HIV-1 Neutralizing Antibodies Research Articles

Teaching a Clone to Walk, One Step at a Time

Development of broad HIV neutralizing antibodies during infection requires prolonged exposure toviral variants. A series of three studies published in this issue of Cell and a related paper in Immunity report new HIV structure-based design efforts focused on sequential boosting regimens with antigens of increasing maturation that point the way forward for an effective HIV vaccine strategy.

Site-Selective Chemoenzymatic Glycosylation of an HIV-1 Polypeptide Antigen with Two Distinct N-Glycans via an Orthogonal Protecting Group Strategy.

A convergent chemoenzymatic approach for sequential installation of different N-glycans in a polypeptide is described. The method includes introduction of distinguishably protected GlcNAc-Asn building blocks during automated solid phase peptide synthesis (SPPS), followed by orthogonal deprotection of the GlcNAc primers and site-selective sequential extension of the sugar chains through glycosynthase-catalyzed transglycosylation reactions. It was observed that the protecting groups on one neighboring GlcNAc moiety have an impact on the substrate activity of another GlcNAc acceptor toward some endoglycosynthases in transglycosylation. The usefulness of this synthetic strategy was exemplified by an efficient synthesis of the glycopeptide neutralizing epitope of broadly HIV-neutralizing antibody PG9. The method should be generally applicable for the synthesis of complex glycopeptides carrying multiple different N-glycans.

Isolation and characterization of a novel neutralizing antibody targeting the CD4-binding site of HIV-1 gp120

Isolation and characterization of novel HIV-1 neutralizing antibodies assists the development of effective AIDS vaccines and immune therapeutics. In this study, we constructed a phage display antibody library by using the PBMC samples of a clade B′ HIV-1-infected long-term nonprogressor (LTNP) whose sera exhibited broadly neutralizing activity. A novel human monoclonal antibody (hMAb), termed A16, was identified by panning the library with two clades of HIV-1 Env glycoproteins. We demonstrated that A16 neutralized 32% of 73 tested HIV-1 isolates and it targeted the CD4-binding site (CD4bs) of gp120 with high affinity. By selecting the peptide mimotopes in combination with computational algorithms and site-directed mutagenesis, the epitope of A16 was mapped to the structurally conserved sites located within the β1-α1, loop D, β20-β21 (bridging sheet) and β24-α5 of gp120, which critically determine the CD4 binding and are involved in the epitopes of CD4bs-directed antibodies. Our studies have shed new insights for the immune response of HIV-1 infection and offered a new tool for designing vaccine immunogens and antibody-based immune therapy.

Engineered Bispecific Antibodies with Exquisite HIV-1-Neutralizing Activity

While the search for an efficacious HIV-1 vaccine remains elusive, emergence of a new generation of virus-neutralizing monoclonal antibodies (mAbs) has re-ignited the field of passive immunization for HIV-1 prevention. However, the plasticity of HIV-1 demands additional improvements to these mAbs to better ensure their clinical utility. Here, we report engineered bispecific antibodies that are the mostpotent and broad HIV-neutralizing antibodies to date. One bispecific antibody, 10E8V2.0/iMab, neutralized 118 HIV-1 pseudotyped viruses tested with a mean 50% inhibitory concentration (IC50) of 0.002μg/mL. 10E8V2.0/iMab also potently neutralized 99% of viruses in a second panel of 200 HIV-1 isolates belonging to clade C, the dominant subtype accounting for ∼50% of new infections worldwide. Importantly, 10E8V2.0/iMab reduced virus load substantially in HIV-1-infected humanized mice and also provided complete protection when administered prior to virus challenge. These bispecific antibodies hold promise as novel prophylactic and/or therapeutic agents in the fight against HIV-1.

Detection of HIV Env-mediated B cell receptor aggregation on engineered B cells by flow cytometry.

