Native-like Trimers Research Articles

Enhancing glycan occupancy of soluble HIV-1 envelope trimers to mimic the native viral spike

SUMMARYArtificial glycan holes on recombinant Env-based vaccines occur when a potential N-linked glycosylation site (PNGS) is under-occupied, but not on their viral counterparts. Native-like SOSIP trimers, including clinical candidates, contain such holes in the glycan shield that induce strain-specific neutralizing antibodies (NAbs) or non-NAbs. To eliminate glycan holes and mimic the glycosylation of native BG505 Env, we replace all 12 NxS sequons on BG505 SOSIP with NxT. All PNGS, except N133 and N160, are nearly fully occupied. Occupancy of the N133 site is increased by changing N133 to NxS, whereas occupancy of the N160 site is restored by reverting the nearby N156 sequon to NxS. Hence, PNGS in close proximity, such as in the N133-N137 and N156-N160 pairs, affect each other’s occupancy. We further apply this approach to improve the occupancy of several Env strains. Increasing glycan occupancy should reduce off-target immune responses to vaccine antigens.

Cryo-EM structures of HIV-1 trimer bound to CD4-mimetics BNM-III-170 and M48U1 adopt a CD4-bound open conformation

Human immunodeficiency virus-1 (HIV-1), the causative agent of AIDS, impacts millions of people. Entry into target cells is mediated by the HIV-1 envelope (Env) glycoprotein interacting with host receptor CD4, which triggers conformational changes allowing binding to a coreceptor and subsequent membrane fusion. Small molecule or peptide CD4-mimetic drugs mimic CD4’s Phe43 interaction with Env by inserting into the conserved Phe43 pocket on Env subunit gp120. Here, we present single-particle cryo-EM structures of CD4-mimetics BNM-III-170 and M48U1 bound to a BG505 native-like Env trimer plus the CD4-induced antibody 17b at 3.7 Å and 3.9 Å resolution, respectively. CD4-mimetic-bound BG505 exhibits canonical CD4-induced conformational changes including trimer opening, formation of the 4-stranded gp120 bridging sheet, displacement of the V1V2 loop, and formation of a compact and elongated gp41 HR1C helical bundle. We conclude that CD4-induced structural changes on both gp120 and gp41 Env subunits are induced by binding to the gp120 Phe43 pocket.

A broadly neutralizing macaque monoclonal antibody against the HIV-1 V3-Glycan patch.

A small fraction of HIV-1- infected humans develop broadly neutralizing antibodies (bNAbs) against HIV-1 that protect macaques from simian immunodeficiency HIV chimeric virus (SHIV). Similarly, a small number of macaques infected with SHIVs develop broadly neutralizing serologic activity, but less is known about the nature of simian antibodies. Here, we report on a monoclonal antibody, Ab1485, isolated from a macaque infected with SHIVAD8 that developed broadly neutralizing serologic activity targeting the V3-glycan region of HIV-1 Env. Ab1485 neutralizes 38.1% of HIV-1 isolates in a 42-pseudovirus panel with a geometric mean IC50 of 0.055 µg/mLl and SHIVAD8 with an IC50 of 0.028 µg/mLl. Ab1485 binds the V3-glycan epitope in a glycan-dependent manner. A 3.5 Å cryo-electron microscopy structure of Ab1485 in complex with a native-like SOSIP Env trimer showed conserved contacts with the N332gp120 glycan and gp120 GDIR peptide motif, but in a distinct Env-binding orientation relative to human V3/N332gp120 glycan-targeting bNAbs. Intravenous infusion of Ab1485 protected macaques from a high dose challenge with SHIVAD8. We conclude that macaques can develop bNAbs against the V3-glycan patch that resemble human V3-glycan bNAbs.

Conjugation of Native-Like HIV-1 Envelope Trimers onto Liposomes Using EDC/Sulfo-NHS Chemistry: Requirements and Limitations.

