Germinal center enhancement by extended antigen availability
Introduction
Most licensed human vaccines rely on antibody-mediated responses for protection. Those responses are primarily dependent on CD4+ T cells and germinal centers (GCs). GCs are sites within lymphoid organs where B cells undergo B cell receptor (BCR) somatic hypermutation (SHM) to enhance BCR affinity for antigen. Knowledge of the GC processes can almost certainly improve rational vaccine design, if parameters that modulate those processes can be understood. The use of model protein antigens has provided considerable insight into the mechanisms underlying GC and antibody responses. However, the use of simple model antigens most likely does not reflect the immunological challenges presented by more complex pathogen antigens, which have potently been driven by eons of evolution to be difficult for host B cells to recognize and neutralize. Few mutations are required for development of high-affinity antibodies against most simple model antigens, including the most commonly studied model antigen 4-hydroxy-3-nitrophenyl acetyl (NP), which only requires a single BCR amino acid mutation to develop high affinity antibodies [1]. Protective antibodies against some pathogens, including HIV-1, contain high numbers of amino acid mutations (>10) and develop over extended periods of time during infection [2, 3]. Lastly, the lifespan of GCs elicited by model antigens can also be short compared to even acute natural infections, where there is frequently a prolonged supply of antigen and GC reactions can last many weeks [4]. Thus, experimental studies of more complex antigens are necessary to study the importance of GC parameters involved in the development of potent antibodies against difficult epitopes on pathogens [5].
One example of a difficult pathogen antigen for B cell recognition and neutralization is HIV envelope (Env). Approximately 10% of HIV+ individuals develop potent broadly neutralizing antibodies (bnAbs) targeting HIV Env [3]. These bnAbs take multiple years to develop and accumulate more amino acid mutations than antibodies generated during conventional immunizations. Many HIV bnAbs require rare SHM events, including deletions or combinatorial mutations (e.g. addition of a new disulfide bond across a CDR loop). Longitudinal analyses of BCR and viral lineages throughout HIV infection has provided clear evidence that bnAbs undergo high amounts of affinity maturation before obtaining their broadly neutralizing activity [6, 7•]. The development of bnAbs via immunization is a major challenge and it is likely that certain conditions that resemble natural infection, including persistent antigen presence, are required for HIV bnAb development [8].
A promising avenue in rational vaccine design for modulating GCs is the sustained delivery of antigen, which can more mimic natural infection. Sporadic studies more than a decade ago found that controlled release of antigen over a longer period of time could result in stronger immune responses than conventional bolus injections [9, 10, 11]. More recent studies have revisited this concept with substantial success [12••, 14••]. Here we describe several mechanisms through which sustained antigen availability may modulate the GC response to enhance the humoral response. These mechanisms include 1) increased availability of native antigen, 2) increased immune complex deposition, 3) modulation of Tfh help and affinity maturation, and 4) modulation of memory B cell formation. Lastly, we discuss the implications of these immunological processes and extended antigen strategies for vaccine design.
Section snippets
Availability of intact protein antigen
GC B cells with the highest affinity for antigen are selected to survive and proliferate based on the ability of the B cell to strip antigen from follicular dendritic cells (FDCs) and subsequently receive help from Tfh cells. One should consider how that process aligns with conventional immunizations. Conventional protein immunizations deliver antigen and adjuvant in a single bolus injection. A potential shortcoming of that strategy is that it is not synchronized with the GC response. The GC
Immune complex deposition
FDCs concentrate and present intact antigen in the form of immune complexes to B cells in the light zone (LZ) of the GC [18, 19]. B cells test their evolving affinity on FDC immune complexes. B cells with BCRs with higher affinity to the antigen are selected to survive and undergo an additional round of affinity maturation or develop into plasma cells. The resulting higher affinity IgG from newly generated plasma cells can then form new immune complexes, replacing the lower affinity immune
Tfh cell help and affinity maturation
T follicular helper (Tfh) cells are required for GC development and function [22]. GC B cells with the highest affinity capture and present the most antigen, in the form of peptide-MHCII complexes to GC Tfh cells in the LZ. GC Tfh cells selectively help B cells that present the most antigen by providing signals essential for GC B cell survival, proliferation, and mutation [23, 24]. Surviving GC B cells migrate to the dark zone (DZ), where they proliferate and undergo SHM. Mutated GC B cells
Development of memory
Vaccination strategies to elicit memory B cells (MBCs) that can reenter GCs are attractive, as it is highly unlikely a single conventional immunization will allow for the extensive mutations required for neutralization of some pathogens. Few, if any, studies have explored the effect of vaccination with persistent antigen on the development of memory. Antigen kinetics may affect the pathways that control the decisions to differentiate into MBCs or PCs. B cells with lower affinity preferentially
Delivery systems and adjuvants
Osmotic pumps have been used for sustained immunization delivery in several proof-of-principle animal studies. While surgical implantation of osmotic pumps may be feasible for a Phase I human vaccine trial, osmotic pumps are clearly impractical for large scale human vaccine trials. Near daily repeated injections are also impractical for large scale human vaccine trials. More pragmatic delivery systems include slow release microneedles implanted through the skin with a patch and injectable
Summary
Protein vaccines safely elicit protective responses in humans and have greatly improved public health globally. For some pathogens with difficult neutralizing epitopes, nontraditional vaccination strategies may be required for long-lived immunity. It will be important to further understand how modulating immunogen kinetics impacts Tfh cell help, SHM, and MBC formation. Insights into the kinetics of the GC response, particularly in humans and NHPs, will benefit the efforts to optimize
Conflict of interest
We wish to confirm that there are no known conflicts of interest associated with this publication.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We thank Crotty lab members for helpful discussions. This work was supported by UM1 AI100663 (CHAVI-ID) and R01 AI134796 (S.C.) from the NIH.
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