Abstract Approximately 20% of individuals infected with HIV-1 produce broadly neutralizing antibodies (bNAbs) against the virus. Understanding how bNAb linked B cell receptors (BCRs) are initially stimulated by the HIV-1 envelope (Env) in the context of natural HIV infection is a major focus of HIV research. While germline-reverted BCR forms of bNAbs do not efficiently recognize Env, recombinant Env proteins have been recently designed to engage and activate some germline BCRs. To better understand how such modified Envs engage BCRs and to further optimize the activation of B cells, we (a) created a stable Ramos cell line that expresses the germline BCR form of a potent and broadly neutralizing antibody, VRC01 and (b) developed a fluorescence-based assay that allows for the monitoring of Env-mediated BCR aggregation. B cells were incubated with different multimeric forms of Env (valency ranging from 1–24) and determined the extent of BCR aggregation and compared it with the extent of B cell activation (measured by Ca2+flux assays). Significant increases in BCR aggregation and B cell-activation were detected in response to heptameric and 24-meric forms of Env. In contrast, lower valency multimeric forms of Env activated B cells inefficiently. No aggregation or calcium flux was detected in response to monomeric Env. A positive correlation was observed between the magnitudes of calcium flux and BCR aggregation. Thus, either BCR aggregation or Ca2+ flux assays can be used to monitor the activation of B cells by recombinant Env proteins that are being evaluated as possible immunogens to elicit bNAbs. Our results are relevant to optimizing the design and formulation of recombinant Env proteins for the elicitation of broadly neutralizing HIV-1 antibodies.

CD277 reduces B cell Activation by HIV Env Immunogens

Abstract CD277 is a human B7-like molecule. involved in activation of Vγ9Vδ2 T cells. Its counter-receptor is unknown and its role in B cell activation has not been described. B cell lines were stably transduced with the IgG isotype of the inferred germline B cell receptor (BCR) form of the broad and potent HIV-1 neutralizing antibody, VRC01, and stimulated with recombinant HIV Env in Ca2+-dependent activation assays. CD277 ectodomain proteins partially inhibited Ca2+ flux in response to heptameric and trimeric HIV Env. Similar observations were made with B cells expressing an anti-HA BCR, when stimulated with recombinant HA. We conclude that the reduction in B cell activation recorded in the presence of CD277 was not specific for the HIV Env. In contrast, CD277 completely abolished B cell activation that was induced by cross-linking the same BCRs with anti-Fab antibodies. To identify molecules that may serve as counter-receptors for CD277 binding, a fluorescently coupled CD277 ectodomain was utilized to conduct a FACs-based negative selection screen from a pooled lentiCRISPR-Cas9 knockout library (GeCKO). This strategy resulted in a significant reduction in library diversity with the overall sgRNA pool by the end of the screen restricted to a few, highly abundant sgRNAs. Specific binding assays among putative counter-receptors are ongoing. Once identified, CD277 counter-receptors could be targeted for inhibition in vaccination studies, to enhance B cell responses to antigen such as HIV Env. These data indicate an important physiological role for CD277 in inhibiting B cell activation and its role in influencing the B cell repertoire should be pursued.

761. Targeted Killing of HIV Infected Cells Using CCR5-Disrupted Anti-HIV-CAR T Cells

A cure for HIV remains an important treatment goal for 30 million HIV-infected individuals worldwide. Long term control of HIV following a single treatment will require a mechanism to eradicate the latent reservoir of HIV infected cells that are capable of reactivating. A previous phase II clinical trial using an anti-HIV chimeric antigen receptor (αHIV-CAR) targeting the CD4-binding site on HIV envelope was partially effective. To optimize this approach we have developed a series of CARs based on the scFV of broadly neutralizing HIV antibodies targeting four different structural regions of the HIV envelope: the V1/V2 loop, the V3 loop, the CD4 binding site, and the membrane-proximal external region. αHIV-CAR T cells targeting different epitopes were compared and were able to kill > 80% of HIV-infected cells grown in the presence of ART. However, a limitation of αHIV-CAR T cells is that the αHIV-CAR also serves as a receptor for HIV and allows HIV infection of αHIV-CAR T cells. Therefore we disrupted the major co-receptor required for HIV cell entry, CCR5, using a megaTAL nuclease, as a means of protecting the CAR-expressing cells from HIV infection. We used two strategies for achieving these dual modifications in primary human T cells. For both strategies, the CCR5 megaTAL nuclease was delivered by mRNA electroporation, which has previously been shown to induce a high rate of bi-allelic NHEJ-mediated gene disruption. αHIV-CAR expression cassettes were delivered into the host genome via lentiviral vectors (LV), or were targeted to the megaTAL nuclease cleavage site in CCR5 using an adeno-associated virus (AAV) that included CCR5 homology arms. Both strategies resulted in stable expression of the CAR construct and specific activation of CAR+ T cells in the presence of an HIV+ cell line. In the presence of actively replicating virus, CCR5-megaTAL treated CAR+ T cells out-performed CAR+ T cells generated by LV delivery alone as measured by reduction in HIV capsid protein. To enable testing in non-human primates (NHP), we re-optimized the cell-editing protocol with NHP lymphocytes. Primary T cells from pigtail macaques were successfully transfected with CCR5 megaTAL mRNA and achieved a CCR5 disruption rate of 50%. Cells were subsequently transduced with αHIV-CAR LV resulting in ~70% of manipulated cells with αHIV-CAR. Expansion of the cells in vitro resulted in 60-fold expansion over 8 days. CAR+ NHP cells specifically killed HIV infected human cells demonstrating that NHP-derived αHIV-CAR+ T cells retained killing function. In conclusion, it is feasible to construct αHIV-CAR+ T cells that are protected from HIV infection in human and NHP cells, and warrants further study in vivo.

