Fusion of the molecular adjuvant C3d to cleavage-independent native-like HIV-1 Env trimers improves the elicited antibody response.

An effective HIV vaccine likely requires the elicitation of neutralizing antibodies (NAbs) against multiple HIV-1 clades. The recently developed cleavage-independent native flexibly linked (NFL) envelope (Env) trimers exhibit well-ordered conformation and elicit autologous tier 2 NAbs in multiple animal models. Here, we investigated whether the fusion of molecular adjuvant C3d to the Env trimers can improve B- cell germinal center (GC) formation and antibody responses. To generate Env-C3d trimers, we performed a glycine-serine- based (G4S) flexible peptide linker screening and identified a linker range that allowed native folding. A 30-60- amino- acid- long linker facilitates Env-to-C3d association and achieves the secretion of well-ordered trimers and the structural integrity and functional integrity of Env and C3d. The fusion of C3d did not dramatically affect the antigenicity of the Env trimers and enhanced the ability of the Env trimers to engage and activate B cells in vitro. In mice, the fusion of C3d enhanced germinal center formation, the magnitude of Env-specific binding antibodies, and the avidity of the antibodies in the presence of an adjuvant. The Sigma Adjuvant System (SAS) did not affect the trimer integrity in vitro but contributed to altered immunogenicity in vivo, resulting in increased tier 1 neutralization, likely by increased exposure of variable region 3 (V3). Taken together, the results indicate that the fusion of the molecular adjuvant, C3d, to the Env trimers improves antibody responses and could be useful for Env-based vaccines against HIV.

HIV-1 gp120-CD4-Induced Antibody Complex Elicits CD4 Binding Site-Specific Antibody Response in Mice.

Elicitation of broadly neutralizing Ab (bNAb) responses toward the conserved HIV-1 envelope (Env) CD4 binding site (CD4bs) by vaccination is an important goal for vaccine development and yet to be achieved. The outcome of previous immunogenicity studies suggests that the limited accessibility of the CD4bs and the presence of predominant nonneutralizing determinants (nND) on Env may impede the elicitation of bNAbs and their precursors by vaccination. In this study, we designed a panel of novel immunogens that 1) preferentially expose the CD4bs by selective elimination of glycosylation sites flanking the CD4bs, and 2) minimize the nND immune response by engineering fusion proteins consisting of gp120 Core and one or two CD4-induced (CD4i) mAbs for masking nND epitopes, referred to as gp120-CD4i fusion proteins. As expected, the fusion proteins possess improved antigenicity with retained affinity for VRC01-class, CD4bs-directed bNAbs and dampened affinity for nonneutralizing Abs. We immunized C57BL/6 mice with these fusion proteins and found that overall the fusion proteins elicit more focused CD4bs Ab response than prototypical gp120 Core by serological analysis. Consistently, we found that mice immunized with selected gp120-CD4i fusion proteins have higher frequencies of germinal center-activated B cells and CD4bs-directed memory B cells than those inoculated with parental immunogens. We isolated three mAbs from mice immunized with selected gp120-CD4i fusion proteins and found that their footprints on Env are similar to VRC01-class bNAbs. Thus, using gp120-CD4i fusion proteins with selective glycan deletion as immunogens could focus Ab response toward CD4bs epitope.

Overcoming Steric Restrictions of VRC01 HIV-1 Neutralizing Antibodies through Immunization.

Broadly HIV-1 neutralizing VRC01 class antibodies target the CD4-binding site of Env. They are derived from VH1-2∗02 antibody heavy chains paired with rare light chains expressing 5-amino acid-long CDRL3s. They have been isolated from infected subjects but have not yet been elicited by immunization. Env-derived immunogens capable of binding the germline forms of VRC01 B cell receptors on naive B cells have been designed and evaluated in knockin mice. However, the elicited antibodies cannot bypass glycans present on the conserved position N276 of Env, which restricts access to the CD4-binding site. Efforts to guide the appropriate maturation of these antibodies by sequential immunization have not yet been successful. Here, we report on a two-step immunization scheme that leads to the maturation of VRC01-like antibodies capable of accommodating the N276 glycan and displaying autologous tier 2 neutralizing activities. Our results are relevant to clinical trials aiming to elicit VRC01 antibodies.

Vaccination with Glycan-Modified HIV NFL Envelope Trimer-Liposomes Elicits Broadly Neutralizing Antibodies to Multiple Sites of Vulnerability.

The elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are occluded by self-N-glycan shielding, limiting B cell recognition of the underlying polypeptide surface. The exceptions to the contiguous glycan shield include the conserved receptor CD4 binding site (CD4bs) and glycoprotein (gp)41 elements proximal to the furin cleavage site. Accordingly, we performed heterologous trimer-liposome prime:boosting in rabbits to drive B cells specific for cross-conserved sites. To preferentially expose the CD4bs to B cells, we eliminated proximal N-glycans while maintaining the native-like state of the cleavage-independent NFL trimers, followed by gradual N-glycan restoration coupled with heterologous boosting. This approach successfully elicited CD4bs-directed, cross-neutralizing Abs, including one targeting a unique glycan-protein epitope and a bNAb (87% breadth) directed to the gp120:gp41 interface, both resolved by high-resolution cryoelectron microscopy. This study provides proof-of-principle immunogenicity toward eliciting bNAbs by vaccination.

Sudan Ebolavirus VP35-NP Crystal Structure Reveals a Potential Target for Pan-Filovirus Treatment.

The filoviruses are etiological agents of life-threatening hemorrhagic fever with high mortality rate and risk of potential outbreak. Among members of this family, the Ebola (EBOV), Sudan (SUDV), and Marburg (MARV) viruses are considered the most pathogenic for humans. The ebolavirus nucleoprotein (NP) is the most abundant protein in infected cells and is essential for viral transcription and replication; thus, it represents an attractive target for therapeutic intervention. Here, we present the structure of SUDV NP in complex with the amino-terminal portion of the phosphoprotein VP35 at 2.3 Å. This structure captures VP35 chaperoning SUDV NP in a monomeric and RNA-free state. This transient state has been proposed to be key to maintaining a pool of monomeric and RNA-free NPs prior to NP-NP polymerization and encapsidation of the viral RNA genome. This structure also reveals a newly visualized interaction between NP and VP35, a well-defined beta sheet that is not present in previous structures. Affinity binding assays demonstrate that this beta sheet is essential for maintaining the high-affinity interaction between VP35 and a hydrophobic pocket on SUDV NP, and electron microscopy indicates the importance of this binding interaction to the oligomeric state and assembly of NP in human cells. Complementary structure-directed mutagenesis identifies critical residues conserved across the filovirus family that could be targeted by broadly effective antivirals.IMPORTANCE Outbreaks of the filoviruses can be unpredictable in timing, location, and identity of the causative virus, with each of Ebola virus, Sudan virus, Bundibugyo virus, and Marburg virus reemerging in the last several years to cause human disease with 30 to 90% lethality. The 2014-2016 outbreak in particular, with nearly 30,000 patients, highlighted the ability of these viruses to emerge unexpectedly and spread rapidly. Two ebolavirus outbreaks have emerged this year, yet we still lack FDA-approved drugs with pan-filovirus activity to treat existing and emergent ebolaviruses. For all filoviruses, the interaction between the nucleoprotein and the phosphoprotein is essential for the virus life cycle and is a potential target for therapeutic intervention. In this report, we describe the crystal structure of the SUDV nucleoprotein with the interacting domain of the viral phosphoprotein, and we identify residues critical for high-affinity interaction and for control of the oligomeric state of the nucleoprotein. Structural comparison of this heterodimer with other members of the filovirus family allowed us to find conserved and essential atomic features that will facilitate understanding of the virus life cycle and the rational design of antivirals.

Differential processing of HIV envelope glycans on the virus and soluble recombinant trimer.

As the sole target of broadly neutralizing antibodies (bnAbs) to HIV, the envelope glycoprotein (Env) trimer is the focus of vaccination strategies designed to elicit protective bnAbs in humans. Because HIV Env is densely glycosylated with 75-90 N-glycans per trimer, most bnAbs use or accommodate them in their binding epitope, making the glycosylation of recombinant Env a key aspect of HIV vaccine design. Upon analysis of three HIV strains, we here find that site-specific glycosylation of Env from infectious virus closely matches Envs from corresponding recombinant membrane-bound trimers. However, viral Envs differ significantly from recombinant soluble, cleaved (SOSIP) Env trimers, strongly impacting antigenicity. These results provide a benchmark for virus Env glycosylation needed for the design of soluble Env trimers as part of an overall HIV vaccine strategy.

Phosphoserine acidic cluster motifs bind distinct basic regions on the µ subunits of clathrin adaptor protein complexes.

