During infection, the fusion peptide (FP) of HIV envelope glycoprotein (Env) serves a central role in viral fusion with the host cell. As such, the FP is highly conserved and therefore an attractive epitope for vaccine design. Here, we describe a vaccination study in non-human primates (NHPs) where glycan deletions were made on soluble HIV Env to increase FP epitope exposure. When delivered via implantable osmotic pumps, this immunogen primed immune responses against the FP, which were then boosted with heterologous trimers resulting in a focused immune response targeting the conserved FP epitope. Although autologous immunizations did not elicit high affinity FP-targeting antibodies, the conserved FP epitope on a heterologous trimer further matured the lower affinity, FP-targeting B cells. This study suggests using epitope conservation strategies on distinct Env trimer immunogens can focus humoral responses on desired neutralizing epitopes and suppress immune-distracting antibody responses against non-neutralizing epitopes

Darwinian evolution of immunoglobulin genes within germinal centers (GC) underlies the progressive increase in antibody affinity following antigen exposure. Whereas the mechanics of how competition between GC B cells drives increased affinity are well established, the dynamical evolutionary features of this process remain poorly characterized. We devised an experimental evolution model in which we “replay” over one hundred instances of a clonally homogenous GC reaction and follow the selective process by assigning affinities to all cells using deep mutational scanning. Our data reveal how GCs achieve predictable evolutionary outcomes through the cumulative effects of many rounds of imperfect selection, acting on a landscape shaped heavily by somatic hypermutation (SHM) targeting biases. Using time-calibrated models, we show that apparent features of GC evolution such as permissiveness to low-affinity lineages and early plateauing of affinity are best explained by survivorship biases that distort our view of how affinity progresses over time.

A vaccine capable of inducing broadly neutralizing antibodies (bnAbs) is essential for effective prevention against HIV in children and adolescents. Germline-targeting vaccine strategies aim to stimulate bnAb precursor B cells through carefully designed immunogens, such as the stabilized SOSIP trimers, which mimic native HIV envelope (Env) proteins while presenting key neutralizing epitopes to germline B cell receptors. Given the ability of children living with HIV to develop bnAbs earlier and at a higher frequency than adults, we compared the immunogenicity of a CD4 binding site (CD4bs) bnAb germline-targeting SOSIP trimer immunization strategy in infant (n = 5) and juvenile (n = 4) rhesus macaques (RMs). Animals received 3 doses of the germline-targeting BG505 GT1.1 immunogen, followed by 3 boosts of wild-type BG505 SOSIP, each adjuvanted with the TLR7/8 agonist, 3M-052-SE. After 1.5 years, the RMs were further boosted with a mixed clade B Env trimer nanoparticle to enhance heterologous virus neutralization responses. This germline-targeting strategy induced equivalent titers of neutralizing antibodies in both groups of RMs, yet the infants exhibited a higher magnitude of vaccine-specific IgG binding. Notably, after 3 doses of BG505 GT1.1 SOSIP, infants had higher BG505 GT1.1-specific IgD- B cells. Upon completion of the vaccine regimen, 4 of 5 infants developed a CD4bs bnAb precursor response detectable in serum compared to only 1 of 4 juveniles. Finally, administration of the mixed clade B nanoparticle was able to increase the breadth of antibody responses in 3 of 5 infants and 2 of 4 juveniles. These results suggest that immunization in early-life may enhance bnAb induction and highlight the potential for future pediatric HIV-1 vaccine strategies. Vaccines that can generate antibody responses that have breadth and potency in neutralization can protect children and adolescents from acquiring multi-strain viruses like HIV. Increasingly, vaccine strategies have become better tailored to elicit these broadly neutralizing antibodies (bnAbs), such as the germline-targeting HIV envelope antigens, which have shown great promise. To determine if we could elicit protective antibody responses against HIV in early life, we immunized infant and juvenile monkeys using the same vaccine strategy. In this study, we found that while both infants and juveniles developed similar levels of antibodies that could neutralize HIV, the infants showed stronger antibody responses in several key areas. Moreso, infants developed the type of early antibody response that researchers believe is needed to eventually evolve into broadly neutralizing antibodies. These findings suggest that initiating HIV vaccination earlier in life may offer a better chance at generating the bnAbs needed to protect against various HIV strains. Our work highlights the potential benefits of initiating an HIV vaccine regimen in childhood.

