Nanoparticles for multivalent display and delivery of vaccine antigens have emerged as\n\n\na promising avenue for enhancing B cell responses to protein subunit vaccines. Here,\n\n\nwe evaluated B cell responses in rhesus macaques immunized with prefusion stabilized\n\n\nRespiratory Syncytial Virus (RSV) F glycoprotein trimer compared to\n\n\nnanoparticles displaying 10 or 20 copies of the same antigen. We show that multivalent\n\n\ndisplay skews antibody specificities and drives epitope-focusing of responding B cells.\n\n\nAntibody cloning and repertoire sequencing revealed that focusing was driven by\n\n\nexpansion of clonally distinct B cells through recruitment of diverse precursors. We\n\n\nidentified two antibody lineages that developed either ultrapotent neutralization or\n\n\npneumovirus cross-neutralization from precursor B cells with low initial affinity for the\n\n\nRSV-F immunogen. This suggests that increased avidity by multivalent display\n\n\nfacilitates the activation and recruitment of these cells. Diversification of the B cell\n\n\nresponse by multivalent nanoparticle immunogens has broad implications for vaccine\n\n\ndesign.

Atypical B cells (ABCs), characterized by CD11c expression, expand during infection, but their developmental routes and functions remain unclear. Using flow cytometry, single-cell transcriptomics, V(D)J sequencing, and functional assays in human malaria, we mapped ABC differentiation. Two intermediate states emerged: CD27+ switched memory-derived and CD24+ non-switched memory-derived populations. Both converged toward a late ABC phenotype defined by loss of CD21, and reduced plasma cell potential. Pseudotime and clonal analyses showed distinct, isotype-dependent trajectories accompanied by progressive CD24/CD27 loss. In vitro stimulation recapitulated this progressive loss of CD24 and CD27. Similar subsets were also present in healthy blood and tonsils, where class switching and CD86 expression were more frequent, consistent with recent T cell interaction. Thus, combined CD11c, CD24, and CD27 profiling resolves ABC heterogeneity and reveals origin-dependent pathways across tissues, providing a framework for future studies in health and disease, and addressing limitations of previous nomenclature approaches. Main novelty ### Competing Interest Statement The authors have declared no competing interest. Swedish Research Council, https://ror.org/03zttf063, 2019-0194, 2023-01943, 521-2012-3311, 2015-02977, 2018-02688, 2018-04468, 2021-03105 M-CM-^Eke Wibergs Stiftelse, M18-0076 Magnus Bergvalls Stiftelse, 2017-02043, 2018-02656 Stockholm County Council, https://ror.org/02zrae794, 20150135, 20180409, 960104, 986923

Nanoparticles for multivalent display and delivery of vaccine antigens have emerged as a promising avenue for enhancing B cell responses to protein subunit vaccines. Here, we evaluated B cell responses in rhesus macaques immunized with prefusion-stabilized respiratory syncytial virus (RSV) F glycoprotein trimer compared with nanoparticles displaying 10 or 20 copies of the same antigen. We show that multivalent display skews antibody specificities and drives epitope-focusing of responding B cells. Antibody cloning and repertoire sequencing revealed that focusing was driven by the expansion of clonally distinct B cells through recruitment of diverse precursors. We identified two antibody lineages that developed either ultrapotent neutralization or pneumovirus cross-neutralization from precursor B cells with low initial affinity for the RSV-F immunogen. This suggests that increased avidity by multivalent display facilitates the activation and recruitment of these cells. Diversification of the B cell response by multivalent nanoparticle immunogens has broad implications for vaccine design.

