Publications by authors named "Sergey Menis"

36 Publications

Targeting HIV Env immunogens to B cell follicles in nonhuman primates through immune complex or protein nanoparticle formulations.

NPJ Vaccines 2020 Aug 5;5(1):72. Epub 2020 Aug 5.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

Following immunization, high-affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble human immunodeficiency virus (HIV) envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here, we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single LN within two days after immunization. Imaging of LNs collected seven days postimmunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent GCs. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with ICs or nanoparticles.
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http://dx.doi.org/10.1038/s41541-020-00223-1DOI Listing
August 2020

B cells expressing authentic naive human VRC01-class BCRs can be recruited to germinal centers and affinity mature in multiple independent mouse models.

Proc Natl Acad Sci U S A 2020 09 1;117(37):22920-22931. Epub 2020 Sep 1.

Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology, La Jolla, CA 92037;

Animal models of human antigen-specific B cell receptors (BCRs) generally depend on "inferred germline" sequences, and thus their relationship to authentic naive human B cell BCR sequences and affinities is unclear. Here, BCR sequences from authentic naive human VRC01-class B cells from healthy human donors were selected for the generation of three BCR knockin mice. The BCRs span the physiological range of affinities found in humans, and use three different light chains (VK3-20, VK1-5, and VK1-33) found among subclasses of naive human VRC01-class B cells and HIV broadly neutralizing antibodies (bnAbs). The germline-targeting HIV immunogen eOD-GT8 60mer is currently in clinical trial as a candidate bnAb vaccine priming immunogen. To attempt to model human immune responses to the eOD-GT8 60mer, we tested each authentic naive human VRC01-class BCR mouse model under rare human physiological B cell precursor frequency conditions. B cells with high (HuGL18) or medium (HuGL17) affinity BCRs were primed, recruited to germinal centers, and they affinity matured, and formed memory B cells. Precursor frequency and affinity interdependently influenced responses. Taken together, these experiments utilizing authentic naive human VRC01-class BCRs validate a central tenet of germline-targeting vaccine design and extend the overall concept of the reverse vaccinology approach to vaccine development.
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http://dx.doi.org/10.1073/pnas.2004489117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502816PMC
September 2020

Multifaceted Effects of Antigen Valency on B Cell Response Composition and Differentiation In Vivo.

Immunity 2020 09 27;53(3):548-563.e8. Epub 2020 Aug 27.

Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology (LJI), La Jolla, CA 92037, USA; Scripps Consortium for HIV/AIDS Vaccine Development (CHAVD), La Jolla, CA 92037, USA; Department of Medicine, Division of Infectious Diseases and Global Public Health, University of California, San Diego (UCSD), La Jolla, CA 92037, USA. Electronic address:

How antigen valency affects B cells in vivo during immune responses is not well understood. Here, using HIV immunogens with defined valencies ranging from 1 to 60, we investigated the role of antigen valency during different phases of B cell responses in vivo. Highly multimerized immunogens preferentially rapidly activated cognate B cells, with little affinity discrimination. This led to strong early induction of the transcription factors IRF4 (interferon regulatory factor 4) and Bcl6, driving both early extrafollicular plasma cell and germinal center responses, in a CD4 T-cell-dependent manner, involving B cells with a broad range of affinities. Low-valency antigens induced smaller effector B cell responses, with preferential recruitment of high-affinity B cells. Thus, antigen valency has multifaceted effects on B cell responses and can dictate affinity thresholds and competitive landscapes for B cells in vivo, with implications for vaccine design.
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http://dx.doi.org/10.1016/j.immuni.2020.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7451196PMC
September 2020

Targeting HIV Env immunogens to B cell follicles in nonhuman primates through immune complex or protein nanoparticle formulations.

NPJ Vaccines 2020 5;5:72. Epub 2020 Aug 5.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139 USA.

Following immunization, high-affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble human immunodeficiency virus (HIV) envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here, we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single LN within two days after immunization. Imaging of LNs collected seven days postimmunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent GCs. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with ICs or nanoparticles.
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http://dx.doi.org/10.1038/s41541-020-00223-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406516PMC
August 2020

In Vivo Assembly of Nanoparticles Achieved through Synergy of Structure-Based Protein Engineering and Synthetic DNA Generates Enhanced Adaptive Immunity.