Filoviruses pose a significant threat to human health with frequent outbreaks and high mortality. Although two vector-based vaccines are available for Ebola virus, a broadly protective filovirus vaccine remains elusive. In this study, we evaluate a general strategy for stabilizing glycoprotein (GP) structures of Ebola, Sudan, and Bundibugyo ebolaviruses and Ravn marburgvirus. A 3.2 Å-resolution crystal structure provides atomic details for the redesigned Ebola virus GP, and cryo-electron microscopy reveals how a pan-ebolavirus neutralizing antibody targets a conserved site on the Sudan virus GP (3.13 Å-resolution), in addition to a low-resolution model of antibody-bound Ravn virus GP. A self-assembling protein nanoparticle (SApNP), I3-01v9, is redesigned at the N-terminus to allow the optimal surface display of filovirus GP trimers. Following detailed in vitro characterization, the lymph node dynamics of Sudan virus GP and GP-presenting SApNPs are investigated in a mouse model. Compared with soluble GP trimer, SApNPs show ~112 times longer retention in lymph node follicles, up-to-28 times greater presentation on follicular dendritic cell dendrites, and up-to-3 times stronger germinal center reactions. Functional antibody responses induced by filovirus GP trimers and SApNPs bearing wildtype and modified glycans are assessed in mice. Our study provides a foundation for next-generation filovirus vaccine development.

Human B cell immunity to the influenza hemagglutinin (HA) stem region, a universal influenza vaccine target, is often stereotyped and immunogenetically restricted, posing challenges for study outside humans. Here, we show that macaques vaccinated with a HA stem immunogen elicit human-like public B cell lineages targeting two major conserved sites of vulnerability, the central stem and anchor epitopes. Central stem antibodies were predominantly derived from VH1-138, the macaque homolog of human VH1-69, a VH-gene preferentially used in human central stem broadly neutralizing antibodies (bnAbs). Similarly, macaques produced anchor bnAbs with the human-like NWP motif. Both bnAb lineages were functionally and structurally analogous to their human counterparts, with recognition mediated largely by germline-encoded motifs. Thus the macaque immunoglobulin repertoire supports human-like public bnAb responses to influenza HA. Moreover, this underscores the utility of homologous germline-encoded immunity, suggesting that immune repertoires of macaques and humans may have been similarly shaped during evolution. ●Functional human-like public antibody lineages can be elicited to HA stem supersites in macaques. ●Macaque central stem bnAbs are predominantly derived from VH1-138, a VH-gene homologous to human VH1-69. ●The human-like CDR L3 NWP anchor epitope-targeting lineage can be elicited in macaques. ●Central stem and anchor bnAbs from humans and macaques engage their respective epitopes with atomic level similarity. Functional human-like public antibody lineages can be elicited to HA stem supersites in macaques. Macaque central stem bnAbs are predominantly derived from VH1-138, a VH-gene homologous to human VH1-69. The human-like CDR L3 NWP anchor epitope-targeting lineage can be elicited in macaques. Central stem and anchor bnAbs from humans and macaques engage their respective epitopes with atomic level similarity.

A protective vaccine against HIV will likely need to induce broadly neutralizing antibodies (bnAbs) that engage relatively conserved epitopes on the HIV envelope glycoprotein (Env) trimer. Nearly all vaccine strategies to induce bnAbs require the use of relatively complex immunization regimens involving a series of different immunogens, most of which are Env trimers. Producing protein-based clinical material to evaluate such relatively complex regimens in humans presents major challenges in cost and time. Furthermore, immunization with HIV trimers as soluble proteins induces strong non-neutralizing responses to the trimer base, which is normally occluded on the virion. These base responses could potentially detract from the induction of nAbs and the eventual induction of bnAbs. mRNA vaccine platforms offer potential advantages over protein delivery for HIV vaccine development, including increased production speed, reduced cost, and the ability to deliver membrane-bound trimers that might facilitate improved immuno-focusing to non-base epitopes. We report the design of mRNA-delivered soluble and membrane-bound forms of a stabilized native-like Env trimer (BG505 MD39.3), initial immunogenicity evaluation in rabbits that triggered clinical evaluation, and more comprehensive evaluation of B cell, T cell, and antibody responses in non-human primates. mRNA-encoded membrane-bound Env immunization elicited reduced off-target base-directed Env responses and stronger neutralizing antibody responses, compared with mRNA-encoded soluble Env. Overall, mRNA delivery of membrane-bound Env appears promising for enhancing B cell responses to subdominant epitopes and facilitating rapid translation to clinical testing, which should assist HIV vaccine development. HIV envelope trimer mRNA enables membrane-bound expression and represents a functional immunogen in pre-clinical mammalian models