The display of native-like human immunodeficiency virus type 1 envelope (HIV-1 Env) trimers on liposomes has gained wide attention over the last few years. Currently, available methods have enabled the preparation of Env-liposome conjugates of unprecedented quality. However, these protocols require the Env trimer to be tagged and/or to carry a specific functional group. For this reason, we have investigated N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide/N-Hydroxysulfosuccinimide (EDC/Sulfo-NHS) chemistry for its potential to covalently conjugate tag-free, non-functionalized native-like Env trimers onto the surface of carboxyl-functionalized liposomes. The preservation of the liposome’s physical integrity and the immunogen’s conformation required a fine-tuned two-step approach based on the controlled use of β-mercaptoethanol. The display of Env trimers was strictly limited to activated liposomes of positive charge, i.e., liposomes with a positive zeta potential that carry amine-reactive Sulfo-NHS esters on their surface. In agreement with that, conjugation was found to be highly ionic strength- and pH-dependent. Overall, we have identified electrostatic pre-concentration (i.e., close proximity between negatively charged Env trimers and positively charged liposomes established through electrostatic attraction) to be crucial for conjugation reactions to proceed. The present study highlights the requirements and limitations of potentially scalable EDC/Sulfo-NHS-based approaches and represents a solid basis for further research into the controlled conjugation of tag-free, non-functionalized native-like Env trimers on the surface of liposomes, and other nanoparticles.

Progress in HIV-1 Env trimer design

Currently, HIV-1 vaccine development has still been a hot pot in the AIDS research. HIV-1 glycoprotein Env is the sole target in the virion surface that mediates the membrane fusion between the virion and cell in the HIV-1 infection process. Env protein is the significant immunogen for HIV-1 vaccine development. In recent years, there have been breakthroughs in the Env trimer research. For example, the strategies including SOSIP, NFL2P, and UFO had been applied to design and generate HIV-1 Env trimer. The improvement of quantity and stability is beneficial to achieve the HIV-1 native-like Env trimer for elicitation of strong neutralizing antibody responsing in animal immunization. This review focuses on the different strategies for Env trimer design and compares their advantages and disadvantages, combining with our work to give some advice, which might provide relevant information for the future HIV-1 immunogen design.

Efficiently cleaved HIV-1 envelopes: can they be important for vaccine immunogen development?

The enormous diversity of HIV-1 is a significant impediment in selecting envelopes (Envs) that can be suitable for designing vaccine immunogens. While tremendous progress has been made in developing soluble, trimeric, native-like Env proteins, those that have elicited neutralizing antibodies (Abs) in animal models are relatively few. A strategy of selecting naturally occurring Envs suitable for immunogen design by studying the correlation between efficient cleavage on the cell surface and their selective binding to broadly neutralizing Abs (bNAbs) and not to non-neutralizing Abs (non-NAbs), properties essential in immunogens, may be useful. Here we discuss some of the challenges of developing an efficacious HIV-1 vaccine and the work done in generating soluble immunogens. We also discuss the study of naturally occurring, membrane-bound, efficiently cleaved (naturally more sensitive to furin) Envs and how they may positively add to the repertoire of HIV-1 Envs that can be used for vaccine immunogen design. However, even with such Envs, the challenges of developing well-folded, native-like trimers as soluble proteins or using other immunogen strategies such as virus-like particles with desirable antigenic properties remain, and are formidable. In spite of the progress that has been made in the HIV-1 vaccine field, an immunogen that elicits neutralizing Abs with significant breadth and potency in vaccines has still not been developed. Efficiently cleaved Envs may increase the number of available Envs suitable for immunogen design and should be studied further.

SOS and IP Modifications Predominantly Affect the Yield but Not Other Properties of SOSIP.664 HIV-1 Env Glycoprotein Trimers.