Adv Sci (Weinh) 2020 Apr 27;7(8):1902802. Epub 2020 Feb 27.

The Vaccine and Immunotherapy Center The Wistar Institute Philadelphia PA 19104 USA.

Nanotechnologies are considered to be of growing importance to the vaccine field. Through decoration of immunogens on multivalent nanoparticles, designed nanovaccines can elicit improved humoral immunity. However, significant practical and monetary challenges in large-scale production of nanovaccines have impeded their widespread clinical translation. Here, an alternative approach is illustrated integrating computational protein modeling and adaptive electroporation-mediated synthetic DNA delivery, thus enabling direct in vivo production of nanovaccines. DNA-launched nanoparticles are demonstrated displaying an HIV immunogen spontaneously self-assembled in vivo. DNA-launched nanovaccines induce stronger humoral responses than their monomeric counterparts in both mice and guinea pigs, and uniquely elicit CD8+ effector T-cell immunity as compared to recombinant protein nanovaccines. Improvements in vaccine responses recapitulate when DNA-launched nanovaccines with alternative scaffolds and decorated antigen are designed and evaluated. Finally, evaluation of functional immune responses induced by DLnanovaccines demonstrates that, in comparison to control mice or mice immunized with DNA-encoded hemagglutinin monomer, mice immunized with a DNA-launched hemagglutinin nanoparticle vaccine fully survive a lethal influenza challenge, and have substantially lower viral load, weight loss, and influenza-induced lung pathology. Additional study of these next-generation in vivo-produced nanovaccines may offer advantages for immunization against multiple disease targets.
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http://dx.doi.org/10.1002/advs.201902802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175333PMC
April 2020

Engineered immunogen binding to alum adjuvant enhances humoral immunity.

Nat Med 2020 03 17;26(3):430-440. Epub 2020 Feb 17.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.

Adjuvants are central to the efficacy of subunit vaccines. Aluminum hydroxide (alum) is the most commonly used vaccine adjuvant, yet its adjuvanticity is often weak and mechanisms of triggering antibody responses remain poorly understood. We demonstrate that site-specific modification of immunogens with short peptides composed of repeating phosphoserine (pSer) residues enhances binding to alum and prolongs immunogen bioavailability. The pSer-modified immunogens formulated in alum elicited greatly increased germinal center, antibody, neutralizing antibody, memory and long-lived plasma cell responses compared to conventional alum-adsorbed immunogens. Mechanistically, pSer-immunogen:alum complexes form nanoparticles that traffic to lymph nodes and trigger B cell activation through multivalent and oriented antigen display. Direct uptake of antigen-decorated alum particles by B cells upregulated antigen processing and presentation pathways, further enhancing B cell activation. These data provide insights into mechanisms of action of alum and introduce a readily translatable approach to significantly improve humoral immunity to subunit vaccines using a clinical adjuvant.
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http://dx.doi.org/10.1038/s41591-020-0753-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069805PMC
March 2020

Immunogenicity of RNA Replicons Encoding HIV Env Immunogens Designed for Self-Assembly into Nanoparticles.

Mol Ther 2019 12 19;27(12):2080-2090. Epub 2019 Aug 19.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA, USA; Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Department of Biological Engineering, MIT, Cambridge, MA, USA; Materials Science & Engineering, MIT, Cambridge, MA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA. Electronic address:

RNA replicons are a promising platform technology for vaccines. To evaluate the potential of lipid nanoparticle-formulated replicons for delivery of HIV immunogens, we designed and tested an alphavirus replicon expressing a self-assembling protein nanoparticle immunogen, the glycoprotein 120 (gp120) germline-targeting engineered outer domain (eOD-GT8) 60-mer. The eOD-GT8 immunogen is a germline-targeting antigen designed to prime human B cells capable of evolving toward VRC01-class broadly neutralizing antibodies. Replicon RNA was encapsulated with high efficiency in 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based lipid nanoparticles, which provided effective delivery in the muscle and expression of luciferase lasting ∼30 days in normal mice, contrasting with very brief and low levels of expression obtained by delivery of equivalent modified mRNA (modRNA). eOD-GT8 60-mer-encoding replicons elicited high titers of gp120-specific antibodies following a single injection in mice, and increased levels of antigen-specific germinal center B cells compared with protein immunization. Immunization of transgenic mice expressing human inferred-germline VRC01 heavy chain B cell receptors that are the targets of the eOD antigen led to priming of B cells and somatic hypermutation consistent with VRC01-class antibody development. Altogether, these data suggest replicon delivery of Env immunogens may be a promising avenue for HIV vaccine development.
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http://dx.doi.org/10.1016/j.ymthe.2019.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904793PMC
December 2019

Enhancing humoral immunity via sustained-release implantable microneedle patch vaccination.

