Is the choice of vaccine adjuvant critical for long-term memory development?

Keywords::

• adjuvant
• B memory cells
• cholera toxin
• CTA1-DD
• germinal center
• long-lived plasma cells

Immunological memory following vaccination

Immunological memory is the ability to raise a faster, stronger and qualitatively better immune response upon re-exposure to an antigen compared with that seen after the initial encounter with the antigen [1]. This is a hallmark of the adaptive immune response and a critical goal for effective vaccination. However, despite its central role, little focus has been given to the identification of parameters that could be decisive for the long-term protective effect of a vaccine. In fact, we are only beginning to understand the mechanisms responsible for long-term memory development. Increasingly, the literature is now addressing the impact of vaccine composition on the long-term protective function [2]. Whereas liveattenuated antiviral vaccines have proven effective in stimulating lifelong protection, non-living vaccines have demonstrated only a relatively short-term duration of protection, with a requirement for repeated vaccination to renew the protective level of specific antibodies [3]. An attractive strategy is therefore to study memory development after using livettenuated vaccines to gain information on how non-living vaccines may be formulated to mimic the efficiency of antiviral attenuated vaccines. Such studies have given important insights into how to achieve a better duration of protection from non-living vaccines [4]. The key component of most non-living vaccines that is critically required for stimulating a significant immune response is the adjuvant [5,6]. Adjuvare comes from latin and means ‘to help’, and this is exactly what these substances do. However, only very few adjuvants have been approved for clinical use and there appears to be a remarkable sluggishness in taking new, more effective adjuvants to the market [7]. This can partly be explained by the cost of development and regulatory restrictions posed on vaccine adjuvants today [8]. Nevertheless, few studies have addressed whether the choice of adjuvant can impact on long-term memory development following vaccination. We also lack critical information about what this impact could be, let alone a detailed understanding of which mechanisms or factors may be involved. There is no general agreement on how to compare adjuvants with regard to their ability to influence the longevity of the immune response. Live-attenuated vaccines stimulate lifelong memory that can be detected as protective serum antibody levels and maintained over 50 years or more in the majority of vaccinated people [9]. Similarly, cell-mediated immunity was maintained in smallpox-immunized people for decades and, interestingly, booster doses did not change the increased frequencies of memory T- and B-cell populations [10]. These are remarkable findings and may serve as hallmarks of what a successful vaccine adjuvant should do.

Assessing adjuvant impact on critical parameters for memory development

We do not have a complete understanding of the signals that invoke memory B-cell development or the recruitment of long-lived plasma cells. Even less is known about which factors regulate the quality and quantity of the B-cell memory and plasma cell populations [11]. However, it would be important to find parameters that could predict the impact of adjuvants on long-term memory development at an early stage of an immune response. Efforts to establish the early impact of live-attenuated vaccines on innate immunity using a systems biology approach have given support for this method [4]. It was reported that the yellow fever YF-17D vaccine targeted several Toll-like receptors (TLRs) and distinct subsets of dendritic cells (DCs), which could be responsible for the effective induction of long-term protection. Another example is the association between effective priming and differentiation of CD4⁺ T cells and long-term persistence of protective antibodies following vaccination [12].

The study of memory cells has been hampered by the relative lack of reliable markers to identify these cells in mice. Only in recent years have several markers been associated with memory function in mice, such as CD80, CD73 and PD-L2 on B cells [13]. By contrast, CD72 has been found to be a relatively good marker for memory B cells in humans, although it appears that not all CD72⁺ cells are memory cells and that CD27^- memory B cells have also been identified [14]. The corresponding markers for CD4⁺ T cells are CD44⁺, CCR7^- and CD62L^- (for effector memory) and CD44⁺, CCR7⁺ and CD62L⁺ (for central memory) cells [15]. Identifying the magnitude and tissue distribution of specific memory T and B cells could be another critical component for assessments of vaccine efficacy with regard to a long-term protective effect [16]. For humoral immunity, it is well known that long-lived plasma cells, but not memory B cells, reside in the bone marrow (BM), while memory B cells are primarily located in the spleen in both mice and humans [17–19]. Recent discoveries have demonstrated that the BM is a site where memory CD4⁺ T cells reside, while the exact location of memory CD8⁺ T cells is still insufficiently known [20]. The memory cells are maintained in survival niches in the BM by specialized stromal cells expressing CXC-chemokine ligand 12, vascular cell adhesion molecule 1 and IL-7 [21].