Protein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the µ subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of furin binds the µ subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a potential PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the µ subunits in vitro The sites of these serines vary among mammals in a manner suggesting host-pathogen conflict, yet the Serinc3 PSAC seems dispensable for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu and furin and the PSAC-containing loop of Serinc3 each bind the µ subunit of AP-2 (µ2) with similar affinities, but they appear to utilize different basic regions on µ2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol 4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the µ surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.

Structure-Guided Redesign Improves NFL HIV Env Trimer Integrity and Identifies an Inter-Protomer Disulfide Permitting Post-Expression Cleavage.

Soluble HIV-1 envelope glycoprotein (Env) trimers are under active investigation as vaccine candidates in relevant pre-clinical models. Like SOSIPs, the cleavage-independent native flexibly linked (NFL) trimers are faithful mimics of the Env spike. Here, we analyzed multiple new designs to explore alternative modifications, informing tertiary interactions, while maintaining NFL trimer homogeneity and integrity. Accordingly, we performed a proline (P) substitution screen in the gp41 heptad repeat 1 region, identifying other trimer-enhancing Ps, including L555P. This P improved trimer integrity compared to I559P in selected properties. Next, we screened 15 structure-guided potential cysteine pairs in gp140 and found that A501C-L663C ("CC2") forms an inter-protomer disulfide bond that demonstrably increased NFL trimer thermostability. We combined these two approaches with trimer-derived substitutions, coupled with glycine substitutions at helix-to-coil transitions, developed by our group. To increase the exposure of the fusion peptide (FP) N-terminus, we engineered an enterokinase (EK) cleavage site upstream of the FP for controlled post-expression cleavage. In combination, the redesigns resulted in highly stable and homogeneous NFL mimics derived from different clades. Following recombinant EK cleavage, the NFL trimers retained covalent linkage, maintaining a native-like structure while displaying enhanced stability and favorable antigenic features. These trimers also displayed increased exposure of neutralizing epitopes in the FP and gp120/gp41 interface, while retaining other neutralizing epitopes and occluding non-neutralizing elements. This array of Env-structure-guided designs reveals additional interactive regions in the prefusion state of the HIV Env spike, affording the development of novel antigens and immunogens.

Ligand accessibility to the HIV-1 Env co-receptor binding site can occur prior to CD4 engagement and is independent of viral tier category.

HIV-1 virus entry into target cells requires the envelope glycoprotein (Env) to first bind the primary receptor, CD4 and subsequently the co-receptor. Antibody access to the co-receptor binding site (CoRbs) in the pre-receptor-engaged state, prior to cell attachment, remains poorly understood. Here, we have demonstrated that for tier-1 Envs, the CoRbs is directly accessible to full-length CD4-induced (CD4i) antibodies even before primary receptor engagement, indicating that on these Envs the CoRbs site is either preformed or can conformationally sample post-CD4-bound state. Tier-2 and tier-3 Envs, which are resistant to full-length CD4i antibody, are neutralized by m36.4, a lower molecular mass of CD4i-directed domain antibody. In some tier-2 and tier-3 Envs, CoRbs is accessible to m36.4 even prior to cellular attachment in an Env-specific manner independent of their tier category. These data suggest differential structural arrangements of CoRbs and varied masking of ligand access to the CoRbs in different Env isolates.

Rational design of a trispecific antibody targeting the HIV-1 Env with elevated anti-viral activity.

HIV-1 broadly neutralizing antibodies (bNAbs) are being explored as passively administered therapeutic and preventative agents. However, the extensively diversified HIV-1 envelope glycoproteins (Env) rapidly acquire mutations to evade individual bNAbs in monotherapy regimens. The use of a "single" agent to simultaneously target distinct Env epitopes is desirable to overcome viral diversity. Here, we report the use of tandem single-chain variable fragment (ScFv) domains of two bNAbs, specific for the CD4-binding site and V3 glycan patch, to form anti-HIV-1 bispecific ScFvs (Bi-ScFvs). The optimal Bi-ScFv crosslinks adjacent protomers within one HIV-1 Env spike and has greater neutralization breadth than its parental bNAbs. Furthermore, the combination of this Bi-ScFv with a third bNAb recognizing the Env membrane proximal external region (MPER) results in a trispecific bNAb, which has nearly pan-isolate neutralization breadth and high potency. Thus, multispecific antibodies combining functional moieties of bNAbs could achieve outstanding neutralization capacity with augmented avidity.