Proc Natl Acad Sci U S A 2019 08 29;116(33):16473-16478. Epub 2019 Jul 29.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139;

Sustained exposure of lymphoid tissues to vaccine antigens promotes humoral immunity, but traditional bolus immunizations lead to rapid antigen clearance. We describe a technology to tailor vaccine kinetics in a needle-free platform translatable to human immunization. Solid pyramidal microneedle (MN) arrays were fabricated with silk fibroin protein tips encapsulating a stabilized HIV envelope trimer immunogen and adjuvant, supported on a dissolving polymer base. Upon brief skin application, vaccine-loaded silk tips are implanted in the epidermis/upper dermis where they release vaccine over a time period determined by the crystallinity of the silk matrix. Following MN immunization in mice, Env trimer was released over 2 wk in the skin, correlating with increased germinal center (GC) B cell responses, a ∼1,300-fold increase in serum IgG titers and a 16-fold increase in bone marrow (BM) plasma cells compared with bolus immunization. Thus, implantable MNs provide a practical means to substantially enhance humoral immunity to subunit vaccines.
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http://dx.doi.org/10.1073/pnas.1902179116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697788PMC
August 2019

Rapid and Focused Maturation of a VRC01-Class HIV Broadly Neutralizing Antibody Lineage Involves Both Binding and Accommodation of the N276-Glycan.

Immunity 2019 07;51(1):141-154.e6

International AIDS Vaccine Initiative Neutralizing Antibody Center, La Jolla, CA 92037, USA; International AIDS Vaccine Initiative, New York, NY 10004, USA; Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

The VH1-2 restricted VRC01-class of antibodies targeting the HIV envelope CD4 binding site are a major focus of HIV vaccine strategies. However, a detailed analysis of VRC01-class antibody development has been limited by the rare nature of these responses during natural infection and the lack of longitudinal sampling of such responses. To inform vaccine strategies, we mapped the development of a VRC01-class antibody lineage (PCIN63) in the subtype C infected IAVI Protocol C neutralizer PC063. PCIN63 monoclonal antibodies had the hallmark VRC01-class features and demonstrated neutralization breadth similar to the prototype VRC01 antibody, but were 2- to 3-fold less mutated. Maturation occurred rapidly within ∼24 months of emergence of the lineage and somatic hypermutations accumulated at key contact residues. This longitudinal study of broadly neutralizing VRC01-class antibody lineage reveals early binding to the N276-glycan during affinity maturation, which may have implications for vaccine design.
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http://dx.doi.org/10.1016/j.immuni.2019.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6642152PMC
July 2019

Slow Delivery Immunization Enhances HIV Neutralizing Antibody and Germinal Center Responses via Modulation of Immunodominance.

Cell 2019 05 9;177(5):1153-1171.e28. Epub 2019 May 9.

Division of Vaccine Discovery, La Jolla Institute for Immunology (LJI), La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (Scripps CHAVI-ID), The Scripps Research Institute, La Jolla, CA 92037, USA; Department of Medicine, University of California, San Diego, La Jolla, CA 92037, USA. Electronic address:

Conventional immunization strategies will likely be insufficient for the development of a broadly neutralizing antibody (bnAb) vaccine for HIV or other difficult pathogens because of the immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. We found that two independent methods of slow delivery immunization of rhesus monkeys (RMs) resulted in more robust T follicular helper (T) cell responses and GC B cells with improved Env-binding, tracked by longitudinal fine needle aspirates. Improved GCs correlated with the development of >20-fold higher titers of autologous nAbs. Using a new RM genomic immunoglobulin locus reference, we identified differential IgV gene use between immunization modalities. Ab mapping demonstrated targeting of immunodominant non-neutralizing epitopes by conventional bolus-immunized animals, whereas slow delivery-immunized animals targeted a more diverse set of epitopes. Thus, alternative immunization strategies can enhance nAb development by altering GCs and modulating the immunodominance of non-neutralizing epitopes.
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http://dx.doi.org/10.1016/j.cell.2019.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6619430PMC
May 2019

Innate immune recognition of glycans targets HIV nanoparticle immunogens to germinal centers.