Mechanisms for adjuvant actions

Dendritic cells are critical for stimulating immune responses against T-dependent antigen [22,23]. Adjuvants can act directly or indirectly on immature DCs, which then undergo maturation and migrate to draining lymph nodes where T-cell priming occurs and an immune response is elicited [24,25]. The direct interaction between adjuvants and DCs in general involves recognition through TLRs or NOD-like receptors [26]. Some adjuvant substances, perhaps most notably the potent and closely related bacterial enterotoxins cholera toxin (CT) and Escherichia coli heat-labile toxin (LT), appear to act independently of both TLRs and NOD-like receptors [5,27,28]. CT and LT are perhaps the best studied and most effective experimental adjuvants known today, but unfortunately they are also very toxic, which precludes their clinical use. There is a vast amount of literature on their structure and function [29]. They consist of an AB₅ complex with the ADP-ribosyltransferase active A1 linked to a pentamer of B subunits via the A2 fragment. The B subunit of CT is responsible for binding to the GM1 receptor, a glycosphingolipid found ubiquitously on the membranes of most mammalian cells. Several studies have shown that ADP-ribosyltransferase activity is required for optimal adjuvanticity, and that binding to cells of the immune system is mediated via the B subunit. To circumvent the toxicity problem, we developed the CTA1-DD adjuvant [30]. This is a promising novel vaccine adjuvant, which carries the ADP-ribosylating CTA1-moiety of CT, while the receptor-binding B subunit has been replaced by a dimer of the D fragment from Staphylococcus aureus protein A [31]. Hence, CTA1-DD does not bind GM1 ganglioside receptors present on all nucleated cells and does not interact with the central nervous tissues, as has been observed for CT and LT following intranasal administration, which was also the reason [32,33] for taking an LT-adjuvanted intranasal influenza vaccine off the market a few years ago [34]. CTA1-DD has been found to host a broad range of adjuvant functions, greatly augmenting cell-mediated and humoral immune responses to admixed antigen [35]. This effect was TLR/MyD88-independent and comparable in strength to that of CT [36]. However, contrary to CT, CTA1-DD was found to be nontoxic and noninflammatory in mice and monkeys and a promising candidate for use in human vaccines.

The germinal center reaction is a prime target for effective vaccine adjuvants

The best-documented effect of vaccine-induced protection against infection and disease appears to be the ability to induce long-term production of specific antibodies [37]. Indeed, there is good correlation between maintenance of high specific serum antibody titers and protection for some human vaccines [38]. However, recent studies demonstrated that high serum antibody titers per se, without concomitant germinal center (GC) reactions, may not be protective, suggesting that the GC reaction, rather than the titers, is critical for long-term vaccine efficacy [24,39]. It was concluded that high-affinity antibodies correlated with protection [40]. Thus, the GC reaction is central not only to the magnitude of the antibody response, but also to somatic hypermutations (SHMs) and affinity maturation of specific responses against T-cell-dependent antigens [41]. The literature on the GC reaction is extensive and its role for the development of long-lived plasma cells and memory B cells is well established [1,42]. Emerging data support the idea that memory B cells escape the GC prior to the plasma cell emigration to the BM, as memory B cells were in general less mutated than plasma cells in the BM [43]. However, whether all memory B cells emerge from the GC is currently being debated, as Bcl-6-deficient mice lacking GC reactions also have low-affinity memory B cells [44]. To what extent the adjuvant can influence SHM, affinity maturation or the longevity of the response is poorly known. Thus, no appreciation exists as to the impact of adjuvants on the GC reaction and how this may influence the long-term effect of vaccination. Recent data indicate that long-lived plasma cell and memory B-cell development may be differentially influenced by a number of key regulators of the GC reaction [13,45,46]. Several factors could be involved in the GC reaction and the selection process. There are established and emerging data to suggest that CD40, ICOS, IL-21, PD-1, CD95, IRF4 and Bcl-6 are among the factors that critically affect the GC formation [13,45,46]. In addition, enhanced production of B-cell-activating factor of the TNF family (BAFF) or a proliferation-inducing ligand (APRIL) could upregulate B-cell and plasma cell survival [47]. In fact, we are only beginning to understand the critical interplay between DCs, CD4⁺ follicular helper T cells and B cells in the formation of GC and how the former cells influence B-cell memory and long-lived plasma cell development. While it is clear that adjuvants can critically influence several of these cells and factors and may, therefore, selectively affect plasma or memory B-cell development, no systematic analysis has been performed on their influence on the development of long-term plasma and memory B cells in the GC.