Common variable immune deficiency (CVID) is a heterogeneous disorder characterized by recurrent infections, low levels of serum immunoglobulins, and impaired vaccine responses. Autoimmune manifestations are common, but B cell central and peripheral selection mechanisms in CVID are incompletely understood. Here, we find that receptor editing, a measure of central tolerance, is increased in transitional B cells from CVID patients and that these cells have a higher immunoglobulin κ:λ ratio in CVID patients with autoimmune manifestations than in those with infection only. Contrariwise, the selection pressure in the germinal center on CD27bright memory B cells is decreased in CVID patients with autoimmune manifestations. Finally, functionally, T cell-dependent activation showed that naive B cells in CVID patients are badly equipped for activation and induction of mismatch repair genes. We conclude that central tolerance is functional whereas peripheral selection is defective in CVID patients with autoimmune manifestations, which could underpin the development of autoimmunity.

Memory B cells persist for a lifetime and rapidly differentiate into antibody-producing plasmablasts and plasma cells upon antigen re-encounter. The clonal relationship and evolution of memory B cells and circulating plasmablasts is not well understood. Using single-cell sequencing combined with isolation of specific antibodies, we found that in two healthy donors, the memory B cell repertoire was dominated by large IgM, IgA and IgG2 clonal families, whereas IgG1 families, including those specific for recall antigens, were of small size. Analysis of multiyear samples demonstrated stability of memory B cell clonal families and revealed that a large fraction of recently generated plasmablasts was derived from long-term memory B cell families and was found recurrently. Collectively, this study provides a systematic description of the structure, stability and dynamics of the human memory B cell pool and suggests that memory B cells may be active at any time point in the generation of plasmablasts.

Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 ug/ml (IC 50 ). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a β-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184-186) that is unique to the 16055 strain. They also provide an explanation for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies. Author summary NHPs immunizations with an HIV-1 immunogen (native-like tier 2 clade C 16055 strain) elicit potent HIV-1 tier 2 autologous polyclonal neutralizing antibodies. To understand the basis of the autologous neutralization, we determined structures of antibodies isolated from the vaccinated NHPs in complex with their epitopes. Our structural analysis reveals that the V2 hypervariable region, unique to 16055, is immunodominant and targeted by antibodies from diverse lineages. Additionally, vaccine-elicited V2 NAbs use different binding angles to avoid Env N-glycan shield and the more negatively charged paratope displays potent autologous neutralizing function. In summary, detailed analysis of how vaccine-elicited monoclonal antibodies interact with the target antigen provide valuable information for the design of immunogens aimed to elicit more broadly HIV-neutralizing antibodies. The use of cocktail/prime-boost sequential regimens that include a range of sequence variation combined with the removal/shielding of unwanted immunodominant epitopes will likely be needed to reach this goal.

Well-ordered HIV-1 envelope glycoprotein (Env) trimers are prioritized for clinical evaluation, and there is a need for an improved understanding about how elicited B cell responses evolve following immunization. To accomplish this, we prime-boosted rhesus macaques with clade C NFL trimers and identified 180 unique Ab lineages from ∼1,000 single-sorted Env-specific memory B cells. We traced all lineages in high-throughput heavy chain (HC) repertoire (Rep-seq) data generated from multiple immune compartments and time points and expressed several as monoclonal Abs (mAbs). Our results revealed broad dissemination and high levels of somatic hypermutation (SHM) of most lineages, including tier 2 virus neutralizing lineages, following boosting. SHM was highest in the Ab complementarity determining regions (CDRs) but also surprisingly high in the framework regions (FRs), especially FR3. Our results demonstrate the capacity of the immune system to affinity-mature large numbers of Env-specific B cell lineages simultaneously, supporting the use of regimens consisting of repeated boosts to improve each Ab, even those belonging to less expanded lineages.