Science 2019 02 20;363(6427):649-654. Epub 2018 Dec 20.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

In vaccine design, antigens are often arrayed in a multivalent nanoparticle form, but in vivo mechanisms underlying the enhanced immunity elicited by such vaccines remain poorly understood. We compared the fates of two different heavily glycosylated HIV antigens, a gp120-derived mini-protein and a large, stabilized envelope trimer, in protein nanoparticle or "free" forms after primary immunization. Unlike monomeric antigens, nanoparticles were rapidly shuttled to the follicular dendritic cell (FDC) network and then concentrated in germinal centers in a complement-, mannose-binding lectin (MBL)-, and immunogen glycan-dependent manner. Loss of FDC localization in MBL-deficient mice or via immunogen deglycosylation significantly affected antibody responses. These findings identify an innate immune-mediated recognition pathway promoting antibody responses to particulate antigens, with broad implications for humoral immunity and vaccine design.
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http://dx.doi.org/10.1126/science.aat9120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420719PMC
February 2019

Enhancing Humoral Responses Against HIV Envelope Trimers via Nanoparticle Delivery with Stabilized Synthetic Liposomes.

Sci Rep 2018 11 8;8(1):16527. Epub 2018 Nov 8.

Koch Institute, MIT, Massachusetts, USA.

An HIV vaccine capable of eliciting durable neutralizing antibody responses continues to be an important unmet need. Multivalent nanoparticles displaying a high density of envelope trimers may be promising immunogen forms to elicit strong and durable humoral responses to HIV, but critical particle design criteria remain to be fully defined. To this end, we developed strategies to covalently anchor a stabilized gp140 trimer, BG505 MD39, on the surfaces of synthetic liposomes to study the effects of trimer density and vesicle stability on vaccine-elicited humoral responses in mice. CryoEM imaging revealed homogeneously distributed and oriented MD39 on the surface of liposomes irrespective of particle size, lipid composition, and conjugation strategy. Immunization with covalent MD39-coupled liposomes led to increased germinal center and antigen-specific T follicular helper cell responses and significantly higher avidity serum MD39-specific IgG responses compared to immunization with soluble MD39 trimers. A priming immunization with liposomal-MD39 was important for elicitation of high avidity antibody responses, regardless of whether booster immunizations were administered with either soluble or particulate trimers. The stability of trimer anchoring to liposomes was critical for these effects, as germinal center and output antibody responses were further increased by liposome compositions incorporating sphingomyelin that exhibited high in vitro stability in the presence of serum. Together these data highlight key liposome design features for optimizing humoral immunity to lipid nanoparticle immunogens.
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http://dx.doi.org/10.1038/s41598-018-34853-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224390PMC
November 2018

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

Nat Commun 2018 09 12;9(1):3693. Epub 2018 Sep 12.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, 92037, USA.

As the sole target of broadly neutralizing antibodies (bnAbs) to HIV, the envelope glycoprotein (Env) trimer is the focus of vaccination strategies designed to elicit protective bnAbs in humans. Because HIV Env is densely glycosylated with 75-90 N-glycans per trimer, most bnAbs use or accommodate them in their binding epitope, making the glycosylation of recombinant Env a key aspect of HIV vaccine design. Upon analysis of three HIV strains, we here find that site-specific glycosylation of Env from infectious virus closely matches Envs from corresponding recombinant membrane-bound trimers. However, viral Envs differ significantly from recombinant soluble, cleaved (SOSIP) Env trimers, strongly impacting antigenicity. These results provide a benchmark for virus Env glycosylation needed for the design of soluble Env trimers as part of an overall HIV vaccine strategy.
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http://dx.doi.org/10.1038/s41467-018-06121-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135743PMC
September 2018

Glycan Masking Focuses Immune Responses to the HIV-1 CD4-Binding Site and Enhances Elicitation of VRC01-Class Precursor Antibodies.

Immunity 2018 08 31;49(2):301-311.e5. Epub 2018 Jul 31.