Soluble recombinant native-like (NL) envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design, designated BG505 SOSIP.664, incorporates an intersubunit disulfide bond (SOS) to covalently link the gp120 and gp41 ectodomain (gp41ECTO) subunits and a point substitution, I559P (IP), to further stabilize the gp41ECTO components. Without the SOS and IP changes, proteolytically cleaved trimers tend to disintegrate into their constituent gp120 and gp41ECTO subunits. We show, however, that NL trimers lacking the SOS and/or IP change can be affinity purified in amounts sufficient for analyses of their antigenicity and thermal stability. In general, these trimer variants have properties highly comparable to those of the fully stabilized SOSIP.664 version. We conclude that the major effect of the SOS and IP changes is to substantially increase trimer stability during and after the expression process, thereby allowing useful amounts to be produced. However, once the trimers have been purified, the SOS and IP changes have only subtle impacts on thermostability and the antigenicity of bNAb and other epitopes.IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. One vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. A commonly used design is designated SOSIP.664, a term reflecting the sequence changes that are used to stabilize the trimers and allow their production in practically useful amounts. Here, we show that these stabilizing changes act to increase trimer yield during the biosynthesis process within the producer cell but have little impact on the properties of purified trimers.

Asymmetric opening of HIV-1 Env bound to CD4 and a coreceptor-mimicking antibody.

The HIV-1 envelope (Env) glycoprotein, a (gp120-gp41)3 trimer, mediates fusion of viral and host cell membranes after gp120 binding to host receptor CD4. Receptor binding triggers conformational changes allowing coreceptor (CCR5) recognition through CCR5’s tyrosine-sulfated N-terminus, release of the gp41 fusion peptide, and fusion. We present 3.3Å and 3.5Å cryo-EM structures of E51, a tyrosine-sulfated coreceptor-mimicking antibody, complexed with a CD4-bound open HIV-1 native-like Env trimer. Two classes of asymmetric Env interact with E51, revealing tyrosine-sulfated interactions with gp120 mimicking CCR5 interactions, and two conformations of gp120-gp41 protomers (A and B protomers in AAB and ABB trimers) that differ in their degree of CD4-induced trimer opening and induction of changes to the fusion peptide. By integrating the new structural information with previous closed and open envelope trimer structures, we modeled the order of conformational changes on the path to coreceptor binding site exposure and subsequent viral–host cell membrane fusion.

Enhancing and shaping the immunogenicity of native-like HIV-1 envelope trimers with a two-component protein nanoparticle

The development of native-like HIV-1 envelope (Env) trimer antigens has enabled the induction of neutralizing antibody (NAb) responses against neutralization-resistant HIV-1 strains in animal models. However, NAb responses are relatively weak and narrow in specificity. Displaying antigens in a multivalent fashion on nanoparticles (NPs) is an established strategy to increase their immunogenicity. Here we present the design and characterization of two-component protein NPs displaying 20 stabilized SOSIP trimers from various HIV-1 strains. The two-component nature permits the incorporation of exclusively well-folded, native-like Env trimers into NPs that self-assemble in vitro with high efficiency. Immunization studies show that the NPs are particularly efficacious as priming immunogens, improve the quality of the Ab response over a conventional one-component nanoparticle system, and are most effective when SOSIP trimers with an apex-proximate neutralizing epitope are displayed. Their ability to enhance and shape the immunogenicity of SOSIP trimers make these NPs a promising immunogen platform.

Neutralization-guided design of HIV-1 envelope trimers with high affinity for the unmutated common ancestor of CH235 lineage CD4bs broadly neutralizing antibodies.