Optimal Combinations of Broadly Neutralizing Antibodies for Prevention and Treatment of HIV-1 Clade C Infection.

The identification of a new generation of potent broadly neutralizing HIV-1 antibodies (bnAbs) has generated substantial interest in their potential use for the prevention and/or treatment of HIV-1 infection. While combinations of bnAbs targeting distinct epitopes on the viral envelope (Env) will likely be required to overcome the extraordinary diversity of HIV-1, a key outstanding question is which bnAbs, and how many, will be needed to achieve optimal clinical benefit. We assessed the neutralizing activity of 15 bnAbs targeting four distinct epitopes of Env, including the CD4-binding site (CD4bs), the V1/V2-glycan region, the V3-glycan region, and the gp41 membrane proximal external region (MPER), against a panel of 200 acute/early clade C HIV-1 Env pseudoviruses. A mathematical model was developed that predicted neutralization by a subset of experimentally evaluated bnAb combinations with high accuracy. Using this model, we performed a comprehensive and systematic comparison of the predicted neutralizing activity of over 1,600 possible double, triple, and quadruple bnAb combinations. The most promising bnAb combinations were identified based not only on breadth and potency of neutralization, but also other relevant measures, such as the extent of complete neutralization and instantaneous inhibitory potential (IIP). By this set of criteria, triple and quadruple combinations of bnAbs were identified that were significantly more effective than the best double combinations, and further improved the probability of having multiple bnAbs simultaneously active against a given virus, a requirement that may be critical for countering escape in vivo. These results provide a rationale for advancing bnAb combinations with the best in vitro predictors of success into clinical trials for both the prevention and treatment of HIV-1 infection.

Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice.

VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype.

Harnessing the protective potential of HIV-1 neutralizing antibodies.

Recent biological, structural, and technical advances are converging within the HIV-1 vaccine field to harness the power of antibodies for prevention and therapy. Numerous monoclonal antibodies with broad neutralizing activity against diverse HIV-1 isolates have now been identified, revealing at least five sites of vulnerability on the envelope (Env) glycoproteins. While there are practical and technological barriers blocking a clear path from broadly neutralizing antibodies (bNAb) to a protective vaccine, this is not a dead end. Scientists are revisiting old approaches with new technology, cutting new trails through unexplored territory, and paving new roads in the hopes of preventing HIV-1 infection. Other promising avenues to capitalize on the power of bNAbs are also being pursued, such as passive antibody immunotherapy and gene therapy approaches. Moreover, non-neutralizing antibodies have inhibitory activities that could have protective potential, alone or in combination with bNAbs. With a new generation of bNAbs, and a clinical trial that associated antibodies with reduced acquisition, the field is closer than ever to developing strategies to use antibodies against HIV-1.

D-101 HIV-1 neutralizing antibodies induced by native-like envelope trimers

D-101 HIV-1 neutralizing antibodies induced by native-like envelope trimers

Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition.

Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89) on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv) molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells.

Low frequency of broadly neutralizing HIV antibodies during chronic infection even in quaternary epitope targeting antibodies containing large numbers of somatic mutations.

