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Review
. 2020 Feb 24;11:283.
doi: 10.3389/fimmu.2020.00283. eCollection 2020.

Designs of Antigen Structure and Composition for Improved Protein-Based Vaccine Efficacy

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Free PMC article
Review

Designs of Antigen Structure and Composition for Improved Protein-Based Vaccine Efficacy

Kyle Saylor et al. Front Immunol. .
Free PMC article

Abstract

Today, vaccinologists have come to understand that the hallmark of any protective immune response is the antigen. However, it is not the whole antigen that dictates the immune response, but rather the various parts comprising the whole that are capable of influencing immunogenicity. Protein-based antigens hold particular importance within this structural approach to understanding immunity because, though different molecules can serve as antigens, only proteins are capable of inducing both cellular and humoral immunity. This fact, coupled with the versatility and customizability of proteins when considering vaccine design applications, makes protein-based vaccines (PBVs) one of today's most promising technologies for artificially inducing immunity. In this review, we follow the development of PBV technologies through time and discuss the antigen-specific receptors that are most critical to any immune response: pattern recognition receptors, B cell receptors, and T cell receptors. Knowledge of these receptors and their ligands has become exceptionally valuable in the field of vaccinology, where today it is possible to make drastic modifications to PBV structure, from primary to quaternary, in order to promote recognition of target epitopes, potentiate vaccine immunogenicity, and prevent antigen-associated complications. Additionally, these modifications have made it possible to control immune responses by modulating stability and targeting PBV to key immune cells. Consequently, careful consideration should be given to protein structure when designing PBVs in the future in order to potentiate PBV efficacy.

Keywords: antigen; epitope; immunity; modification; vaccine; vaccine composition; vaccine structure.

