The FMP2.1/AS02A malaria vaccine appears to induce detectable cellular as well as humoral responses in malaria semi-immune adults. Measurable T cell-mediated immune responses (IL-5 production and lymphocyte proliferative responses) were detected in semi-immune malaria-exposed adults who received the AMA-1 vaccine. A balanced Th1/T2 cytokine response has been described in preclinical monkey trials after vaccination with AS02A adjuvanted to P. falciparum
antigen as compared to other adjuvants (AS01B, AS05, and AS08) formulated to MSP142
which invoked an enhanced Th1 response [33
]. Likewise, both IFN-γ and IL-5 production has been described in preclinical studies with RTS,S/AS02A [34
], and Phase I studies in malaria-naïve U.S. volunteers vaccinated with FMP2.1/AS02A [3
]. Enhanced IL-5 production was noted in the half-dose FMP2.1/AS02A group as compared to the full dose FMP2.1/AS02A group. While the significance of this is unclear, the paradoxical findings of higher CMI responses in response to lower doses of adjuvanted protein, was also observed in U.S. malaria-naïve volunteers receiving AMA-1/AS02A [3
] as well as volunteers receiving AMA-1/AS01B, Liver stage antigen 1 (LSA-1)/AS02A and LSA-1/AS01B (M. Spring, J. Cummings and D.G. Heppner, personal communication). In contrast to the results seen in the U.S.-based trial, no differences in IFN-γ production were noted between recipients of FMP2.1/AS02A and the comparator rabies vaccine [3
Protective immunity to malaria has been shown to correlate with detectable cellular immune responses. B and T cell responses have been measured in response to defined AMA-1 epitopes in volunteers naturally exposed to malaria [17
]. Moreover, proliferative responses to parasitized RBC (pRBC) in U.S. volunteers immunized with attenuated falciparum sporozoites correlated with protection against subsequent sporozoite challenge [24
]. We detected measurable increases in cellular immunologic responses to AMA-1 after vaccination in an adult Malian population. This is encouraging in that responses to vaccination are detectable against a high background of acquired immunity. Differences in immune responses between vaccine and control groups may be more pronounced in the target population of children who have less acquired immunity. The paucity of long-lived T cell memory responses that develop in response to malaria antigens even in the setting of repetitive antigen exposure has long been noted. The immune response to blood stage malaria antigens appears to evolve over years and acquisition of immunity may continue into adulthood [17
]. While T helper cells may facilitate AMA-1-specific antibody production [9
], other effector T cell functions may contribute to host immunity independently of antibody. Immunization with recombinant P. chabaudi
AMA-1 in mice stimulates immune protection against homologous P. chabaudi
infection. This appears to be partially mediated by CD4+
cells in an antibody-independent fashion but in synergism with anti-AMA-1 antibodies [22
]. In this model, IFN-γ was not detected in response to vaccination and challenge. This parallels our findings showing no difference in IFN-γ production as measured by ELISpot between vaccine groups at Day 90 but contrasts with the findings in malaria-naïve adults in which potent IFN-γ production was measured in response to vaccination [3
]. Of note, in the latter study vaccination also stimulated dose dependent IL-5 production in malaria-naïve adults. In areas of ongoing malaria transmission, IFN-γ production to this vaccine as measured by ELISpot may not serve as a relevant measure of CMI acquisition. Whether a Th2-biased CMI, as evidenced by IL-5 production, correlates with enhanced protective efficacy will be assessed in greater detail in upcoming Phase 2 vaccine trials.
Vaccinations occurred between December 2004 and February 2005 and Day 90 serum was collected in March 2005 at the height of the dry season when malaria transmission is virtually undetectable. Samples were processed in random order, with representative Day 0 and 90 samples included in each experiment using identical reagents and lots of fetal calf serum for cell cryopreservation and for processing of immunologic assays. Negligible IL-5 was detected pre-vaccination (Day 0). In contrast, detectable IFN-γ was noted at Day 0 in both vaccine recipients and RabAvert® controls. This observation is likely the result of CMI acquired by ongoing exposure to parasites during the transmission season. Similarly, 20/56 (35.7%) volunteers had detectable lymphocyte proliferative responses at Day 0 as well as antibody responses with significant vaccine-induced boosting peaking by Day 74 [25
].Modest increases in IFN-γ production were detected after stimulation of post-vaccination PBMC (collected during the dry season) with AMA-1 in all groups, including the rabies comparator. The mechanism(s) for these increases in the absence of ongoing malaria transmission are unclear. It is possible that during the malaria transmission season, persistent sub-clinical infection, which is common in semi-immune adults, results in continuous exposure to high levels of malaria antigens leading to down-regulation of IFN-γ responses to these antigens and to decreased inflammatory responses. Similarly, it is known that inducible T regulatory cells may influence the immunologic response to malaria infection during transmission season [35
]. These phenomena might not occur during the dry season when exposure to malaria antigens is greatly diminished. Blood smears were not done on asymptomatic individuals so we cannot investigate these theories. Potential explanations for the absence of a measurable vaccine-induced increase in IFN-γ production as detected by ELISpot may have been the failure of the vaccine to induce persistent cellular response in this population. Additionally, we sampled PBMC 28-day post-dose three in this study whereas in the previous study in malaria naïve adults, we sampled PBMC at 14 days post-dose three and did find significant increases in IFN-γ by ELISpot. Other potential factors include a technical difference in the assay whereby PBMC were plated at 50,000 and 100,000 per well from the Malian adult study (to reduce background) compared to 200,000 per well PBMC from the malaria naïve adults.
Both the lymphocyte proliferation and the IL-5 ELISpot assays demonstrated a clear increase in CMI correlating with vaccination. A mild but significant increase in IL-5 production in the control group receiving rabies vaccine at Day 90 was noted in response to antigenic stimulation by AMA-1 (10µg/ml). This increase was modest in comparison to the vaccine recipients and was not observed in cultures stimulated with 1µg/ml AMA-1. The results suggest that Th2 cytokine responses may be a better indicator of this vaccine’s “take” than Th1 responses represented by IFN-γ although more research needs to be performed including measurement of other Th2 cytokine responses to validate this contention and to assess the impact of the adjuvant in this response. Furthermore, the fact that close correlations were observed between antibody response, lymphocyte proliferation responses and IL-5 production argues strongly for this vaccine construct being able to elicit a wide array of host immune responses. An ongoing Phase 2 trial of this vaccine in children will assess associations between these immune responses and protection against disease and measure the impact of parasite diversity on CMI and antibody responses.
We have demonstrated a CMI response in relation to vaccination with FMP2.1/AS02A in malaria-experienced Malian adults. Our studies demonstrate AMA-1-specific lymphocyte proliferation and IL-5 production in response to vaccination and a close correlation of these CMI responses to antibody production. Whether these CMI responses are mainly supporting antibody production or represent other important mechanisms of CMI effector immunity (e.g., expansion of T memory subsets, production of other cytokines not evaluated in the present study, etc.) remains to be determined. Nevertheless, the finding that a multifaceted immune response is elicited after a three-dose vaccination series with AMA-1 is encouraging for future development of this protein construct alone or as part of a vaccine comprised of multiple malaria antigens. If protective efficacy is demonstrated in the Phase 2 pediatric trial of the FMP2.1/AS02A vaccine that is presently underway in Mali the cellular immunity associated with this protection will be studied.