The CD4 binding site (CD4bs) of the HIV-1 envelope glycoprotein is susceptible to multiple lineages of broadly neutralizing antibodies (bnAbs) that are attractive to elicit with vaccines. The CH235 lineage (VH1-46) of CD4bs bnAbs is particularly attractive because the most mature members neutralize 90% of circulating strains, do not possess long HCDR3 regions, and do not contain insertions and deletions that may be difficult to induce. We used virus neutralization to measure the interaction of CH235 unmutated common ancestor (CH235 UCA) with functional Env trimers on infectious virions to guide immunogen design for this bnAb lineage. Two Env mutations were identified, one in loop D (N279K) and another in V5 (G458Y), that acted synergistically to render autologous CH505 transmitted/founder virus susceptible to neutralization by CH235 UCA. Man5-enriched N-glycans provided additional synergy for neutralization. CH235 UCA bound with nanomolar affinity to corresponding soluble native-like Env trimers as candidate immunogens. A cryo-EM structure of CH235 UCA bound to Man5-enriched CH505.N279K.G458Y.SOSIP.664 revealed interactions of the antibody light chain complementarity determining region 3 (CDR L3) with the engineered Env loops D and V5. These results demonstrate that virus neutralization can directly inform vaccine design and suggest a germline targeting and reverse engineering strategy to initiate and mature the CH235 bnAb lineage.

Similarities and differences between native HIV-1 envelope glycoprotein trimers and stabilized soluble trimer mimetics.

The HIV-1 envelope glycoprotein (Env) trimer is located on the surface of the virus and is the target of broadly neutralizing antibodies (bNAbs). Recombinant native-like soluble Env trimer mimetics, such as SOSIP trimers, have taken a central role in HIV-1 vaccine research aimed at inducing bNAbs. We therefore performed a direct and thorough comparison of a full-length unmodified Env trimer containing the transmembrane domain and the cytoplasmic tail, with the sequence matched soluble SOSIP trimer, both based on an early Env sequence (AMC011) from an HIV+ individual that developed bNAbs. The structures of the full-length AMC011 trimer bound to either bNAb PGT145 or PGT151 were very similar to the structures of SOSIP trimers. Antigenically, the full-length and SOSIP trimers were comparable, but in contrast to the full-length trimer, the SOSIP trimer did not bind at all to non-neutralizing antibodies, most likely as a consequence of the intrinsic stabilization of the SOSIP trimer. Furthermore, the glycan composition of full-length and SOSIP trimers was similar overall, but the SOSIP trimer possessed slightly less complex and less extensively processed glycans, which may relate to the intrinsic stabilization as well as the absence of the membrane tether. These data provide insights into how to best use and improve membrane-associated full-length and soluble SOSIP HIV-1 Env trimers as immunogens.

Optimizing the production and affinity purification of HIV-1 envelope glycoprotein SOSIP trimers from transiently transfected CHO cells.

We describe methods to improve the efficiency with which HIV-1 Envelope glycoprotein SOSIP trimer immunogens can be produced by transient transfection of ExpiCHO-S cells and then affinity purified using the trimer-specific human monoclonal antibody PGT145. The specificity of PGT145 for properly folded trimers allows for the facile, one-step, isolation of these immunogens in research laboratories. PGT145 columns are also valuable as a component of more complex purification processes in current Good Manufacturing Practice programs. However, we found that PGT145 purification was highly variable and markedly inefficient when used to process supernatants from transiently transfected ExpiCHO-S cells expressing the BG505 SOSIP.664 and other trimeric Env proteins. In contrast, no such problems arose when the same Env proteins derived from a stable CHO cell line were processed on the same PGT145 columns, or with transient transfection supernatants from 293F cells. An investigation of the ExpiCHO-S transfection system identified the presence of polyanions, including but perhaps not limited to dextran sulfate, in the Enhancer component of the transfection system. We hypothesized that these polyanions bound to the cationic PGT145 epitope on the trimers and impeded their ability to bind to the PGT145 affinity column. We found that replacing the Enhancer component with alternative culture medium supplements substantially increased the yield of PGT145-purifiable trimers, and we also confirmed that both dextran sulfate and the Enhancer component were indeed inhibitors of PGT145 binding to BG505 SOSIP.664 trimers in immunoassays. The presence of polyanions, including but not limited to nucleic acids, should be considered in other circumstances where PGT145 columns are less efficient than expected at purifying native-like trimers.

