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One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions, and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next-generation sequencing of immune repertoires, we were able to specifically identify clonal family members, synthesize the monoclonal antibodies, and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design., Description, CryoEM and next-generation sequencing were used to identify monoclonal antibodies elicited by HIV Env vaccine candidates.

Saponins are potent and safe vaccine adjuvants, but their mechanisms of action remain incompletely understood. Here, we explored the properties of several saponin formulations, including immunostimulatory complexes (ISCOMs) formed by the self-assembly of saponin and phospholipids in the absence or presence of the Toll like receptor (TLR) 4 agonist monophosphoryl lipid A (MPLA). We found that MPLA self-assembles with saponins to form particles physically resembling ISCOMs, which we termed saponin/MPLA nanoparticles (SMNP). Saponin-containing adjuvants exhibited distinctive mechanisms of action, altering lymph flow in a mast cell-dependent manner and promoting antigen entry into draining lymph nodes. SMNP was particularly effective, exhibiting even greater potency than the compositionally-related adjuvant AS01(B) in mice, and primed robust germinal center B cell, T(FH), and HIV tier 2 neutralizing antibodies in non-human primates. Altogether, these findings shed new light on mechanisms by which saponin adjuvants act to promote the immune response and suggest SMNP may be a promising adjuvant in the setting of HIV, SARS-CoV-2, and other pathogens.

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 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 lymph node within 2 days after immunization. Imaging of LNs collected 7 days post-immunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent germinal centers. 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 immune complexes or nanoparticles.

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.

The first immunization in a protein prime-boost vaccination is likely to be a critical event for how the immune response unfolds. However, priming immunizations generally produce little circulating antibody (Ab) or T cell responses in blood and therefore have been poorly studied in large animal models and humans. Using fine needle aspirates (FNA) of draining lymph nodes (LN), we closely tracked the primary immune response in rhesus monkeys immunized intramuscularly (IM) or subcutaneously (SubQ) with eOD-GT8 60mer nanoparticle, an epitope-directed HIV Env CD4 binding site immunogen entering human clinical trial. Significant numbers of germinal center (GC) B cells and antigen-specific CD4 T cells were detectable in the draining LN as early as 7 days post-immunization and peaked at approximately day 21. Strikingly, SubQ immunization resulted in 10-fold larger antigen-specific BGC cell responses compared to IM immunization. Antibody responses were only modestly higher after SubQ immunization. Lymphatic drainage studies revealed that SubQ immunization at the deltoid results in faster, larger, and more consistent LN drainage than IM immunization, which paralleled results from drainage studies of upper leg LNs that indicated more efficient LN drainage after SubQ administration. Together, these data indicate that, in a large animal model, robust antigen-specific GC responses can occur rapidly to a single immunization with a nanoparticle immunogen and differential injection site drainage of a vaccine substantially impacts immune responses in local LNs.

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 (TFH) 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. An integrated immunological, bioinformatic and imaging approach demonstrates how slow delivery immunization enhances neutralizing antibody and germinal center reactions over conventional strategies in response to HIV Env protein immunization in non-human primates., National Institutes of Health (Grant AI125068), National Institutes of Health (Grant Al100663)

SUMMARYThe observation that humans can produce broadly neutralizing antibodies (bnAbs) against HIV-1 has generated enthusiasm about the potential for a bnAb vaccine against HIV-1. Conventional immunization strategies will likely be insufficient for the development of a bnAb HIV vaccine and vaccines to other difficult pathogens, due to the significant immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. Using longitudinal lymph node fine needle aspirates, we found that two independent methods of slow delivery immunization of rhesus macaques (RM) resulted in larger GCs, more robust and sustained GC Tfh cell responses, and GC B cells with improved Env-binding, which correlated with the development of ~20 to 30-fold higher titers of tier 2 HIV-1 nAbs. Using a new RM genomic immunoglobulin loci reference sequence, we identified differential IgV gene usage between slow delivery immunized and conventional bolus immunized animals. The most immunodominant IgV gene used by conventionally immunized animals was associated with many GC B cell lineages. Ab mapping of those GC B cell specificities demonstrated targeting of an immunodominant non-neutralizing trimer base epitope, while that response was muted in slow delivery immunized animals. Thus, alternative immunization strategies appear to enhance nAb development by altering GCs and modulating immunodominance of non-neutralizing epitopes.

A major obstacle to development of an effective AIDS vaccine is that along with the intended beneficial responses, the immunization regimen may activate CD4 + T cells that can facilitate acquisition of human immunodeficiency virus (HIV) by serving as target cells for the virus. Lu et al. (W. Lu et al., Cell Rep 2: 1736–1746, 2012, https://doi.org/10.1016/j.celrep.2012.11.016 ) reported that intragastric administration of chemically inactivated simian immunodeficiency virus SIV mac239 and Lactobacillus plantarum (iSIV- L. plantarum ) protected 15/16 Chinese-origin rhesus macaques (RMs) from high-dose intrarectal SIV mac239 challenge at 3 months postimmunization. They attributed the observed protection to induction of immune tolerance, mediated by “MHC-Ib/E-restricted CD8 + regulatory T cells that suppressed SIV-harboring CD4 + T cell activation and ex vivo SIV replication in 15/16 animals without inducing SIV-specific antibodies or cytotoxic T.” J.-M. Andrieu et al. (Front Immunol 5:297, 2014, https://doi.org/10.3389/fimmu.2014.00297 ) subsequently reported protection from infection in 23/24 RMs immunized intragastrically or intravaginally with iSIV and Mycobacterium bovis BCG, L. plantarum , or Lactobacillus rhamnosus , which they ascribed to the same tolerogenic mechanism. Using vaccine materials obtained from our coauthors, we conducted an immunization and challenge experiment with 54 Indian RMs and included control groups receiving iSIV only or L. plantarum only as well as unvaccinated animals. Intrarectal challenge with SIV mac239 resulted in rapid infection in all groups of vaccinated RMs as well as unvaccinated controls. iSIV- L. plantarum- vaccinated animals that became SIV infected showed viral loads similar to those observed in animals receiving iSIV only or L. plantarum only or in unvaccinated controls. The protection from SIV transmission conferred by intragastric iSIV- L. plantarum administration reported previously for Chinese-origin RMs was not observed when the same experiment was conducted in a larger cohort of Indian-origin animals. IMPORTANCE Despite an increased understanding of immune responses against HIV, a safe and effective AIDS vaccine is not yet available. One obstacle is that immunization may activate CD4 + T cells that may act as target cells for acquisition of HIV. An alternative strategy may involve induction of a tolerance-inducing response that limits the availability of activated CD4 + T cells, thus limiting the ability of virus to establish infection. In this regard, exciting results were obtained for Chinese-origin rhesus macaques by using a “tolerogenic” vaccine, consisting of intragastric administration of Lactobacillus plantarum and 2,2′-dithiodipyridine-inactivated SIV, which showed highly significant protection from virus transmission. In the present study, we administered iSIV- L. plantarum to Indian-origin rhesus macaques and failed to observe any protective effect on virus acquisition in this experimental setting. This work is important because it contributes to the overall assessment of the clinical potential of a new candidate AIDS vaccine platform based on iSIV- L. plantarum .