Squalene-Based Influenza Vaccine Adjuvants and Their Impact on the Hemagglutinin-Specific B Cell Response

Abstract

Influenza infections continue to cause significant annual morbidity and mortality despite ongoing influenza vaccine research. Adjuvants are administered in conjunction with influenza vaccines to enhance the immune response and strengthen protection against disease. Squalene-based emulsion adjuvants including MF59, AS03, and AF03, are registered for administration with influenza vaccines and are widely used in many countries. Squalene-based emulsion adjuvants induce a strong innate immune response, enhancing antigen presentation both quantitively and qualitatively to generate strong B cell responses and antibody production. They also diversify the reactivity profiles and strengthen the affinities of antibodies against the influenza hemagglutinin, increasing protection across virus clades. In this review, we consider the mechanisms of the enhancement of innate and adaptive immune responses by squalene-based emulsionSE adjuvants and the resulting increase in magnitude and breadth of hemagglutinin-specific B cell responses. We relate observed effects of SE adjuvants and current mechanistic understandings to events in responding lymph nodes. These insights will guide the rational design and optimization of influenza vaccines to provide broad and effective protection.

Keywords: antibody; germinal center; hemagglutinin; influenza A virus; memory B cells; squalene-based adjuvant.

Figure 1

Immunocompetent environment induced by squalene-based emulsion (SE) adjuvants at the injection site. SE adjuvants cause local muscle cell damage (e.g., cell lysis by detergent-like molecules in the formulation), inducing the transient release of damage-associated molecular pattern (DAMP) signals such as ATP and double-stranded DNA, and the change in expression of as many as 891 genes in mice, compared to 312 genes by Alum at the injection site [35,42,43,44]. These DAMP signals bind to receptors on antigen-presenting cells (APCs) cells, trigger cytokine and chemokine secretion cascades as early as 3–6 h post-injection in mice [45]. This process leads to chemokine-driven infiltration of innate cells to the site of vaccine administration, generally peaking in 24 h, followed by a rapid decline by 72 h [38,45]. The signaling pathways triggered by SE adjuvants that drive the activation, APC differentiation, and lymph node migration of resident and recruited leukocytes remain unclear. Evidence suggests that these processes are inflammasome-independent and involve MyD88 and apoptosis-associated speck-like protein containing a caspase recruitment domain [46]. Activated innate cells secrete additional inflammatory molecules [32,33], continuing as a positive feedback loop at the injected muscle to generate a robust innate immune response. This mechanism allows SE adjuvants to create a localized transient ‘immunocompetent environment’ at the injection site, potently enhancing the antigen uptake and the activation of the APCs to migrate to the local lymph nodes and efficiently prime the adaptive system for B cell response [42]. The induction of an immunocompetent environment by SE adjuvants, however, is independent of immunogens [36]. The o/w emulsion vehicle itself appears to suffice [36].

Figure 2

Overview of the HA-specific B cell response in a draining lymph node after administration of vaccine with squalene-based emulsion (SE) adjuvant. SE adjuvants promote antigen transport to lymph nodes by monocyte-derived cells. These cells, which cross sinus linings to enter lymph node parenchyma, have the potential to deliver intact antigen and also to serve as highly efficient antigen-presenting cells for CD4 T cell activation. Free lymph-borne antigen enters lymph node parenchyma via multiple routes such as trapping by subcapsular sinus macrophages and transfer for memory CD4 T cell activation or recognition by memory B cells (MBCs) and naïve B cells. Direct antigen transfer across lymphatic endothelial cells into lymph nodes also occurs. Abundant antigen availability in draining lymph nodes after administration with SE adjuvants diminishes B cell competition (for example, between MBCs and naïve B cells) for cognate antigen required for activation. MBCs and naïve B cells that “see” cognate antigen receive T cell help to drive activation and proliferation. Pathway options for activated B cells (A, red arrows) include (i) formation of extrafollicular antibody-secreting cells (ASCs); (ii) formation of extrafollicular MBCs; and (iii) entry into germinal center (GC) reactions. After maturation of immunoglobulin V region genes in GCs, GC B cells “test” their receptor affinity by competing for antigen held by follicular dendritic cells (FDCs). Successful acquisition of antigen from FDCs enables GC B cells to be stimulated by T cells and to repeat the maturation/selection cycle or differentiate into MBCs or antibody-secreting plasma cells and exit the GC (B, dark red arrows). The abundant free antigen in the lymph node is shuttled to FDCs, reducing the stringency of GC B cell selection after somatic mutation. In addition, strong CD4 T cell activation and formation of T follicular helper (T_FH) cells promoted by SE adjuvants increases the availability of help for GC B cells and facilitates selection. Overall, SE adjuvants enhance the activation and affinity maturation of naïve B cells.