Next generation sequencing (NGS) of immunoglobulin (Ig) repertoires (Rep-seq) enables examination of the adaptive immune system at an unprecedented level. Applications include studies of expressed repertoires, gene usage, somatic hypermutation levels, Ig lineage tracing and identification of genetic variation within the Ig loci through inference methods. All these applications require starting libraries that allow the generation of sequence data with low error rate and optimal representation of the expressed repertoire. Here, we provide detailed protocols for the production of libraries suitable for human Ig germline gene inference and Ig repertoire studies. Various parameters used in the process were tested in order to demonstrate factors that are critical to obtain high quality libraries. We demonstrate an improved 5'RACE technique that reduces the length constraints of Illumina MiSeq based Rep-seq analysis but allows for the acquisition of sequences upstream of Ig V genes, useful for primer design. We then describe a 5' multiplex method for library preparation, which yields full length V(D)J sequences suitable for genotype identification and novel gene inference. We provide comprehensive sets of primers targeting IGHV, IGKV, and IGLV genes. Using the optimized protocol, we produced IgM, IgG, IgK, and IgL libraries and analyzed them using the germline inference tool IgDiscover to identify expressed germline V alleles. This process additionally uncovered three IGHV, one IGKV, and six IGLV novel alleles in a single individual, which are absent from the IMGT reference database, highlighting the need for further study of Ig genetic variation. The library generation protocols presented here enable a robust means of analyzing expressed Ig repertoires, identifying novel alleles and producing individualized germline gene databases from humans.

