine and epithelial-basal lamina interactions.