Current COVID-19 vaccines are largely limited in their ability to induce broad, durable immunity against emerging viral variants. Design and development of improved vaccines utilizing existing platforms requires an in-depth understanding of the antigenic and immunogenic properties of available vaccines. Here we examined the antigenicity of two of the original COVID-19 vaccines, mRNA-1273 and NVX-CoV2373, by electron microscopy-based polyclonal epitope mapping (EMPEM) of serum from immunized non-human primates (NHPs) and clinical trial donors. Both vaccines induce diverse polyclonal antibody (pAb) responses to the N-terminal domain (NTD) in addition to the receptor-binding domain (RBD) of the Spike protein, with the NTD supersite being an immunodominant epitope. High-resolution cryo-EMPEM studies revealed extensive pAb responses to and around the supersite with unique angles of approach and engagement. NTD supersite pAbs were also the most susceptible to variant mutations compared to other specificities, indicating that ongoing Spike ectodomain-based vaccine design strategies should consider immuno-masking this site to prevent induction of these strain-specific responses.

The JN.1-sublineage KP.3.1.1 recently emerged as the globally prevalent SARS-CoV-2 variant, demonstrating increased infectivity and antibody escape. We investigated how mutations and a deletion in the KP.3.1.1 spike protein (S) affect ACE2 binding and antibody escape. Mass spectrometry revealed a new glycan site at residue N30 and altered glycoforms at neighboring N61. Cryo-EM structures showed that the N30 glycan and rearrangement of adjacent residues did not significantly change the overall spike structure, up-down ratio of the receptor-binding domains (RBDs), or ACE2 binding. Furthermore, a KP.3.1.1 S structure with hACE2 further confirmed an epistatic effect between F456L and Q493E on ACE2 binding. Our analysis shows SARS-CoV-2 variants that emerged after late 2023 are now incorporating reversions to residues found in other sarbecoviruses, including the N30 glycan, Q493E, and others. Overall, these results inform on the structural and functional consequences of the KP.3.1.1 mutations, the current SARS-CoV-2 evolutionary trajectory, and immune evasion

Antibodies are crucial therapeutics, comprising a significant portion of approved drugs due to their safety and clinical efficacy. Traditional antibody discovery methods are labor-intensive, limiting scalability and high-throughput analysis. Here, we improved upon our streamlined approach combining structural analysis and bioinformatics to infer heavy and light chain sequences from electron potential maps of serum-derived polyclonal antibodies (pAbs) bound to antigens. Using ModelAngelo, an automated structure-building tool, we accelerated pAb sequence determination and identified sequence matches in B cell repertoires via ModelAngelo derived Hidden Markov Models (HMMs) associated with pAb structures. Benchmarking against results from a non-human primate HIV vaccine trial, our pipeline reduced analysis time from weeks to under a day with higher precision. Validation with murine immune sera from influenza vaccination revealed multiple protective antibodies. This workflow enhances antibody discovery, enabling faster, more accurate mapping of polyclonal responses with broad applications in vaccine development and therapeutic antibody discovery.

