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.
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•Slow delivery immunization enhances HIV neutralizing antibody development in monkeys•Slow delivery immunization alters immunodominance of the responding B cells•Weekly longitudinal germinal center (GC) B and TFH analyses provides new GC insights•High-resolution rhesus immunoglobulin locus genomic reference sequence
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.
The first immunization in a protein prime-boost vaccination is likely to be critical for how the immune response unfolds. Using fine needle aspirates (FNAs) of draining lymph nodes (LNs), we tracked ...the kinetics of the primary immune response in rhesus monkeys immunized intramuscularly (IM) or subcutaneously (s.c.) with an eOD-GT8 60-mer nanoparticle immunogen to facilitate clinical trial design. Significant numbers of germinal center B (BGC) cells and antigen-specific CD4 T cells were detectable in the draining LN as early as 7 days post-immunization and peaked near day 21. Strikingly, s.c. immunization results in 10-fold larger antigen-specific BGC cell responses compared to IM immunization. Lymphatic drainage studies revealed that s.c. immunization resulted in faster and more consistent axillary LN drainage than IM immunization. These data indicate robust antigen-specific germinal center responses can occur rapidly to a single immunization with a nanoparticle immunogen and vaccine drainage substantially impacts immune responses in local LNs.
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•Rapid immunogen-specific lymph node responses detected at day 7 post-immunization•Lymph node immune response kinetics can be longitudinally tracked•Greater germinal center responses after subcutaneous versus intramuscular immunization•Immunization injection method substantially impacts immune responses in local LNs
The first immunization of protein prime-boost vaccination is likely critical but has been understudied in large animals and humans. Havenar-Daughton et al. use lymph node fine needle aspirates to determine primary germinal center response kinetics in rhesus monkeys immunized intramuscularly or subcutaneously with a clinical trial candidate nanoparticle immunogen.
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 ...immune-stimulatory complexes (ISCOMs) formed by the self-assembly of saponin and phospholipids in the absence or presence of the Toll-like receptor 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
in mice, and primed robust germinal center B cell, T
, and HIV tier 2 neutralizing antibodies in nonhuman primates. Together, these findings shed new light on mechanisms by which saponin adjuvants act to promote the immune response and suggest that SMNP may be a promising adjuvant in the setting of HIV, SARS-CoV-2, and other pathogens.
T follicular helper (T
) cells are powerful regulators of affinity matured long-lived plasma cells. Eliciting protective, long-lasting antibody responses to achieve persistent immunity is the goal of ...most successful vaccines. Thus, there is potential in manipulating T
cell responses. Herein, we describe an HIV vaccine development approach exploiting the cytokine activin A to improve antibody responses against recombinant HIV Envelope (Env) trimers in non-human primates. Administration of activin A improved the magnitude of Env-specific antibodies over time and promoted a significant increase in Env-specific plasma cells in the bone marrow. The boost in antibody responses was associated with reduced frequencies of T follicular regulatory (T
) cells and increased germinal center T follicular helper (GC-T
) to T
cell ratios. Overall, these findings suggest that adjuvants inducing activin A production could potentially be incorporated in future rational design vaccine strategies aimed at improving germinal centers, long-lived plasma cells, and sustained antibody responses.
Abstract Immunodominance of antibodies targeting non-neutralizing epitopes and the high level of somatic hypermutation within germinal centers (GCs) required for most HIV broadly neutralizing ...antibodies (bnAbs) are major impediments to the development of an effective HIV vaccine. Rational protein vaccine design and non-conventional immunization strategies are potential avenues to overcome these hurdles. Here, we report using implantable osmotic pumps to continuously deliver a series of epitope-targeted immunogens to rhesus macaques over the course of six months to prime and elicit antibody responses against the conserved fusion peptide (FP). GC responses and antibody specificities were tracked longitudinally using lymph node fine-needle aspirates and electron microscopy polyclonal epitope mapping (EMPEM), respectively, to show antibody responses to the FP/N611 glycan hole region were primed, although exhibited limited neutralization breadth. Application of cryoEMPEM delineated key residues for on-target and off-target responses that can drive the next round of structure-based vaccine design.
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
1736-1746, 2012, https://doi.org/10.1016/j.celrep.2012.11.016) reported that intragastric administration of chemically inactivated simian immunodeficiency virus SIV
and
(iSIV-
) protected 15/16 Chinese-origin rhesus macaques (RMs) from high-dose intrarectal SIV
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
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
BCG,
, or
, 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
only as well as unvaccinated animals. Intrarectal challenge with SIV
resulted in rapid infection in all groups of vaccinated RMs as well as unvaccinated controls. iSIV-
vaccinated animals that became SIV infected showed viral loads similar to those observed in animals receiving iSIV only or
only or in unvaccinated controls. The protection from SIV transmission conferred by intragastric iSIV-
administration reported previously for Chinese-origin RMs was not observed when the same experiment was conducted in a larger cohort of Indian-origin animals.
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
and 2,2'-dithiodipyridine-inactivated SIV, which showed highly significant protection from virus transmission. In the present study, we administered iSIV-
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-
.
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.
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.
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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