Neutralizing antibodies (Abs) are thought to be a critical component of an appropriate HIV vaccine response. It has been proposed that Abs recognizing conformationally dependent quaternary epitopes on the HIV envelope (Env) trimer may be necessary to neutralize diverse HIV strains. A number of recently described broadly neutralizing monoclonal Abs (mAbs) recognize complex and quaternary epitopes. Generally, many such Abs exhibit extensive numbers of somatic mutations and unique structural characteristics. We sought to characterize the native antibody (Ab) response against circulating HIV focusing on such conformational responses, without a prior selection based on neutralization. Using a capture system based on VLPs incorporating cleaved envelope protein, we identified a selection of B cells that produce quaternary epitope targeting Abs (QtAbs). Similar to a number of broadly neutralizing Abs, the Ab genes encoding these QtAbs showed extensive numbers of somatic mutations. However, when expressed as recombinant molecules, these Abs failed to neutralize virus or mediate ADCVI activity. Molecular analysis showed unusually high numbers of mutations in the Ab heavy chain framework 3 region of the variable genes. The analysis suggests that large numbers of somatic mutations occur in Ab genes encoding HIV Abs in chronically infected individuals in a non-directed, stochastic, manner.

HIV-1 Antibody Neutralization Breadth Is Associated with Enhanced HIV-Specific CD4+ T Cell Responses.

Antigen-specific CD4(+) T helper cell responses have long been recognized to be a critical component of effective vaccine immunity. CD4(+) T cells are necessary to generate and maintain humoral immune responses by providing help to antigen-specific B cells for the production of antibodies. In HIV infection, CD4(+) T cells are thought to be necessary for the induction of Env-specific broadly neutralizing antibodies. However, few studies have investigated the role of HIV-specific CD4(+) T cells in association with HIV neutralizing antibody activity in vaccination or natural infection settings. Here, we conducted a comprehensive analysis of HIV-specific CD4(+) T cell responses in a cohort of 34 untreated HIV-infected controllers matched for viral load, with and without neutralizing antibody breadth to a panel of viral strains. Our results show that the breadth and magnitude of Gag-specific CD4(+) T cell responses were significantly higher in individuals with neutralizing antibodies than in those without neutralizing antibodies. The breadth of Gag-specific CD4(+) T cell responses was positively correlated with the breadth of neutralizing antibody activity. Furthermore, the breadth and magnitude of gp41-specific, but not gp120-specific, CD4(+) T cell responses were significantly elevated in individuals with neutralizing antibodies. Together, these data suggest that robust Gag-specific CD4(+) T cells and, to a lesser extent, gp41-specific CD4(+) T cells may provide important intermolecular help to Env-specific B cells that promote the generation or maintenance of Env-specific neutralizing antibodies. One of the earliest discoveries related to CD4(+) T cell function was their provision of help to B cells in the development of antibody responses. Yet little is known about the role of CD4(+) T helper responses in the setting of HIV infection, and no studies to date have evaluated the impact of HIV-specific CD4(+) T cells on the generation of antibodies that can neutralize multiple different strains of HIV. Here, we addressed this question by analyzing HIV-specific CD4(+) T cell responses in untreated HIV-infected persons with and without neutralizing antibodies. Our results indicate that HIV-infected persons with neutralizing antibodies have significantly more robust CD4(+) T cell responses targeting Gag and gp41 proteins than individuals who lack neutralizing antibodies. These associations suggest that Gag- and gp41-specific CD4(+) T cell responses may provide robust help to B cells for the generation or maintenance of neutralizing antibodies in natural HIV-infection.

Common Characteristics of HIV-Neutralizing Antibodies with a Fondness for Sugars

Broadly neutralizing antibodies targeting quaternary epitopes on the apex of the HIV-1 envelope spike are an attractive vaccine target, yet engineering immunogens that recapitulate such epitopes has proven difficult. In this issue of Immunity, Andrabi and colleagues (2015) identify an exciting new candidate immunogen that could initiate the production of these types of antibodies through vaccination.