The recognition of intracellular antigens by CD8+ T cells through T-cell receptors (TCRs) is central to adaptive immunity, enabling responses against infections and cancer. The recent approval of TCR-gene-edited T cells for cancer therapy demonstrates the therapeutic advantage of using pMHC recognition to eliminate cancer. However, identification and selection of TCRs from patient material is complex and influenced by the TCR repertoire of the donors used. To overcome these limitations, we here present a rapid and robust de novo binder design platform leveraging state-of-the-art generative models, including RFdiffusion, ProteinMPNN, and AlphaFold2, to engineer minibinders (miBds) targeting the cancer-associated pMHC complex, NY-ESO-1(157-165)/HLA-A*02:01. By incorporating in silico cross-panning and molecular dynamics simulations, we enhanced specificity screening to minimise off-target interactions. We identified a miBd that exhibited high specificity for the NY-ESO-1-derived peptide SLLMWITQC in complex with HLA-A*02:01 and minimal cross-reactivity in mammalian display assays. We further demonstrate the therapeutic potential of this miBd by integrating it into a chimeric antigen receptor, as de novo Binders for Immune-mediated Killing Engagers (BIKEs). BIKE-transduced T cells selectively and effectively killed NY-ESO-1+ melanoma cells compared to non-transduced controls, demonstrating the promise of this approach in precision cancer immunotherapy. Our findings underscore the transformative potential of generative protein design for accelerating the discovery of high-specificity pMHC-targeting therapeutics. Beyond CAR-T applications, our workflow establishes a foundation for developing miBds as versatile tools, heralding a new era of precision immunotherapy.

Influenza viruses evolve rapidly, driving seasonal epidemics and posing global pandemic threats. While neuraminidase (NA) has emerged as a vaccine target, shared molecular features of NA antibody responses are still not well understood. Here, we describe cryo-electron microscopy structures of the broadly protective human antibody DA03E17, which was previously identified from an H1N1-infected donor, in complex with NA from A/H1N1, A/H3N2, and B/Victoria-lineage viruses. DA03E17 targets the highly conserved NA active site using its long CDR H3, which features a DR (Asp–Arg) motif that engages catalytic residues and mimics sialic acid interactions. We further demonstrate that this motif is conserved among several NA active site-targeting antibodies, indicating a common receptor mimicry strategy. We also identified potential antibody precursors containing this DR motif in all donors of a healthy human donor BCR database, highlighting the prevalence of this motif and its potential as vaccine targeting. Our findings reveal shared molecular features in NA active site-targeting antibodies, offering insights for NA-based universal influenza vaccine design.

The quest for a universal influenza vaccine holds great promise for mitigating the global burden of influenza-related morbidity and mortality. However, challenges persist in identifying conserved epitopes capable of inducing protection. In this study, we explore the influence of glycan evolution on H3 hemagglutinin from 1968 to present day and its impacts on antigenicity and immunogenicity. We observe that the appearance of potential N-linked glycosylation sites in Sing/16 hemagglutinin head domain reduces the binding of broadly neutralizing antibodies and shifts the polyclonal immune response upon vaccination to target the stem. Furthermore, structural characterization of HK/68 and Sing/16 by cryo-electron microscopy shows that while HK/68 is resistant to enzymatic deglycosylation, removal of glycans destabilizes the hyperglycosylated head and membrane-proximal region in Sing/16. These insights expand our understanding of glycans beyond their role in protein folding and highlight the interplay among glycan integration and immune recognition to design a universal influenza vaccine

Vaccines incorporating slow delivery, multivalent antigen display, or immunomodulation through adjuvants have an important role to play in shaping the humoral immune response. Here we analyzed mechanisms of action of a clinically relevant combination adjuvant strategy, where phosphoserine (pSer)-tagged immunogens bound to aluminum hydroxide (alum) adjuvant (promoting prolonged antigen delivery to draining lymph nodes) are combined with a potent saponin nanoparticle adjuvant termed SMNP (which alters lymph flow and antigen entry into lymph nodes). When employed with a stabilized HIV Env trimer antigen in mice, this combined adjuvant approach promoted substantial enhancements in germinal center (GC) and antibody responses relative to either adjuvant alone. Using scRNA-seq and scBCR-seq, we found that the alum-pSer/SMNP combination both increased the diversity of GC B cell clones and increased GC B cell clonal expansion, coincident with increases in the expression of Myc and the proportion of S-phase GC B cells. To gain insight into the source of these changes in the GC response, we analyzed antigen biodistribution and structural integrity in draining lymph nodes and found that the combination adjuvant approach, but not alum-pSer delivery or SMNP alone, promoted accumulation of highly intact antigen on follicular dendritic cells, reflecting an integration of the slow antigen delivery and altered lymph node uptake effects of these two adjuvants. These results demonstrate how adjuvants with complementary mechanisms of action impacting vaccine biodistribution and kinetics can synergize to enhance humoral immunity.

