The toxicity of wP vaccines, including the induction of fever and rarer cases of seizures, has been linked with LPS in the vaccine [21], and efforts are being made to develop less toxic wP vaccines by reducing the LPS content [269]. However, one of the reasons the wP vaccines are so efficacious is due to the presence of LPS, which activates TLR4 signalling pathways, and has been shown to be important for protection against B. pertussis infection [20]. Indeed, immunisation of TLR4-defective mice with a wP vaccine failed to confer protective immunity against B. pertussis [20]. LPS is too toxic for human use, but a derivative, monophosphoryl lipid A (MPL), which binds to TLR4 and retains certain of the immunostimulatory properties of LPS without the toxicity issues, can promote similar immune responses [270]. AS04, a combination of MPL and alum, has been approved for use in human vaccines since 2005 and is currently used in some viral vaccines, such as hepatitis B (Table 1.3) [271]. Geurtsen et al. demonstrated that mice immunised with an aP vaccine adjuvanted with MPL had reduced lung colonisation after challenge when compared with mice immunised with the aP vaccine containing alum [272]. Furthermore, spleen cells from mice immunised with the MPL-containing aP vaccine had decreased production of B. pertussis-specific IL-5 ex vivo when compared with the mice given the alum-adjuvanted vaccine, indicating that MPL can skew the immune response away from Th2 (Figure 1.5). Moreover, Brummelman and colleagues demonstrated that addition of a meningococcal LPS derivative, LpxL1, to a commercial alum-adjuvanted aP vaccine enhanced the antigen-specific IFN-γ response and IgG2a to IgG1 ratio in mice, compared with mice immunised with the commercial alum-adjuvanted aP vaccine alone, indicating an overall enhancement of Th1 responses (Figure 1.5) [273].
Evidence of the benefits of using TLR agonists as adjuvants for pertussis vaccines is not confined to TLR4, CpG oligonucleotides from bacterial DNA, that signal through TLR9, are potent inducers of Th1 responses [274], and also promote induction of Th17 cells (Figure 1.5). Asokanathan et al. demonstrated that immunisation of mice with dPT, FHA and Prn in combination with both CpG and alum enhanced IFN-γ production by spleen cells and NO production by peritoneal macrophages when compared with mice immunised with the antigens in combination with alum alone [244]. Furthermore, mice that had been immunised with the aP vaccine that included CpG and alum had no detectable bacteria in the lungs 7 days after aerosol challenge, whereas bacteria were still present in mice immunised with the antigens in combination with alum [244]. Similarly, Kindrachuk et al. showed that mice immunised i.n. with dPT formulated with a complex of CpG and the synthetic innate defence regulator peptide HH2 (CpG-HH2) produced high levels of PT-specific IgG1 and IgG2a
antibodies [275]. In contrast, mice that received dPT with CpG alone (dPT-CpG) produced modest IgG1 and IgG2a production, and mice immunised with dPT-HH2 alone produced high levels of IgG1 antibody, but no IgG2a antibodies [275]. These results indicate that the CpG-HH2 combination induced a balanced Th1/Th2 response.
A novel TLR1/2 binding lipoprotein from B. pertussis, BP1569, was recently identified by the Mills lab. BP1569 was shown to have immunostimulatory properties in vivo and in vitro in TLR4 defective mice and cells, and in addition, was shown to be an antigenic component of B. pertussis [276]. Due to an inability to completely remove LPS from the lipoprotein, an LPS-free lipopeptide version of BP1569, LP1569, was synthesised. If LP1569 retains the immunostimulatory capacity of BP1569, it could have the potential to act as an adjuvant for a pertussis vaccine.
The ability of TLR agonists to redirect the immune response induced by alum-adjuvanted aP vaccines in mice is promising for the future development of pertussis vaccines, as addition of a TLR agonist to an existing vaccine would be logistically simpler than starting with a completely new formulation. A number of TLR agonists have already been tested in phase II and phase III clinical trials [277]. These molecules are chemically stable and have low production costs, and therefore are ideal as adjuvants for future generation vaccines against B. pertussis.
Figure 1.5 Mechanisms of natural and vaccine-induced immunity to B. pertussis
Upon B. pertussis infection, APCs such as DCs phagocytose B. pertussis and break down antigen into antigenic peptides. These activated DCs present B. pertussis antigens to naïve T cells, and produce the Th polarising cytokines IL-1β, IL-23, and IL-12, to induce Th17 and Th1 cell differentiation respectively. The resultant Th1 cells produce IFN-γ which activates macrophages to produce NO and effectively kill intracellular pathogens, and induces production of the IgG2a antibody isotype by B cells to efficiently opsonise B. pertussis. Th17 cells produce IL-17 which facilitates neutrophil recruitment and activation, and these granulocytes are proficient at phagocytosing and killing bacteria. Similar to natural infection, the wP vaccine induces a mixed Th1 and Th17 cell response which explains its high efficacy. In contrast, the aP vaccine predominantly induces a Th2 cell response due to the presence of the adjuvant alum in the vaccine, although it also promotes Th17 cell differentiation. Th2 cells produce IL-4 which induces antibody class switching to the IgG1 isotype, but no macrophage activation which is crucial for B. pertussis clearance. As described in sections 1.9.3, 1.9.5 and 1.12, substituting alum in the aP vaccine for certain TLR agonists including CpG, MPL or LpxL1, the cytokine IL-12, or LTK63 from E. coli, enhances protection against B. pertussis by directing the immune response towards Th1, and inducing potent cell-mediated immunity. (Figure adapted from [278]).