GAP immunization in combination with exogenous adjuvants provides useful information about mechanisms underlying protective immunity. Induction of protective immune responses by GAP immunization is dependent on sporozoites migrating to the liver and invading hepatocytes. However, the administration of adjuvants at the site of GAP injection will result in systemic distribution of the adjuvant which will therefore be considerably diluted at the sites where parasite antigens are taken up by antigen presenting cells (APCs), i.e. the liver, spleen or proximal lymph nodes [26]. In order to maximize the adjuvant effect, i.e. the increase of the number of APCs that have both taken up parasite antigen and have received adjuvant induced stimulatory signals to enhance their function, it is important to maximize the adjuvant effect at the point of antigen uptake and processing [26, 27].
Conclusions and discussion
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We therefore, explored the possibility of creating GAPs that were engineered to express immunomodulatory proteins in sporozoites and liver stages, so called ‘adjuvant GAPs’.
Four different proteins were selected with known adjuvant activity: nontoxic cholera toxin B (CTB) sub-unit, mouse heat shock protein Gp96, Mycobacterium heat shock protein X (HspX) and Salmonella flagellin (FliC), shown to act as adjuvants in other vaccine studies [28-36]. The selected adjuvant molecules are thought to stimulate different Toll-like receptors (TLRs), which can not only improve antibody and CD4+ T cell responses but
also promote the cross-presentation of vaccine antigens directing the immune response towards the formation of cytotoxic (CD8+) T cells. Parasite antigen-specific CD8+ T cells are
considered of particular importance in detection and clearance of Plasmodium-infected hepatocytes [19]. However, immunization with none of the four adjuvant GAP developed in the present study resulted in a significant increase in protective efficacy (more than 10-fold) compared to the unmodified PyGAP in the P. yoelli-BALB/c model employed in this study to measure enhanced protective immunity.
This inability to achieve significantly higher protective immunity with the adjuvant GAPs could be due to a number of factors, but is unlikely to be due to a poor expression of the adjuvant proteins. These proteins were fused to UIS4, a protein associated with the PVM, which surrounds the parasites inside a hepatocyte [37]. UIS4 is strongly expressed during
Plasmodium liver stage development [38]. Our failure to measure enhanced protective immune responses may be due to the inability of the selected adjuvants to induce protective immune responses that can more effectively detect and destroy developing liver stage parasites.
The selected adjuvants are known to stimulate TLR 2 (Gp96), TLR 4 (Gp96/CTB/HSPX) and TLR 5 (FliC) on the plasma membrane of APCs [30, 32, 34, 39] and the selection was based on the hypothesis that when GAP-infected hepatocytes disintegrate and release parasite antigens they will also simultaneously release the adjuvant, with parasite antigens being taken up by APCs and the released adjuvants stimulating TLRs on the same APC. This would then result in increased inflammatory responses against parasite antigens thereby improving and increasing cellular and humoral immune responses. It is possible that the adjuvants selected do not stimulate the most appropriate adaptive response that would result in the recognition and elimination of infected liver cells. In this study we did not directly measure the effect of the TLR-agonists on different immune cell populations in immunized mice and we only measured protective immunity by determination of the prepatent period after challenge with WT parasites. One can speculate that either the adjuvants did not activate the appropriate immune cells or that those that are involved in removal of infected liver cells are activated but this activation is not sufficient to result in a more than 10-fold increase in protective immunity (i.e. 1 day or more delay in patency).
Future studies
The failure of the adjuvant GAPS to greatly enhance protective immune responses may be due to the immunization protocol we employed. In this study, we have used the P.
Conclusions and discussion
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yoelli-BALB/c immunization protocol which involves immunization with a single dose of sporozoites of a late arresting PyGAP followed by a challenge with wild type sporozoites 14 days later [40]. In future studies, the effect of the selected adjuvants on protective immunity may have been better observed after prime-boost immunization strategies where the re-call of expanded immunological memory responses may enhance protective immunity in different strains of mice. Indeed, such strategies might also be used to examine if immunization with the adjuvant GAP results in an increase of the duration of protective immune response compared to non-adjuvanted GAP. Whilst we were not able to detect a higher that 10-fold increase in vaccine potency, we have developed an immunization-challenge protocol, as well as a PyGAP GIMO mother line to rapidly create adjuvant GAPs, which can be used to evaluate other immunization schedules, additional adjuvants and/or novel enhanced GAPs. Novel adjuvant GAPs could be tested that encode other immunomodulatory molecules that have been characterized to enhance both anti- microbial and tumor vaccines. In our study we focused on adjuvants that interact with TLRs on the APC cell surface; in case of take-up of GAP-infected hepatocytes by APC this may be an issue since both the adjuvant and parasites are intracellular within the phagosome of the APC. In this case, adjuvant signaling would be better if it were to trigger cytoplasmic pattern recognition receptors (PPRs) either inside the APC or, indeed, the infected cell. For example, the C-terminus of flagellin has a NAIP5 ligand that would potentially activate intracellular sensing pathways, which could activate hepatocyte death and/or inflammatory cytokine production and perhaps increase the adjuvant potency [41, 42]. In Chapter 5 we only used the FliC portion of Salmonella flagellin that has been demonstrated to interact with TLR5 on the surface of APCs. In future studies we could therefore create and analyze a new adjuvant GAP that encodes full length flagellin to expand the adjuvant potential of this molecule. Manipulation, of the host immune response to direct and increase appropriate adaptive immune responses after vaccination is of value not only to enhance GAP vaccines but also other vaccines that need to generate immune responses to target liver infections.