Cellular and molecular characterization of immune responses elicited by influenza virus infection and seasonal vaccination have informed efforts to improve vaccine efficacy, breadth, and longevity. Here, we use negative stain electron microscopy polyclonal epitope mapping (nsEMPEM) to structurally characterize the humoral IgG antibody responses to hemagglutinin (HA) from human patients vaccinated with a seasonal quadrivalent flu vaccine or infected with influenza A viruses. Our data show that both vaccinated and infected patients had humoral IgGs targeting highly conserved regions on both H1 and H3 subtype HAs, including the stem and anchor, which are targets for universal influenza vaccine design. Responses against H1 predominantly targeted the central stem epitope in infected patients and vaccinated donors, whereas head epitopes were more prominently targeted on H3. Responses against H3 were less abundant, but a greater diversity of H3 epitopes were targeted relative to H1. While our analysis is limited by sample size, on average, vaccinated donors responded to a greater diversity of epitopes on both H1 and H3 than infected patients. These data establish a baseline for assessing polyclonal antibody responses in vaccination and infection, providing context for future vaccine trials and emphasizing the importance of carefully designing vaccines to boost protective responses towards conserved epitopes.

Clade 2.3.4.4b H5N1 is causing an unprecedented outbreak in dairy cows in the United States. To understand if recent H5N1 viruses are changing their receptor use, we screened recombinant hemagglutinin (HA) from historical and recent 2.3.4.4b H5N1 viruses for binding to distinct glycans bearing terminal sialic acids. We found that H5 from A/Texas/37/2024, an isolate from the dairy cow outbreak, has increased binding breadth to glycans bearing terminal α2,3 sialic acids, the avian receptor, compared to historical and recent 2.3.4.4b H5N1 viruses. We did not observe any binding to α2,6 sialic acids, the receptor used by human seasonal influenza viruses. We identified a single mutation outside of the receptor binding site, T199I, was responsible for increased binding breadth, as it increased receptor binding site flexibility. Together, these data show recent H5N1 viruses are evolving increased receptor binding breadth which could impact the host range and cell types infected with H5N1

Stabilized trimers preserving the native-like HIV envelope structure may be key components of a preventive HIV vaccine regimen to induce broadly neutralizing antibodies (bnAbs). We evaluated trimeric BG505 SOSIP.664 gp140, formulated with a novel TLR7/8 signaling adjuvant, 3M-052-AF/Alum, for safety, adjuvant dose-finding and immunogenicity in a first-in-healthy adult (n=17), randomized, placebo-controlled trial (HVTN 137A). The vaccine regimen appeared safe. Robust, trimer-specific antibody, B-cell and CD4+ T-cell responses emerged post-vaccination. Five vaccinees developed serum autologous tier-2 nAbs (ID50 titer, 1:28-1:8647) after 2-3 doses targeting C3/V5 and/or V1/V2/V3 Env regions by electron microscopy and mutated pseudovirus-based neutralization analyses. Trimer-specific, B-cell-derived monoclonal antibody activities confirmed these results and showed weak heterologous neutralization in the strongest responder. Our findings demonstrate the clinical utility of the 3M-052-AF/alum adjuvant and support further improvements of trimer-based Env immunogens to focus responses on multiple broad nAb epitopes. Key takeaway/take-home messages: HIV BG505 SOSIP.664 trimer with novel 3M-052-AF/alum adjuvant in humans appears safe and induces serum neutralizing antibodies to matched clade A, tier 2 virus, that map to diverse Env epitopes with relatively high titers. The novel adjuvant may be an important mediator of vaccine response.