Stabilization of the V2 loop improves the presentation of V2 loop–associated broadly neutralizing antibody epitopes on HIV-1 envelope trimers

A successful HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs) that target the envelope glycoprotein (Env) spike on the virus. Native-like recombinant Env trimers of the SOSIP design now serve as a platform for achieving this challenging goal. However, SOSIP trimers usually do not bind efficiently to the inferred germline precursors of bNAbs (gl-bNAbs). We hypothesized that the inherent flexibilities of the V1 and V2 variable loops in the Env trimer contribute to the poor recognition of gl-bNAb epitopes at the trimer apex that extensively involve V2 residues. To reduce local V2 flexibility and improve the binding of V2-dependent bNAbs and gl-bNAbs, we designed BG505 SOSIP.664 trimer variants containing newly created disulfide bonds intended to stabilize the V2 loop in an optimally antigenic configuration. The first variant, I184C/E190C, contained a new disulfide bond within the V2 loop, whereas the second variant, E153C/R178C, had a new disulfide bond that cross-linked V2 and V1. The resulting engineered native-like trimer variants were both more reactive with and were neutralized by V2 bNAbs and gl-bNAbs, a finding that may be valuable in the design of germline targeting and boosting trimer immunogens to create an antigenic conformation optimal for HIV vaccine development.

PI-3 Single molecule fluorescence approaches to probe the binding of soluble-CD4 to native-like HIV-1 SOSIP trimers

Several lines of evidence suggest that HIV-1 envelope (Env) on virions transition through a series of conformations in solution as well as when they engage CD4 molecules during viral attachment. Moreover, the flexibility and antigenicity of trimers changes as they progressively become saturated with CD4 molecules. HIV-1 Env can potentially engage up to 3 independent CD4 molecules for efficient Env triggering in the virial fusion. However, it is still unknown if one, 2 or 3 CD4 receptor molecules are required for this process. To date, the ensemble techniques and indirect biological assays have been unable to address this question quantitatively. Here, we employed a unique approach utilizing the single molecule detection (SMD) analyses to determine quantitatively the number of sCD4 molecules bound to HIV-1 Env and capture the intermediate interaction states that may be missed by the ensemble methods. A key step in direct counting of single molecules is to ensure single fluorophore labeling of the target molecule. For this purpose, we constructed a C-terminus SNAP-tagged human sCD4 fusion protein and fluorescently labeled it with a single Alexa 488 fluorophore (sCD4-SNAP-A488). This approach was devoid of any genetic or external perturbation to the SOSIP.664 trimers. Our results by SMD confirm that up to 3 sCD4-SNAP-A488 molecules bind a single SOSIP trimer and provide insight into trimer occupancy shifts over time. The experimental data reveals that Env is intrinsically dynamic, transitioning between 3 distinct stoichiometries (1-, 2- and 3-sCD4 bound states). Real-time single molecule association kinetic experiments divulge different states of trimer triggering and stoichiometry of the complexes. We have developed an algorithm for automated analyses of our data to infer the stoichiometry in real-time. Importantly, our single-molecule based association kinetic analysis indicates that assemblies of trimer with 1 or 3 sCD4 molecules are more stable whereas binding of 2 sCD4s seems transitional. Our findings are consistent with structural information available for conformations of HIV-1 Env in its ligand-free, prefusion state and in the CD4-bound states and provide new critical insights into the sCD4 interaction with the Env for better understanding of the complex biology of HIV-1 entry into cells.