Glutaraldehyde Cross-linking of HIV-1 Env Trimers Skews the Antibody Subclass Response in Mice.

Well-ordered soluble HIV-1 envelope glycoprotein (Env) spike mimetics such as Native Flexibly Linked (NFL) trimers display high homogeneity, desired antigenicity, and high in vitro stability compared to previous generation soluble HIV-1 Env trimers. Glutaraldehyde (GLA) cross-linking was shown to further increase the thermostability of clade C 16055 NFL trimers and enhance the induction of tier 2 autologous neutralizing antibodies in guinea pigs. Here, we investigated if GLA fixation affected other aspects of the Env-specific immune response by performing a comparative immunogenicity study in C57BL/6 mice with non-fixed and GLA-fixed 16055 NFL trimers administered in AbISCO-100 adjuvant. We detected lower Env-specific binding antibody titers and increased skewing toward Th2 responses in mice immunized with GLA-fixed trimers compared to mice immunized with unfixed trimers, as shown by a higher Env-specific IgG1:IgG2b antibody subclass ratio. These results suggest that the presence of GLA adducts on Env influences the quality of the induced antibody response.

Targeted N-glycan deletion at the receptor-binding site retains HIV Env NFL trimer integrity and accelerates the elicited antibody response.

Extensive shielding by N-glycans on the surface of the HIV envelope glycoproteins (Env) restricts B cell recognition of conserved neutralizing determinants. Elicitation of broadly neutralizing antibodies (bNAbs) in selected HIV-infected individuals reveals that Abs capable of penetrating the glycan shield can be generated by the B cell repertoire. Accordingly, we sought to determine if targeted N-glycan deletion might alter antibody responses to Env. We focused on the conserved CD4 binding site (CD4bs) since this is a known neutralizing determinant that is devoid of glycosylation to allow CD4 receptor engagement, but is ringed by surrounding N-glycans. We selectively deleted potential N-glycan sites (PNGS) proximal to the CD4bs on well-ordered clade C 16055 native flexibly linked (NFL) trimers to potentially increase recognition by naïve B cells in vivo. We generated glycan-deleted trimer variants that maintained native-like conformation and stability. Using a panel of CD4bs-directed bNAbs, we demonstrated improved accessibility of the CD4bs on the N-glycan-deleted trimer variants. We showed that pseudoviruses lacking these Env PNGSs were more sensitive to neutralization by CD4bs-specific bNAbs but remained resistant to non-neutralizing mAbs. We performed rabbit immunogenicity experiments using two approaches comparing glycan-deleted to fully glycosylated NFL trimers. The first was to delete 4 PNGS sites and then boost with fully glycosylated Env; the second was to delete 4 sites and gradually re-introduce these N-glycans in subsequent boosts. We demonstrated that the 16055 PNGS-deleted trimers more rapidly elicited serum antibodies that more potently neutralized the CD4bs-proximal-PNGS-deleted viruses in a statistically significant manner and strongly trended towards increased neutralization of fully glycosylated autologous virus. This approach elicited serum IgG capable of cross-neutralizing selected tier 2 viruses lacking N-glycans at residue N276 (natural or engineered), indicating that PNGS deletion of well-ordered trimers is a promising strategy to prime B cell responses to this conserved neutralizing determinant.

HIV-1 Cross-Reactive Primary Virus Neutralizing Antibody Response Elicited by Immunization in Nonhuman Primates.