Vaccine Research Center, NIAID, NIH, Bethesda, MD 20892, USA. Electronic address:

An important class of HIV-1 broadly neutralizing antibodies, termed the VRC01 class, targets the conserved CD4-binding site (CD4bs) of the envelope glycoprotein (Env). An engineered Env outer domain (OD) eOD-GT8 60-mer nanoparticle has been developed as a priming immunogen for eliciting VRC01-class precursors and is planned for clinical trials. However, a substantial portion of eOD-GT8-elicited antibodies target non-CD4bs epitopes, potentially limiting its efficacy. We introduced N-linked glycans into non-CD4bs surfaces of eOD-GT8 to mask irrelevant epitopes and evaluated these mutants in a mouse model that expressed diverse immunoglobulin heavy chains containing human IGHV1-202, the germline VRC01 V segment. Compared to the parental eOD-GT8, a mutant with five added glycans stimulated significantly higher proportions of CD4bs-specific serum responses and CD4bs-specific immunoglobulin G B cells including VRC01-class precursors. These results demonstrate that glycan masking can limit elicitation of off-target antibodies and focus immune responses to the CD4bs, a major target of HIV-1 vaccine design.
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http://dx.doi.org/10.1016/j.immuni.2018.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896779PMC
August 2018

The human naive B cell repertoire contains distinct subclasses for a germline-targeting HIV-1 vaccine immunogen.

Sci Transl Med 2018 07;10(448)

Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA.

Traditional vaccine development to prevent some of the worst current pandemic diseases has been unsuccessful so far. Germline-targeting immunogens have potential to prime protective antibodies (Abs) via more targeted immune responses. Success of germline-targeting vaccines in humans will depend on the composition of the human naive B cell repertoire, including the frequencies and affinities of epitope-specific B cells. However, the human naive B cell repertoire remains largely undefined. Assessment of antigen-specific human naive B cells among hundreds of millions of B cells from multiple donors may be used as pre-phase 1 ex vivo human testing to potentially forecast B cell and Ab responses to new vaccine designs. VRC01 is an HIV broadly neutralizing Ab (bnAb) against the envelope CD4-binding site (CD4bs). We characterized naive human B cells recognizing eOD-GT8, a germline-targeting HIV-1 vaccine candidate immunogen designed to prime VRC01-class Abs. Several distinct subclasses of VRC01-class naive B cells were identified, sharing sequence characteristics with inferred precursors of known bnAbs VRC01, VRC23, PCIN63, and N6. Multiple naive B cell clones exactly matched mature VRC01-class bnAb L-CDR3 sequences. Non-VRC01-class B cells were also characterized, revealing recurrent public light chain sequences. Unexpectedly, we also identified naive B cells related to the IOMA-class CD4bs bnAb. These different subclasses within the human repertoire had strong initial affinities () to the immunogen, up to 13 nM, and represent encouraging indications that multiple independent pathways may exist for vaccine-elicited VRC01-class bnAb development in most individuals. The frequencies of these distinct eOD-GT8 B cell specificities give insights into antigen-specific compositional features of the human naive B cell repertoire and provide actionable information for vaccine design and advancement.
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http://dx.doi.org/10.1126/scitranslmed.aat0381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145074PMC
July 2018

Glycoengineering HIV-1 Env creates 'supercharged' and 'hybrid' glycans to increase neutralizing antibody potency, breadth and saturation.

PLoS Pathog 2018 05 2;14(5):e1007024. Epub 2018 May 2.

San Diego Biomedical Research Institute, San Diego, California, United States of America.