In the work presented in this thesis, I characterized B cell responses in rhesus macaques inoculated with HIV-1 envelope glycoprotein (Env)-based vaccines to define genetic and functional properties of the elicited antibodies for an improved understanding of Env immunogenicity. In Paper I, we investigated antibody heavy chain V gene segment expression in the overall B cell repertoire and in response to vaccination by using 2 approaches, 454-pyrosequencing and single-cell RT-PCR of bulk and sorted memory B cells, respectively. Both methods gave highly concordant results and identified differential expression of the Ig gene segments. We further investigated the VH repertoire of antigen-specific memory B cells induced by an HIV-1 Env trimer immunogen. We found that the antibody response against Env was highly polyclonal consisting of most of the expressed VH segments in immunized subject. In Paper II, we isolated and comprehensively characterized genetic properties of a panel of vaccine-induced MAbs from Env immunized subjects. We demonstrated that the MAbs were genetically diverse with no bias in their V gene usage towards the distinct epitope regions they were mapped to, consistent with a highly polyclonal response. MAbs targeting two sub-regions, the CD4 binding site (CD4bs) and variable region 3 (V3), were neutralizing. The combined activity of these MAbs recapitulated the neutralizing activity observed in unfractionated plasma samples of the animals from which they were isolated, consistent with that these were the predominant specificities elicited. In Paper III, we used Illumina highthroughput sequencing (HTS) and developed a novel computational tool, IgDiscover, for germline Ig V allele identification. Previous work using targeted genomic PCR of germline V genes from Chinese rhesus macaques revealed a striking allelic diversity between animals and the presence of many novel alleles, highlighting the limitations of existing macaque V gene databases. By using IgDiscover to construct individualized Ig germline gene databases, we can improve the accuracy of Ab repertoire analyses in outbred populations considerably. In Paper IV, we investigated B cell responses induced by a new generation, well-ordered Env trimers. We used HTS sequencing to investigate how the response evolved over time and whether it disseminated into different immune compartments. We developed an IgDiscover extension module, Clonoquery, to trace hundreds of B cell clonal lineages targeting distinct sub-regions of Env in the peripheral blood, bone marrow, spleen, draining lymph node and gut. We detected broad dissemination of Env-specific B cell clones in all compartments measured, except gut. We showed that some lineages were greatly expanded and that the mean level of somatic hypermutation of the variants within each lineage increased with boosting, replacing previous variants from the same lineage in both blood and bone marrow. In conclusion, this thesis offers a new information about the genetic and functional composition of Env-specific B cell responses in immunized rhesus macaques, a highly relevant model for understanding vaccine-induced response in humans. It also offers valuable information about the rhesus macaque Ig repertoire in general as well as broadly applicable tools for repertoire sequencing. LIST OF PUBLICATIONS I. Christopher Sundling, Zhenhai Zhang, Ganesh E. Phad, Zizhang Sheng, Yimeng Wang, John R. Mascola, Yuxing Li, Richard T. Wyatt, Lawrence Shapiro and Gunilla B. Karlsson Hedestam. Single-cell and deep sequencing of IgG-switched macaque B cells reveal a diverse Ig repertoire following immunization. Journal of Immunology. 2014, 192(8):3637-44. II. f f Ganesh E. Phad, Néstor Vázquez Bernat, Yu Feng, Jidnyasa Ingale, Paola Andrea Martinez Murillo, Sijy O'Dell, Yuxing Li, John R. Mascola, Christopher Sundling, Richard T. Wyatt and Gunilla B. Karlsson Hedestam. Diverse antibody genetic and recognition properties revealed following HIV-1 envelope glycoprotein immunization. Journal of Immunology. 2015, 194(12):5903-14. III. k Martin M. Corcoran*, Ganesh E. Phad*, Néstor Vázquez Bernat, Christiane Stahl-Hennig, Noriyuki Sumida, Mats A.A. Persson, Marcel Martin, Gunilla B. Karlsson Hedestam. Production of individualized V gene databases reveals high levels of immunoglobulin genetic diversity. Nature Communications. 2016, 7:13642. *Shared first authors IV. c Ganesh E. Phad†, Pradeepa Pushparaj, Karen Tran, Paola Martinez-Murillo, Monika Adori, Viktoriya Dubrovskaya, Néstor Vázquez Bernat, Komal Bhullar, Sanjana Narang, Chiara Sorini, Eduardo Villablanca, Sijy O’Dell, John Mascola, Christopher Sundling, Marcel Martin, Ben Murrell, Martin Corcoran, Richard Wyatt and Gunilla B. Karlsson Hedestam†. Clonal lineage tracing reveals dissemination and maturation of vaccine-induced B cell responses in multiple immune compartments (Manuscript) †Corresponding authors PUBLICATIONS NOT INCLUDED IN THIS THESIS I. Martinez-Murillo P*, Tran K*, Guenaga J, Lindgren G, Àdori M, Feng Y, Phad GE, Vázquez Bernat N, Bale S, Ingale J, Dubrovskaya V, O'Dell S, Pramanik L, Spångberg M, Corcoran M, Loré K, Mascola JR, Wyatt RT, Karlsson Hedestam GB. Particulate Array of Well-Ordered HIV Clade C Env Trimers Elicits Neutralizing Antibodies that Display a Unique V2 Cap Approach. Immunity. 2017, 46(5):804817. *Shared first authors II. Wang Y, Sundling C, Wilson R, O'Dell S, Chen Y, Dai K, Phad GE, Zhu J, Xiao Y, Mascola JR, Karlsson Hedestam GB, Wyatt RT, Li Y. High-Resolution Longitudinal Study of HIV-1 Env Vaccine-Elicited B Cell Responses to the Virus Primary Receptor Binding Site Reveals Affinity Maturation and Clonal Persistence. Journal of Immunology. 2016, 196(9):3729-43. III. Madani N, Princiotto AM, Easterhoff D, Bradley T, Luo K, Williams WB, Liao HX, Moody MA, Phad GE, Vázquez Bernat N, Melillo B, Santra S, Smith AB 3rd, Karlsson Hedestam GB, Haynes B, Sodroski J. Antibodies Elicited by Multiple Envelope Glycoprotein Immunogens in Primates Neutralize Primary Human Immunodeficiency Viruses (HIV-1) Sensitized by CD4-Mimetic Compounds. Journal of Virology. 2016, 90(10):5031-46. IV. Dai K, Khan SN, Wang Y, He L, Guenaga J, Ingale J, Sundling C, O'Dell S, McKee K, Phad GE, Corcoran M, Wilson R, Mascola JR, Zhu J, Li Y, Karlsson Hedestam GB, Wyatt RT. HIV-1 Vaccine-elicited Antibodies Reverted to Their Inferred Naive Germline Reveal Associations between Binding Affinity and in vivo Activation. Scientific Reports. 2016, 6:20987. V. Klein F, Nogueira L, Nishimura Y, Phad G, West AP Jr, Halper-Stromberg A, Horwitz JA, Gazumyan A, Liu C, Eisenreich TR, Lehmann C, Fätkenheuer G, Williams C, Shingai M, Martin MA, Bjorkman PJ, Seaman MS, Zolla-Pazner S, Karlsson Hedestam GB, Nussenzweig MC. Enhanced HIV-1 immunotherapy by commonly arising antibodies that target virus escape variants. Journal of Experimental Medicine. 2014, 211(12):2361-72. VI. Navis M, Tran K, Bale S, Phad GE, Guenaga J, Wilson R, Soldemo M, McKee K, Sundling C, Mascola J, Li Y, Wyatt RT, Karlsson Hedestam GB. HIV-1 receptor binding site-directed antibodies using a VH1-2 gene segment orthologue are activated by Env trimer immunization. PLoS Pathology. 2014,10(8):e1004337. VII. Sundling C, Phad G, Douagi I, Navis M, Karlsson Hedestam GB. Isolation of antibody V(D)J sequences from single cell sorted rhesus macaque B cells. Journal of Immunological Methods. 2012, 386(1-2):85-93.