Characterization of native-like HIV-1 gp140 glycoprotein expressed in insect cells

The trimeric HIV-1 envelope glycoprotein (Env) is critical for vaccine development aimed at achieving broadly-neutralizing antibody responses. The use of various recombinant expression systems and construct designs are associated with the resultant nature of produced proteins, especially in terms of glycosylation, antigenicity, and immunogenicity of the glycoprotein. Here, we explored an otherwise baculovirus cassette than classical one designed to express HIV-1 Env protein, including SOSIP mutation and Foldon moiety involvement. This improved design increased the ratio of the Env trimer fraction from ∼40% to ∼60% with respect to that of prototypical design, as indicated by high-performance size-exclusion chromatography and sedimentation velocity analysis. In addition, the design prolonged cell viability and enhanced the final yield (approximately 13–15 mg/L) after affinity purification. gp140 produced from insect cells mimicked the native-like trimer and mainly adopted glycosylation pattern of oligomannose glycans. The native-like Env proteins conferred cross-clade neutralizing antibody production in BALB/c mice. In summary, the expression of Env in insect cells by optimizing the baculovirus vector provides an alternative strategy for HIV-1 immunogen production and may benefit future Env-based HIV vaccine design.

Vaccine-Induced Protection from Homologous Tier 2 SHIV Challenge in Nonhuman Primates Depends on Serum-Neutralizing Antibody Titers

SummaryPassive administration of HIV neutralizing antibodies (nAbs) can protect macaques from hard-to-neutralize (tier 2) chimeric simian-human immunodeficiency virus (SHIV) challenge. However, conditions for nAb-mediated protection after vaccination have not been established. Here, we selected groups of 6 rhesus macaques with either high or low serum nAb titers from a total of 78 animals immunized with recombinant native-like (SOSIP) Env trimers. Repeat intrarectal challenge with homologous tier 2 SHIVBG505 led to rapid infection in unimmunized and low-titer animals. High-titer animals, however, demonstrated protection that was gradually lost as nAb titers waned over time. An autologous serum ID50 nAb titer of ∼1:500 afforded more than 90% protection from medium-dose SHIV infection. In contrast, antibody-dependent cellular cytotoxicity and T cell activity did not correlate with protection. Therefore, Env protein-based vaccination strategies can protect against hard-to-neutralize SHIV challenge in rhesus macaques by inducing tier 2 nAbs, provided appropriate neutralizing titers can be reached and maintained.

Partially Open HIV-1 Envelope Structures Exhibit Conformational Changes Relevant for Coreceptor Binding and Fusion

SummaryHIV-1 Env, a trimer of gp120-gp41 heterodimers, mediates membrane fusion after binding host receptor CD4. Receptor binding displaces V1V2 loops from Env's apex, allowing coreceptor binding and opening Env to enable gp41-mediated fusion. We present 3.54 Å and 4.06 Å cryoelectron microscopy structures of partially open soluble native-like Env trimers (SOSIPs) bound to CD4. One structure, a complex with a coreceptor-mimicking antibody that binds both CD4 and gp120, stabilizes the displaced V1V2 and reveals its structure. Comparing partially and fully open Envs with closed Envs shows that gp41 rearrangements are independent of the CD4-induced rearrangements that result in V1V2 displacement and formation of a 4-stranded bridging sheet. These findings suggest ordered conformational changes before coreceptor binding: (1) gp120 opening inducing side-chain rearrangements and a compact gp41 central helix conformation, and (2) 4-stranded bridging-sheet formation and V1V2 displacement. These analyses illuminate potential receptor-induced Env changes and inform design of therapeutics disrupting viral entry.

Rational Design of DNA-Expressed Stabilized Native-Like HIV-1 Envelope Trimers

SummaryThe HIV-1-envelope glycoprotein (Env) is the main target of antigen design for antibody-based prophylactic vaccines. The generation of broadly neutralizing antibodies (bNAb) likely requires the appropriate presentation of stabilized trimers preventing exposure of non-neutralizing antibody (nNAb) epitopes. We designed a series of membrane-bound Envs with increased trimer stability through the introduction of key stabilization mutations. We derived a stabilized HIV-1 trimer, ConSOSL.UFO.750, which displays a dramatic reduction in nNAb binding while maintaining high quaternary and MPER-specific bNAb binding. Its soluble counterpart, ConSOSL.UFO.664, displays similar antigenicity, and its native-like Env structure is confirmed by negative stain-EM and glycosylation profiling of the soluble ConSOSL.UFO.664 trimer. A rabbit immunization study demonstrated that the ConSOSL.UFO.664 can induce autologous tier 2 neutralization. We have successfully designed a stabilized native-like Env trimer amenable to nucleic acid or viral vector-based vaccination strategies.