The extensive glycosylation of HIV-1 envelope (Env) glycoprotein leaves few glycan-free holes large enough to admit broadly neutralizing antibodies (bnAb). Consequently, most bnAbs must inevitably make some glycan contacts and avoid clashes with others. To investigate how Env glycan maturation regulates HIV sensitivity to bnAbs, we modified HIV-1 pseudovirus (PV) using various glycoengineering (GE) tools. Promoting the maturation of α-2,6 sialic acid (SA) glycan termini increased PV sensitivity to two bnAbs that target the V2 apex and one to the interface between Env surface gp120 and transmembrane gp41 subunits, typically by up to 30-fold. These effects were reversible by incubating PV with neuraminidase. The same bnAbs were unusually potent against PBMC-produced HIV-1, suggesting similar α-2,6 hypersialylated glycan termini may occur naturally. Overexpressing β-galactosyltransferase during PV production replaced complex glycans with hybrid glycans, effectively 'thinning' trimer glycan coverage. This increased PV sensitivity to some bnAbs but ablated sensitivity to one bnAb that depends on complex glycans. Other bnAbs preferred small glycans or galactose termini. For some bnAbs, the effects of GE were strain-specific, suggesting that GE had context-dependent effects on glycan clashes. GE was also able to increase the percent maximum neutralization (i.e. saturation) by some bnAbs. Indeed, some bnAb-resistant strains became highly sensitive with GE-thus uncovering previously unknown bnAb breadth. As might be expected, the activities of bnAbs that recognize glycan-deficient or invariant oligomannose epitopes were largely unaffected by GE. Non-neutralizing antibodies were also unaffected by GE, suggesting that trimers remain compact. Unlike mature bnAbs, germline-reverted bnAbs avoided or were indifferent to glycans, suggesting that glycan contacts are acquired as bnAbs mature. Together, our results suggest that glycovariation can greatly impact neutralization and that knowledge of the optimal Env glycoforms recognized by bnAbs may assist rational vaccine design.
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http://dx.doi.org/10.1371/journal.ppat.1007024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5951585PMC
May 2018

Precursor Frequency and Affinity Determine B Cell Competitive Fitness in Germinal Centers, Tested with Germline-Targeting HIV Vaccine Immunogens.

Immunity 2018 01 26;48(1):133-146.e6. Epub 2017 Dec 26.

Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID), The Scripps Research Institute, La Jolla, CA 92037, USA; Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, CA 92037, USA. Electronic address:

How precursor frequencies and antigen affinities impact interclonal B cell competition is a particularly relevant issue for candidate germline-targeting HIV vaccine designs because of the in vivo rarity of naive B cells that recognize broadly neutralizing epitopes. Knowing the frequencies and affinities of HIV-specific VRC01-class naive human B cells, we transferred B cells with germline VRC01 B cell receptors into congenic recipients to elucidate the roles of precursor frequency, antigen affinity, and avidity on B cell responses following immunization. All three factors were interdependently limiting for competitive success of VRC01-class B cells. In physiological high-affinity conditions using a multivalent immunogen, rare VRC01-class B cells successfully competed in germinal centers (GC), underwent extensive somatic hypermutation, and differentiated into memory B cells. The data reveal dominant influences of precursor frequency, affinity, and avidity for interclonal GC competition and indicate that germline-targeting immunogens can overcome these challenges with high-affinity multimeric designs.
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http://dx.doi.org/10.1016/j.immuni.2017.11.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773359PMC
January 2018

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

Nat Commun 2017 03 28;8:14954. Epub 2017 Mar 28.

Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

HIV-1 envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs) and the focus for design of an antibody-based HIV vaccine. The Env trimer is covered by ∼90N-linked glycans, which shield the underlying protein from immune surveillance. bNAbs to HIV develop during infection, with many showing dependence on glycans for binding to Env. The ability to routinely assess the glycan type at each glycosylation site may facilitate design of improved vaccine candidates. Here we present a general mass spectrometry-based proteomics strategy that uses specific endoglycosidases to introduce mass signatures that distinguish peptide glycosites that are unoccupied or occupied by high-mannose/hybrid or complex-type glycans. The method yields >95% sequence coverage for Env, provides semi-quantitative analysis of the glycosylation status at each glycosite. We find that most glycosites in recombinant Env trimers are fully occupied by glycans, varying in the proportion of high-mannose/hybrid and complex-type glycans.
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http://dx.doi.org/10.1038/ncomms14954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5379070PMC
March 2017

Effects of partially dismantling the CD4 binding site glycan fence of HIV-1 Envelope glycoprotein trimers on neutralizing antibody induction.

Virology 2017 05 6;505:193-209. Epub 2017 Mar 6.