Binding of inferred germline precursors of broadly neutralizing HIV-1 antibodies to native-like envelope trimers

HIV-1 envelope glycoproteins (Env) and Env-based immunogens usually do not interact efficiently with the inferred germline precursors of known broadly neutralizing antibodies (bNAbs). This deficiency may be one reason why Env and Env-based immunogens are not efficient at inducing bNAbs. We evaluated the binding of 15 inferred germline precursors of bNAbs directed to different epitope clusters to three soluble native-like SOSIP.664 Env trimers. We found that native-like SOSIP.664 trimers bind to some inferred germline precursors of bNAbs, particularly ones involving the V1/V2 loops at the apex of the trimer. The data imply that native-like SOSIP.664 trimers will be an appropriate platform for structure-guided design improvements intended to create immunogens able to target the germline precursors of bNAbs.

From gene to harvest: insights into upstream process development for the GMP production of a monoclonal antibody in transgenic tobacco plants

The EU Sixth Framework Programme Integrated Project 'Pharma-Planta' developed an approved manufacturing process for recombinant plant-made pharmaceutical proteins (PMPs) using the human HIV-neutralizing monoclonal antibody 2G12 as a case study. In contrast to the well-established Chinese hamster ovary platform, which has been used for the production of therapeutic antibodies for nearly 30 years, only draft regulations were initially available covering the production of recombinant proteins in transgenic tobacco plants. Whereas recombinant proteins produced in animal cells are secreted into the culture medium during fermentation in bioreactors, intact plants grown under nonsterile conditions in a glasshouse environment provide various 'plant-specific' regulatory and technical challenges for the development of a process suitable for the acquisition of a manufacturing licence for clinical phase I trials. During upstream process development, several generic steps were addressed (e.g. plant transformation and screening, seed bank generation, genetic stability, host plant uniformity) as well as product-specific aspects (e.g. product quantity). This report summarizes the efforts undertaken to analyse and define the procedures for the GMP/GACP-compliant upstream production of 2G12 in transgenic tobacco plants from gene to harvest, including the design of expression constructs, plant transformation, the generation of production lines, master and working seed banks and the detailed investigation of cultivation and harvesting parameters and their impact on biomass, product yield and intra/interbatch variability. The resulting procedures were successfully translated into a prototypic manufacturing process that has been approved by the German competent authority.

A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen.

In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive and memory T-cell immune responses, as well as humoral responses. This novel HIV-1 gp120-14K immunogen might be considered as an HIV vaccine candidate for broad T and B-cell immune responses.

Eliciting neutralizing antibodies against the membrane proximal external region of HIV-1 Env by chimeric live attenuated influenza A virus vaccines

Despite significant efforts directed toward research on HIV-1 vaccines, a truly effective immunogen has not been achieved. However, the broadly neutralizing antibodies (BnAbs) 2F5 and 4E10, targeting the highly conserved membrane proximal external region (MPER) of HIV-1, are two promising tools for vaccine development. Here we engrafted the MPER into the linker domain between the trimeric core structure and the transmembrane domain of influenza A virus HA2 to investigate the potential of such chimeric viruses to elicit HIV-1 neutralizing antibodies. In the context of proliferating attenuated influenza A viruses, these HIV-1 neutralizing antibody epitopes could be continuously expressed and mimicked their native conformation to induce humoral immune responses. While MPER-specific antibodies could be detected in serum of guinea pigs vaccinated with the chimeric viruses, they exhibited only weakly neutralizing activities. These antisera from vaccinated animals neutralized viruses of clades B and BC (tier 1), but not of clades AE (tier 1) and C (tier 2). These results suggest that influenza A virus can be used as a vehicle for displaying MPER and inducing BnAbs, but it provides limited protection against HIV-1 infection. In the future development of HIV-1 vaccines by rational design, a more effective live virus vector or multiple antigens should be chosen to facilitate the process of neutralizing antibody maturation.

HIV-1 VACCINES. HIV-1 neutralizing antibodies induced by native-like envelope trimers.

A challenge for HIV-1 immunogen design is the difficulty of inducing neutralizing antibodies (NAbs) against neutralization-resistant (tier 2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation, BG505 SOSIP.664, induced NAbs potently against the sequence-matched tier 2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (tier 1) viruses. Tier 2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas tier 1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous tier 2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for the development of HIV-1 vaccines aimed at inducing bNAbs.