Immunogenicity in Rabbits of HIV-1 SOSIP Trimers from Clades A, B, and C, Given Individually, Sequentially, or in Combination.

ABSTRACTRecombinant soluble HIV-1 envelope glycoprotein (Env) SOSIP trimers are a design platform for inducing broadly neutralizing antibodies (bNAbs) by vaccination. To date, these and alternative designs of native-like trimers, given singly or in pairs, have not induced bNAbs in test animals such as rabbits or macaques. Here, we have evaluated whether trivalent and tetravalent combinations of SOSIP trimers from clades A, B, and C, delivered simultaneously or sequentially, induce better neutralizing antibody responses in rabbits than when given alone. None of the tested formulations led to the induction of bNAbs. We found that BG505 clade A trimers dominated the autologous NAb responses induced by combinations, which probably relates to the presence of immunodominant glycan holes on the BG505 trimer. Furthermore, autologous NAb responses to all individual trimers were reduced when they were delivered in combinations compared with when delivered alone, suggesting that immunogen interference had occurred. Finally, in a sequential regimen, a heterologous clade C trimer cross-boosted NAb responses that were primed by earlier immunizations with clade A and B trimers. Taken together, these findings should allow us to improve the design of immunization regimens based on native-like HIV-1 Env trimers.IMPORTANCE A successful HIV-1 vaccine most probably requires a trimeric envelope glycoprotein (Env) component, as Env is the only viral protein on the surface of the virus and therefore the only target for neutralizing antibodies. Native-like Env trimers can induce strain-specific neutralizing antibodies but not yet broadly neutralizing antibodies. To try to broaden the antibody response, we immunized rabbits with soluble native-like Env trimers from three different clades using monovalent, multivalent, and sequential regimens. We found that the neutralizing antibody response against each immunogen was reduced when the immunogens were delivered in combination or sequentially compared to the monovalent regimen. In contrast, when the Env trimers from different clades were delivered sequentially, the neutralizing antibody response could be cross-boosted. Although the combination of native-like Env trimers from different clades did not induce broadly neutralizing antibodies, the results provide clues on how to use native-like trimers in vaccination experiments.

Epitopes for neutralizing antibodies induced by HIV-1 envelope glycoprotein BG505 SOSIP trimers in rabbits and macaques.

The native-like, soluble SOSIP.664 trimer based on the BG505 clade A env gene of HIV-1 is immunogenic in various animal species, of which the most studied are rabbits and rhesus macaques. The trimer induces autologous neutralizing antibodies (NAbs) consistently but at a wide range of titers and with incompletely determined specificities. A precise delineation of immunogenic neutralization epitopes on native-like trimers could help strategies to extend the NAb response to heterologous HIV-1 strains. One autologous NAb epitope on the BG505 Env trimer is known to involve residues lining a hole in the glycan shield that is blocked by adding a glycan at either residue 241 or 289. This glycan-hole epitope accounts for the NAb response of most trimer-immunized rabbits but not for that of a substantial subset. Here, we have used a large panel of mutant BG505 Env-pseudotyped viruses to define additional sites. A frequently immunogenic epitope in rabbits is blocked by adding a glycan at residue 465 near the junction of the gp120 V5 loop and β24 strand and is influenced by amino-acid changes in the structurally nearby C3 region. We name this new site the “C3/465 epitope”. Of note is that the C3 region was under selection pressure in the infected infant from whom the BG505 virus was isolated. A third NAb epitope is located in the V1 region of gp120, although it is rarely immunogenic. In macaques, NAb responses induced by BG505 SOSIP trimers are more often directed at the C3/465 epitope than the 241/289-glycan hole.