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.

Comprehensive knowledge of immunoglobulin genetics is required to advance our understanding of B cell biology. Validated immunoglobulin variable (V) gene databases are close to completion only for human and mouse. We present a novel computational approach, IgDiscover, that identifies germline V genes from expressed repertoires to a specificity of 100%. IgDiscover uses a cluster identification process to produce candidate sequences that, once filtered, results in individualized germline V gene databases. IgDiscover was tested in multiple species, validated by genomic cloning and cross library comparisons and produces comprehensive gene databases even where limited genomic sequence is available. IgDiscover analysis of the allelic content of the Indian and Chinese-origin rhesus macaques reveals high levels of immunoglobulin gene diversity in this species. Further, we describe a novel human IGHV3-21 allele and confirm significant gene differences between Balb/c and C57BL6 mouse strains, demonstrating the power of IgDiscover as a germline V gene discovery tool.

The elicitation of HIV-1 broadly neutralizing antibodies following envelope glycoprotein (Env) vaccination is exceedingly difficult. Suboptimal engagement of naïve B cells is suggested to limit these low frequency events, especially at the conserved CD4bs. Here, we analyzed CD4bs-directed monoclonal antibodies (mAbs) elicited by YU2 gp140-foldon trimers in a non-human primate by selective sorting using CD4bs "knock out" trimers. Following two inoculations, the CD4bs-directed mAbs efficiently recognized the eliciting immunogen in their affinity-maturing state but did not recognize CD4bs-defective probes. We reverted these mAbs to their most likely inferred germline (igL) state, leaving the HCDR3 unaltered, to establish correlates of in vitro affinity to in vivo activation. Most igL-reverted mAbs bound the eliciting gp140 immunogen, indicating that CD4bs-directed B cells possessing reasonable affinity existed in the naïve repertoire. We detected relatively high affinities for the majority of the igL mAbs to gp120 and of Fabs to gp140, which, as expected, increased when the antibodies 'matured' following vaccination. Affinity increases were associated with slower off-rates as well as with acquisition of neutralizing capacity. These data reveal in vitro binding properties associated with in vivo activation that result in functional archiving of antigen-specific B cells elicited by a complex glycoprotein antigen following immunization.