San Diego Biomedical Research Institute, 10865 Road to the Cure, San Diego, CA 92121, USA. Electronic address:

Previously, VLPs bearing JR-FL strain HIV-1 Envelope trimers elicited potent neutralizing antibodies (nAbs) in 2/8 rabbits (PLoS Pathog 11(5): e1004932) by taking advantage of a naturally absent glycan at position 197 that borders the CD4 binding site (CD4bs). In new immunizations, we attempted to improve nAb responses by removing the N362 glycan that also lines the CD4bs. All 4 rabbits developed nAbs. One targeted the N197 glycan hole like our previous sera. Two sera depended on the N463 glycan, again suggesting CD4bs overlap. Heterologous boosts appeared to reduce nAb clashes with the N362 glycan. The fourth serum targeted a N362 glycan-sensitive epitope. VLP manufacture challenges prevented us from immunizing larger rabbit numbers to empower a robust statistical analysis. Nevertheless, trends suggest that targeted glycan removal may improve nAb induction by exposing new epitopes and that it may be possible to modify nAb specificity using rational heterologous boosts.
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http://dx.doi.org/10.1016/j.virol.2017.02.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895097PMC
May 2017

HIV Vaccine Design to Target Germline Precursors of Glycan-Dependent Broadly Neutralizing Antibodies.

Immunity 2016 09 8;45(3):483-496. Epub 2016 Sep 8.

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA 02139, USA. Electronic address:

Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040827PMC
http://dx.doi.org/10.1016/j.immuni.2016.08.016DOI Listing
September 2016

Induction of HIV Neutralizing Antibody Lineages in Mice with Diverse Precursor Repertoires.

Cell 2016 Sep;166(6):1471-1484.e18

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Howard Hughes Medical Institute, Boston, MA 02115, USA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2(∗)02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages.
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http://dx.doi.org/10.1016/j.cell.2016.07.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5103708PMC
September 2016

Tailored Immunogens Direct Affinity Maturation toward HIV Neutralizing Antibodies.

Cell 2016 Sep;166(6):1459-1470.e11

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02129, USA. Electronic address:

Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses.
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http://dx.doi.org/10.1016/j.cell.2016.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5018249PMC
September 2016

Priming HIV-1 broadly neutralizing antibody precursors in human Ig loci transgenic mice.

Science 2016 09 8;353(6307):1557-1560. Epub 2016 Sep 8.

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA. International AIDS Vaccine Initiative (IAVI) Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA. Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA. Ragon Institute of Massachusetts General Hospital (MGH), MIT, and Harvard, Cambridge, MA 02129, USA.

A major obstacle to a broadly neutralizing antibody (bnAb)-based HIV vaccine is the activation of appropriate B cell precursors. Germline-targeting immunogens must be capable of priming rare bnAb precursors in the physiological setting. We tested the ability of the VRC01-class bnAb germline-targeting immunogen eOD-GT8 60mer (60-subunit self-assembling nanoparticle) to activate appropriate precursors in mice transgenic for human immunoglobulin (Ig) loci. Despite an average frequency of, at most, about one VRC01-class precursor per mouse, we found that at least 29% of singly immunized mice produced a VRC01-class memory response, suggesting that priming generally succeeded when at least one precursor was present. The results demonstrate the feasibility of using germline targeting to prime specific and exceedingly rare bnAb-precursor B cells within a humanlike repertoire.
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http://dx.doi.org/10.1126/science.aah3945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5404394PMC
September 2016

Holes in the Glycan Shield of the Native HIV Envelope Are a Target of Trimer-Elicited Neutralizing Antibodies.

Cell Rep 2016 08 18;16(9):2327-38. Epub 2016 Aug 18.

Department of Immunology & Microbial Science, IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA 02139, USA. Electronic address:

A major advance in the search for an HIV vaccine has been the development of a near-native Envelope trimer (BG505 SOSIP.664) that can induce robust autologous Tier 2 neutralization. Here, potently neutralizing monoclonal antibodies (nAbs) from rabbits immunized with BG505 SOSIP.664 are shown to recognize an immunodominant region of gp120 centered on residue 241. Residue 241 occupies a hole in the glycan defenses of the BG505 isolate, with fewer than 3% of global isolates lacking a glycan site at this position. However, at least one conserved glycan site is missing in 89% of viruses, suggesting the presence of glycan holes in most HIV isolates. Serum evidence is consistent with targeting of holes in natural infection. The immunogenic nature of breaches in the glycan shield has been under-appreciated in previous attempts to understand autologous neutralizing antibody responses and has important potential consequences for HIV vaccine design.
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http://dx.doi.org/10.1016/j.celrep.2016.07.074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007210PMC
August 2016

Glycan clustering stabilizes the mannose patch of HIV-1 and preserves vulnerability to broadly neutralizing antibodies.

Nat Commun 2015 Jun 24;6:7479. Epub 2015 Jun 24.