Elicitation of broadly neutralizing antibody (bNAb) responses is a major goal for the development of an HIV-1 vaccine. Current HIV-1 envelope glycoprotein (Env) vaccine candidates elicit predominantly tier 1 and/or autologous tier 2 virus neutralizing antibody (NAb) responses, as well as weak and/or sporadic cross-reactive tier 2 virus NAb responses with unknown specificity. To delineate the specificity of vaccine-elicited cross-reactive tier 2 virus NAb responses, we performed single memory B cell sorting from the peripheral blood of a rhesus macaque immunized with YU2gp140-F trimers in adjuvant, using JR-FL SOSIP.664, a native Env trimer mimetic, as a sorting probe to isolate monoclonal Abs (MAbs). We found striking genetic and functional convergence of the SOSIP-sorted Ig repertoire, with predominant VH4 or VH5 gene family usage and Env V3 specificity. Of these vaccine-elicited V3-specific MAbs, nearly 20% (6/33) displayed cross-reactive tier 2 virus neutralization, which recapitulated the serum neutralization capacity. Substantial similarities in binding specificity, neutralization breadth and potency, and sequence/structural homology were observed between selected macaque cross-reactive V3 NAbs elicited by vaccination and prototypic V3 NAbs derived from natural infections in humans, highlighting the convergence of this subset of primate V3-specific B cell repertories. Our study demonstrated that cross-reactive primary virus neutralizing B cell lineages could be elicited by vaccination as detected using a standardized panel of tier 2 viruses. Whether these lineages could be expanded to acquire increased breadth and potency of neutralization merits further investigation.IMPORTANCE Elicitation of antibody responses capable of neutralizing diverse HIV-1 primary virus isolates (designated broadly neutralizing antibodies [bNAbs]) remains a high priority for the vaccine field. bNAb responses were so far observed only in response to natural infection within a subset of individuals. To achieve this goal, an improved understanding of vaccine-elicited responses, including at the monoclonal Ab level, is essential. Here, we isolated and characterized a panel of vaccine-elicited cross-reactive neutralizing MAbs targeting the Env V3 loop that moderately neutralized several primary viruses and recapitulated the serum neutralizing antibody response. Striking similarities between the cross-reactive V3 NAbs elicited by vaccination in macaques and natural infections in humans illustrate commonalities between the vaccine- and infection-induced responses to V3 and support the feasibility of exploring the V3 epitope as a HIV-1 vaccine target in nonhuman primates.

Elicitation of Robust Tier 2 Neutralizing Antibody Responses in Nonhuman Primates by HIV Envelope Trimer Immunization Using Optimized Approaches.

The development of stabilized recombinant HIV envelope trimers that mimic the virion surface molecule has increased enthusiasm for a neutralizing antibody (nAb)-based HIV vaccine. However, there is limited experience with recombinant trimers as immunogens in nonhuman primates, which are typically used as a model for humans. Here, we tested multiple immunogens and immunization strategies head-to-head to determine their impact on the quantity, quality, and kinetics of autologous tier 2 nAb development. A bilateral, adjuvanted, subcutaneous immunization protocol induced reproducible tier 2 nAb responses after only two immunizations 8 weeks apart, and these were further enhanced by a third immunization with BG505 SOSIP trimer. We identified immunogens that minimized non-neutralizing V3 responses and demonstrated that continuous immunogen delivery could enhance nAb responses. nAb responses were strongly associated with germinal center reactions, as assessed by lymph node fine needle aspiration. This study provides a framework for preclinical and clinical vaccine studies targeting nAb elicitation.

Synthetic multivalent V3 glycopeptides display enhanced recognition by glycan-dependent HIV-1 broadly neutralizing antibodies.

We describe here the synthesis of novel multivalent HIV V3 domain glycopeptides and their binding to broadly neutralizing antibodies PGT128 and 10-1074. Our binding data reveal a distinct mode of antigen recognition by the two antibodies and further suggest that multivalent glycopeptides could mimic the neutralizing epitopes more efficiently than the monomeric glycopeptide.

Glycine Substitution at Helix-to-Coil Transitions Facilitates the Structural Determination of a Stabilized Subtype C HIV Envelope Glycoprotein.

Advances in HIV-1 envelope glycoprotein (Env) design generate native-like trimers and high-resolution clade A, B, and G structures and elicit neutralizing antibodies. However, a high-resolution clade C structure is critical, as this subtype accounts for the majority of HIV infections worldwide, but well-ordered clade C Env trimers are more challenging to produce due to their instability. Based on targeted glycine substitutions in the Env fusion machinery, we defined a general approach that disfavors helical transitions leading to post-fusion conformations, thereby favoring the pre-fusion state. We generated a stabilized, soluble clade C Env (16055 NFL) and determined its crystal structure at 3.9 Å. Its overall conformation is similar to SOSIP.664 and native Env trimers but includes a covalent linker between gp120 and gp41, an engineered 201-433 disulfide bond, and density corresponding to 22 N-glycans. Env-structure-guided design strategies resulted in multiple homogeneous cross-clade immunogens with the potential to advance HIV vaccine development.

Particulate Array of Well-Ordered HIV Clade C Env Trimers Elicits Neutralizing Antibodies that Display a Unique V2 Cap Approach.