The CTA1-DD adjuvant effectively promotes long-lived plasma & memory B cells

The adjuvant effects of CT are thought to involve the modulation of antigen-presenting cells (APCs), but it is poorly understood which APCs are functionally targeted by the holotoxins or derivatives thereof. All nucleated cells, including all professional APCs, can bind the toxins via the GM1 ganglioside receptor present in the cell membrane. Previous reports have documented both proinflammatory and anti-inflammatory effects of CT. From several studies, including our own work, it has been demonstrated that CT exposure of APCs has an augmenting effect on IL-1 and IL-6 production, whereas other studies have reported a downregulating effect on IL-12 and TNF-α, and a promoting effect on IL-10 and IL-17 production [35,48–51]. Taken together, these effects would indicate both a proinflammatory and an anti-inflammatory function of CT on innate immunity. Indeed, we recently demonstrated that CT regulated gene expression of STAT3, which is involved in both IL-6 (proinflammation) and IL-10 (anti-inflammation) production and signaling [52]. Nevertheless, CT was found to be an excellent inducer of long-term memory responses following oral immunization. We demonstrated that IgA plasma cells were present in the gut lamina propria for more than 6 months after an oral immunization, and upon rechallenge with CT, even after 24 months, a vigorous antigen-specific IgA antibody response was observed in the gut. Antigen-specific memory B cells could be isolated from the spleen or the mesenteric lymph nodes at 12 months following oral immunization and adoptively transferred to naive syngeneic recipient mice, which were subsequently challenged by oral CT. This elicited a strong recall IgA response in the gut, clearly demonstrating the presence of functional memory B cells in orally immunized mice [53,54].

Moreover, our recent work with the CTA1-DD adjuvant has clearly demonstrated that it effectively stimulated GC formations in a dose-dependent fashion and generated both antigen-specific long-lived plasma cell populations in the BM and memory B cells, primarily located in the spleen [55]. We found that a single priming immunization intraperitoneally with antigen and adjuvant stimulated specific serum IgG responses that were comparable between Ribi (MPL), Alum and CTA1-DD; but only CTA1-DD responses persisted for more than 16 months. The half-life of specific IgG antibodies in serum was roughly three-times longer in CTA1-DD-adjuvanted mice than for Ribi and Alum-adjuvanted immunizations. A CTA1-DD dose-dependent increase in GC size and numbers was found, with more than 60% of the splenic B-cell follicles hosting GC. This effect correlated well with long-lived plasma cells in the BM and memory B cells in the spleen. The CTA1-DD also had a strong qualitative impact on SHM and affinity maturation of specific IgG antibodies. The augmenting effect on SHM and affinity maturation may partly explain why a recent report on the use of CTA1-DD together with chlamydial major outer membrane protein antigen showed greatly enhanced high-affinity antibacterial neutralizing antibodies, an effect that also conferred resistance in the oviduct to immunopathology induced by Chlamydia infection [56]. Interestingly, adoptive transfer of resting splenic CD19⁺ GL7^- CD80⁺, but not CD80^- B cells, at 1 year following immunization, demonstrated functional long-term antigen-specific IgG and IgM memory B cells. Based on these findings, we now believe it will be possible to follow vaccine-induced B-cell memory in greater detail, which will allow us to investigate memory-cell distribution, activation requirements and effector functions.

To conclude, we have reason to believe that adjuvants differ significantly in their ability to promote long-lived plasma and memory B cells following vaccination. Hence, it would be useful to establish criteria to allow distinct comparisons to be made between adjuvants for their memory-inducing ability prior to designing future vaccines. This would strongly help in the rational design of non-living vaccines that, if possible, could be made to match today’s attenuated antiviral vaccines in conveying lifelong protection against infection.

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.