Figures

Figure 1
Figure 1
Recombinant toxins. (A) Diphtheria toxin (DT), when replacing glycine with glutamic acid at position 52, loses its toxicity without affecting its antigenicity. The highlighted residues (red) indicate the exact residue (sphere) and area (licorice) where this substitution would occur on monomeric DT. (B) Cholera toxin (CT) is composed of six subunits; one A subunit and five B subunits. B subunit (monomer in red, remaining subunits in pink), which lacks the toxicity of its partner A subunit, has proven to be extremely immunogenic and is used as a carrier protein and adjuvant. B subunit of heat-labile enterotoxin, which shares much of the same homology as B subunit of cholera toxin, has been similarly investigated (12). (C) Tetanus toxin (TT) is comprised of two chains, a light chain and a heavy chain, of which the light chain is responsible for the protein's toxicity. In the past, proteolytic digestion of TT with papain yielded two fragments, a light chain-containing, toxic B fragment and a non-toxic C fragment (red). Vaccination with the non-toxic C fragment was found to be protective against lethal toxin dose in a mouse model, and today PBVs comprised of recombinant C fragment are being investigated as a potential replacement for TT vaccines. Botulinum toxin, which shares much of the same homology as tetanus toxin, has been similarly investigated (13). The 3D protein structures for DT, CT, and TT used in this image were rendered in PyMOL 2.3.0 and accessed via the Protein Data Bank (–18).
Figure 2
Figure 2
Immunological mechanisms of recombinant, protein-based vaccination. (A) PBV structure, as illustrated here for the model protein hepatitis B core antigen (HBcAg, 183 aa long, non-truncated form, Accession number P03146), is ultimately determined by primary sequence. Vaccine can comprise monomeric antigen (i.e., toxoid protein) or multimeric antigen (i.e., virus-like particles), though multimeric antigen is used for demonstration purposes here. T cell and B cell antigenic determinants can be identified in primary sequence using various in vitro and in silico methods. The linear MHC epitopes illustrated here were predicted using Epitope Analysis Resources on the Immune Epitope Database (IEDB) website. More specifically, MHC epitopes were predicted for HLA-A*02:01 (class I molecules) and all heterodimer combinations of DQB1*02:01 (class II molecules) using IEDB recommended methods. Linear B cell epitopes, on the other hand, were assigned using frequency analysis results from the IEDB website (26). PBV structures are color coded to represent epitope content. (B) Cell processing and activation in response to PBV is generally orchestrated by antigen presenting cells, of which the most important are dendritic cells. APCs sample their environment via endocytosis, specifically via receptor-mediated endocytosis when antigen presents glycan and/or protein pathogen-associated molecular patterns such as high mannose glycans or bacterial flagellin. Depending on the structural and compositional characteristics of the antigen, APCs will either process antigen via MHC class II pathway or MHC class I pathway using a mechanism known as cross-presentation, respectively, resulting in activation of either CD4+ (helper) or CD8+ (cytotoxic) T cell response. CD4+ T cell activation requires co-activating signals and results in the proliferation of effector and memory CD4+ T cell pools. Effector CD4+ T cells go on to assist with the activation of B cells (T cell dependent activation) and provide survival signals to activated CD8+ T cells, whereas CD8+ T cells have immediate effector functionality. B cells can also undergo T cell independent activation when antigen cross-links multiple BCRs on B cells surface (TI-2 activation) or co-signals via PRR (TI-1 activation) (27, 28). (C) Activation results in the proliferation of memory and effector cytotoxic T cell and B cell pools. Memory CD8+ cells remain dormant until they encounter cells presenting MHC class I molecules loaded with cognate epitope, upon which they begin mounting an effector response. Effector CD8+ T cells go on to instruct apoptosis in cells presenting MHC class I molecules loaded with cognate epitope. B cells activated via T cell independent pathway generally proliferate into short-lived plasmablasts that express low affinity IgM antibodies (not shown). B cells activated via T cell dependent pathway, on the other hand, result in the proliferation of memory B cells and long-lived plasma cells expressing high-affinity IgA, IgE, or IgG antibodies. Antibodies secreted by plasms cells (and plasmablasts) go on to bind vaccine and pathogen and initiate antibody effector functions. Memory B cells remain dormant until they encounter antigen presenting cognate epitope, upon which they rapidly proliferate and clones either class switch to become antibody secreting plasma cells or re-enter germinal centers and restart affinity maturation processes (27, 28). The 3D protein structure for HBcAg used in this image was rendered in PyMOL 2.3.0 and accessed via the Protein Data Bank (14, 18, 29).
Figure 3
Figure 3
PBV modification principles. (A) Potential fusion modifications sites for the model protein hepatitis B core antigen (HBcAg, 149 aa long, truncated form of AN P03146 used for vaccine purposes) as represented by primary structure. Residues are color-coded in gray scale, with darker residues indicating more exposed insertion locations. Polypeptide termini and random coil loop regions are primary targets for PBV fusion modification, as they generally have the least effect on protein structure. Within these considerations, surface regions that do not participate in intra- and intermolecular interactions are preferred. Specifically, most HBcAg fusion PBVs presenting foreign B cell epitopes have been modified within the α3α4 loop (AIR between amino acids 75 and 85), though modifications are also routinely made at the C and N termini. A different approach to insertion site selection should be taken when creating fusion PBVs targeting T cell immune responses, as antibody response to epitope becomes detrimental. Toward this goal, inserting epitopes within loop regions that are less exposed and less likely to negatively influence protein stability is optimal (31, 32). (B) Potential fusion modification and conjugation sites for truncated HBcAg model protein as represented by higher order folded and assembled structure. Residues are color-coded in gray scale, with darker residues indicating more exposed insertion locations. Natural conjugation sites (lysine and cysteine) have also been highlighted in red. (C) PBV modifications are generally orchestrated via fusion, conjugation, or encapsulation. Each type of modification occurs at a different level of protein structure, with fusion inserts occurring within primary structure, conjugated inserts occurring within secondary/tertiary structure, and encapsulated inserts occurring within the quaternary structure of proteins that form enclosed, organized matrixes. As such, both monomeric and multimeric protein can accommodate fusion and conjugation modifications, whereas only multimeric protein can accommodate encapsulation. The 3D protein structure for HBcAg used in this image was rendered in PyMOL 2.3.0 and accessed via the Protein Data Bank (14, 18, 29).
Figure 4
Figure 4
The impact of PBV stability on immune response. PBV stability has a profound impact on conformation, immunogenicity, and vaccination outcome. Outside of inherent fold stability that's dictated by protein primary structure, many factors contribute to final PBV conformation and stability. Upstream and downstream processes, such as expression and purification, have a sizeable impact on the capacity of PBV to form higher-order structures and can ultimately lead to unintended PBV surface modification, aggregation, and/or decomposition. These issues can also present during formulation as a result of protein-protein, protein-adjuvant, and protein-container interactions. Incompatibilities between physiological conditions and PBV formulation can result in poor extracellular stability, a phenomenon that often presents as excessive local inflammation and poor transport of antigen to secondary lymphoid organs. Finally, cellular stability, which is a function of all the factors mentioned previously, largely dictates MHC processing and the nature of the immune response orchestrated by APCs (cellular vs. humoral, Th1 vs. Th2 vs. Th17, etc.). The 3D protein structure for HBcAg used in this image was rendered in PyMOL 2.3.0 and accessed via the Protein Data Bank (14, 18, 29).

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