ABSTRACT The human immunodeficiency virus (HIV-1) envelope glycoproteins (Env) mediate virus entry through a series of complex conformational changes triggered by binding to the receptors CD4 and CCR5/CXCR4. Broadly neutralizing antibodies that recognize conserved Env epitopes are thought to be an important component of a protective immune response. However, to date, HIV-1 Env immunogens that elicit broadly neutralizing antibodies have not been identified, creating hurdles for vaccine development. Small-molecule CD4-mimetic compounds engage the CD4-binding pocket on the gp120 exterior Env and induce Env conformations that are highly sensitive to neutralization by antibodies, including antibodies directed against the conserved Env region that interacts with CCR5/CXCR4. Here, we show that CD4-mimetic compounds sensitize primary HIV-1 to neutralization by antibodies that can be elicited in monkeys and humans within 6 months by several Env vaccine candidates, including gp120 monomers. Monoclonal antibodies directed against the gp120 V2 and V3 variable regions were isolated from the immunized monkeys and humans; these monoclonal antibodies neutralized a primary HIV-1 only when the virus was sensitized by a CD4-mimetic compound. Thus, in addition to their direct antiviral effect, CD4-mimetic compounds dramatically enhance the HIV-1-neutralizing activity of antibodies that can be elicited with currently available immunogens. Used as components of microbicides, the CD4-mimetic compounds might increase the protective efficacy of HIV-1 vaccines. IMPORTANCE Preventing HIV-1 transmission is a high priority for global health. Eliciting antibodies that can neutralize transmitted strains of HIV-1 is difficult, creating problems for the development of an effective vaccine. We found that small-molecule CD4-mimetic compounds sensitize HIV-1 to antibodies that can be elicited in vaccinated humans and monkeys. These results suggest an approach to prevent HIV-1 sexual transmission in which a virus-sensitizing microbicide is combined with a vaccine.

Because of the genetic variability of the HIV-1 envelope glycoproteins (Env), the elicitation of neutralizing Abs to conserved neutralization determinants including the primary receptor binding site, CD4 binding site (CD4bs), is a major focus of vaccine development. To gain insight into the evolution of Env-elicited Ab responses, we used single B cell analysis to interrogate the memory B cell Ig repertoires from two rhesus macaques after five serial immunizations with Env/adjuvant. We observed that the CD4bs-specific repertoire displayed unique features in the third CDR of Ig H chains with minor alterations along the immunization course. Progressive affinity maturation occurred as evidenced by elevated levels of somatic hypermutation (SHM) in Ab sequences isolated at the late immunization time point compared with the early time point. Abs with higher SHM were associated with increased binding affinity and virus neutralization capacity. Moreover, a notable portion of the CD4bs-specific repertoire was maintained between early and late immunization time points, suggesting that persistent clonal lineages were induced by Env vaccination. Furthermore, we found that the predominant persistent CD4bs-specific clonal lineages had larger population sizes and higher affinities than that from the rest of the repertoires, underscoring the critical role of Ag affinity selection in Ab maturation and clonal expansion. Genetic and functional analyses revealed that the accumulation of SHM in both framework regions and CDRs contributed to the clonal affinity and antigenicity evolution. Our longitudinal study provides high-resolution understanding of the dynamically evolving CD4bs-specific B cell response after Env immunization in primates.

Isolation of mAbs elicited by vaccination provides opportunities to define the development of effective immunity. Ab responses elicited by current HIV-1 envelope glycoprotein (Env) immunogens display narrow neutralizing activity with limited capacity to block infection by tier 2 viruses. Intense work in the field suggests that improved Env immunogens are forthcoming, and it is therefore important to concurrently develop approaches to investigate the quality of vaccine-elicited responses at a higher level of resolution. In this study, we cloned a representative set of mAbs elicited by a model Env immunogen in rhesus macaques and comprehensively characterized their genetic and functional properties. The mAbs were genetically diverse, even within groups of Abs targeting the same subregion of Env, consistent with a highly polyclonal response. mAbs directed against two subdeterminants of Env, the CD4 binding site and V region 3, could in part account for the neutralizing activity observed in the plasma of the animal from which they were cloned, demonstrating the power of mAb isolation for a detailed understanding of the elicited response. Finally, through comparative analyses of mAb binding and neutralizing capacity of HIV-1 using matched Envs, we demonstrate complex relationships between epitope recognition and accessibility, highlighting the protective quaternary packing of the HIV-1 spike relative to vaccine-induced mAbs.