Monoclonal neutralizing antibodies (mAbs) are considered an important prophylactic against SARS-CoV-2 infection in at-risk populations and a strategy to counteract future sarbecovirus-induced disease. However, most mAbs isolated so far neutralize only a few sarbecovirus strains. Therefore, there is a growing interest in bispecific antibodies (bsAbs) which can simultaneously target different spike epitopes and thereby increase neutralizing breadth and prevent viral escape. Here, we generate and characterize a panel of 30 novel broadly reactive bsAbs using an efficient controlled Fab-arm exchange protocol. We specifically combine some of the broadest mAbs described so far, which target conserved epitopes on the receptor binding domain (RBD). Several bsAbs show superior cross-binding and neutralization compared to the parental mAbs against sarbecoviruses from diverse clades, including recent SARS-CoV-2 variants. BsAbs which include mAb COVA2-02 are among the most potent and broad combinations. As a result, we study the unknown epitope of COVA2-02 and show that this mAb targets a distinct conserved region at the base of the RBD, which could be of interest when designing next-generation bsAb constructs to contribute to a better pandemic preparedness.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as human volunteers challenged with ETEC, diarrheal severity is significantly increased severity in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of human volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected human volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryo-EM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design.

Recent breakthroughs in protein structure prediction have enhanced the precision and speed at which protein configurations can be determined, setting new benchmarks for accuracy and efficiency in the field. However, the fundamental mechanisms of biological processes at a molecular level are often connected to conformational changes of proteins. Molecular dynamics (MD) simulations serve as a crucial tool for capturing the conformational space of proteins, providing valuable insights into their structural fluctuations. However, the scope of MD simulations is often limited by the accessible timescales and the computational resources available, posing challenges to comprehensively exploring protein behaviors. Recently emerging approaches have focused on expanding the capability of AlphaFold2 (AF2) to predict conformational substates of protein structures by manipulating the input multiple sequence alignment (MSA). These approaches operate under the assumption that the MSA also contains information about the heterogeneity of protein structures. Here, we benchmark the performance of various workflows that have adapted AF2 for ensemble prediction focusing on the subsampling of the MSA as implemented in ColabFold and compare the obtained structures with ensembles obtained from MD simulations and NMR. As test cases, we chose four proteins namely the bovine pancreatic inhibitor protein (BPTI), thrombin and two antigen binding fragments (antibody Fv and nanobody), for which reliable experimentally validated structural information (X-ray and/or NMR) was available. Thus, we provide an overview of the levels of performance and accessible timescales that can currently be achieved with machine learning (ML) based ensemble generation. In three out of the four test cases, we find structural variations fall within the predicted ensembles. Nevertheless, significant minima of the free energy surfaces remain undetected. This study highlights the possibilities and pitfalls when generating ensembles with AF2 and thus may guide the development of future tools while informing upon the results of currently available applications.

The study of immunogens capable of eliciting broadly neutralizing antibodies (bnAbs) is crucial for the development of an HIV vaccine. To date, only cows, making use of their ultralong CDRH3 loops, have reliably elicited bnAbs following immunization with HIV Envelope trimers. Antibody responses to the CD4 binding site have been readily elicited by immunization of cows with a stabilized Env trimer of the BG505 strain and, with more difficulty, to the V2-apex region of Env with a cocktail of trimers. Here, we sought to determine whether the BG505 Env trimer could be engineered to generate new bnAb specificities in cows. Since the cow CD4 binding site bnAbs bind to monomeric BG505 gp120, we also sought to determine whether gp120 immunization alone might be sufficient to induce bnAbs. We found that engineering the CD4 binding site by mutation of a key binding residue of BG505 HIV Env resulted in a reduced bnAb response that took more immunizations to develop. Monoclonal antibodies isolated from one animal were directed to the V2-apex, suggesting a re-focusing of the bnAb response. Immunization with monomeric BG505 g120 generated no serum bnAb responses, indicating that the ultralong CDRH3 bnAbs are only elicited in the context of the trimer in the absence of many other less restrictive epitopes presented on monomeric gp120. The results support the notion of a hierarchy of epitopes on HIV Env and suggest that, even with the presence in the cow repertoire of ultralong CDRH3s, bnAb epitopes are relatively disfavored.