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

The envelope spike of HIV-1 employs a 'glycan shield' to protect itself from antibody-mediated neutralization. Paradoxically, however, potent broadly neutralizing antibodies (bnAbs) that target this shield have been isolated. The unusually high glycan density on the gp120 subunit limits processing during biosynthesis, leaving a region of under-processed oligomannose-type structures, which is a primary target of these bnAbs. Here we investigate the contribution of individual glycosylation sites in the formation of this so-called intrinsic mannose patch. Deletion of individual sites has a limited effect on the overall size of the intrinsic mannose patch but leads to changes in the processing of neighbouring glycans. These structural changes are largely tolerated by a panel of glycan-dependent bnAbs targeting these regions, indicating a degree of plasticity in their recognition. These results support the intrinsic mannose patch as a stable target for vaccine design.
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http://dx.doi.org/10.1038/ncomms8479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4500839PMC
June 2015

HIV-1 VACCINES. Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen.

Science 2015 Jul 18;349(6244):156-61. Epub 2015 Jun 18.

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA.

A major goal of HIV-1 vaccine research is the design of immunogens capable of inducing broadly neutralizing antibodies (bnAbs) that bind to the viral envelope glycoprotein (Env). Poor binding of Env to unmutated precursors of bnAbs, including those of the VRC01 class, appears to be a major problem for bnAb induction. We engineered an immunogen that binds to VRC01-class bnAb precursors and immunized knock-in mice expressing germline-reverted VRC01 heavy chains. Induced antibodies showed characteristics of VRC01-class bnAbs, including a short CDRL3 (light-chain complementarity-determining region 3) and mutations that favored binding to near-native HIV-1 gp120 constructs. In contrast, native-like immunogens failed to activate VRC01-class precursors. The results suggest that rational epitope design can prime rare B cell precursors for affinity maturation to desired targets.
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http://dx.doi.org/10.1126/science.aac5894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4669217PMC
July 2015

Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site.

PLoS Pathog 2015 May 29;11(5):e1004932. Epub 2015 May 29.

San Diego Biomedical Research Institute, San Diego, California, United States of America.

Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative "glycan fence" that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine.
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http://dx.doi.org/10.1371/journal.ppat.1004932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4449185PMC
May 2015

Comprehensive sieve analysis of breakthrough HIV-1 sequences in the RV144 vaccine efficacy trial.

PLoS Comput Biol 2015 Feb 3;11(2):e1003973. Epub 2015 Feb 3.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.

The RV144 clinical trial showed the partial efficacy of a vaccine regimen with an estimated vaccine efficacy (VE) of 31% for protecting low-risk Thai volunteers against acquisition of HIV-1. The impact of vaccine-induced immune responses can be investigated through sieve analysis of HIV-1 breakthrough infections (infected vaccine and placebo recipients). A V1/V2-targeted comparison of the genomes of HIV-1 breakthrough viruses identified two V2 amino acid sites that differed between the vaccine and placebo groups. Here we extended the V1/V2 analysis to the entire HIV-1 genome using an array of methods based on individual sites, k-mers and genes/proteins. We identified 56 amino acid sites or "signatures" and 119 k-mers that differed between the vaccine and placebo groups. Of those, 19 sites and 38 k-mers were located in the regions comprising the RV144 vaccine (Env-gp120, Gag, and Pro). The nine signature sites in Env-gp120 were significantly enriched for known antibody-associated sites (p = 0.0021). In particular, site 317 in the third variable loop (V3) overlapped with a hotspot of antibody recognition, and sites 369 and 424 were linked to CD4 binding site neutralization. The identified signature sites significantly covaried with other sites across the genome (mean = 32.1) more than did non-signature sites (mean = 0.9) (p < 0.0001), suggesting functional and/or structural relevance of the signature sites. Since signature sites were not preferentially restricted to the vaccine immunogens and because most of the associations were insignificant following correction for multiple testing, we predict that few of the genetic differences are strongly linked to the RV144 vaccine-induced immune pressure. In addition to presenting results of the first complete-genome analysis of the breakthrough infections in the RV144 trial, this work describes a set of statistical methods and tools applicable to analysis of breakthrough infection genomes in general vaccine efficacy trials for diverse pathogens.
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http://dx.doi.org/10.1371/journal.pcbi.1003973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4315437PMC
February 2015