The development of soluble envelope glycoprotein (Env) mimetics displaying ordered trimeric symmetry has ushered in a new era in HIV-1 vaccination. The recently reported native, flexibly linked (NFL) design allows the generation of native-like trimers from clinical isolates at high yields and homogeneity. As the majority of infections world-wide are of the clade C subtype, we examined responses in non-human primates to well-ordered subtype C 16055 trimers administered in soluble or high-density liposomal formats. We detected superior germinal center formation and enhanced autologous neutralizing antibodies against the neutralization-resistant (tier 2) 16055 virus following inoculation of liposome-arrayed trimers. Epitope mapping of the neutralizing monoclonal antibodies (mAbs) indicated major contacts with the V2 apex, and 3D electron microscopy reconstructions of Fab-trimer complexes revealed a horizontal binding angle to the Env spike. These vaccine-elicited mAbs target the V2 cap, demonstrating a means to accomplish tier 2 virus neutralization by penetrating the dense N-glycan shield.

Global site-specific N-glycosylation analysis of HIV envelope glycoprotein.

HIV-1 envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs) and the focus for design of an antibody-based HIV vaccine. The Env trimer is covered by ∼90N-linked glycans, which shield the underlying protein from immune surveillance. bNAbs to HIV develop during infection, with many showing dependence on glycans for binding to Env. The ability to routinely assess the glycan type at each glycosylation site may facilitate design of improved vaccine candidates. Here we present a general mass spectrometry-based proteomics strategy that uses specific endoglycosidases to introduce mass signatures that distinguish peptide glycosites that are unoccupied or occupied by high-mannose/hybrid or complex-type glycans. The method yields >95% sequence coverage for Env, provides semi-quantitative analysis of the glycosylation status at each glycosite. We find that most glycosites in recombinant Env trimers are fully occupied by glycans, varying in the proportion of high-mannose/hybrid and complex-type glycans.

Evolution of B cell analysis and Env trimer redesign.

HIV-1 and its surface envelope glycoproteins (Env), gp120 and gp41, have evolved immune evasion strategies that render the elicitation of effective antibody responses to the functional Env entry unit extremely difficult. HIV-1 establishes chronic infection and stimulates vigorous immune responses in the human host; forcing selection of viral variants that escape cellular and antibody (Ab)-mediated immune pressure, yet possess contemporary fitness. Successful survival of fit variants through the gauntlet of the human immune system make this virus and these glycoproteins a formidable challenge to target by vaccination, requiring a systematic approach to Env mimetic immunogen design and evaluation of elicited responses. Here, we review key aspects of HIV-1 Env immunogenicity and immunogen re-design, based on experimental data generated by us and others over the past decade or more. We further provide rationale and details regarding the use of newly evolving tools to analyze B cell responses, including approaches to use next generation sequencing for antibody lineage tracing and B cell fate mapping. Together, these developments offer opportunities to address long-standing questions about the establishment of effective B cell immunity elicited by vaccination, not just against HIV-1.

An HIV-1 Env-Antibody Complex Focuses Antibody Responses to Conserved Neutralizing Epitopes.

Elicitation of broadly neutralizing Ab (bNAb) responses to the conserved elements of the HIV-1 envelope glycoproteins (Env), including the primary receptor CD4 binding site (CD4bs), is a major focus of vaccine development yet to be accomplished. However, a large number of CD4bs-directed bNAbs have been isolated from HIV-1-infected individuals. Comparison of the routes of binding used by the CD4bs-directed bNAbs from patients and the vaccine-elicited CD4bs-directed mAbs indicates that the latter fail to neutralize primary virus isolates because they approach the Env spike with a vertical angle and contact the specific surface residues occluded in the native spike, including the bridging sheet on gp120. To preferentially expose the CD4bs and direct the immune response away from the bridging sheet, resulting in an altered angle of approach, we engineered an immunogen consisting of gp120 core in complex with the prototypic CD4-induced Ab, 17b. This mAb directly contacts the bridging sheet but not the CD4bs. The complex was further stabilized by chemical crosslinking to prevent dissociation. Rabbits immunized with the crosslinked complex displayed earlier affinity maturation, achieving tier 1 virus neutralization compared with animals immunized with gp120 core alone. Immunization with the crosslinked complex induced transient Ab responses with binding specificity similar to the CD4bs-directed bNAbs. mAbs derived from complex-immunized rabbits displayed footprints on gp120 more distal from the bridging sheet as compared with previous vaccine-elicited CD4bs Abs, indicating that Env-Ab complexes effectively dampen immune responses to undesired immunodominant bridging sheet determinants.