Fully effective vaccines against malaria will likely require generation of broad-based humoral and cellular immunity. Thus, developing non-live vaccine formulations that can elicit potent antibody and T cell responses offers a platform that can itself induce substantial protection such as the RTS,S vaccine, and also be used with other vaccines to further optimize responses and protection. In this report, we compare the immunogenicity of Pf CSP as a soluble antigen versus targeting the CSP to DCs through the DEC-205 receptor using poly(I:C) as adjuvant in NHP. The data show that targeting CSP to DCs through DEC-205 receptor does not enhance the magnitude or alter the quality of CSP-specific CD4+ T cells generated post-immunization compared to CSP with the same adjuvant. In addition, we failed to detect measurable CSP-specific CD8+ T cells in NHP immunized with CSP or αDEC-CSP + poly(I:C). However, significantly higher titers of Pf sporozoite binding CSP-specific IgG antibodies were generated in NHP immunized with CSP + poly(I:C) as compared to αDEC-CSP + poly(I:C) group. Antibodies from both vaccine groups showed neutralizing activity by limiting Pf sporozoite entry into a hepatocyte cell line in an in vitro assay. Taken together, the data show that CSP delivered as soluble antigen elicits comparable Th1 immunity and more potent antibody response as compared to CSP targeted to DCs trough DEC-205 receptor when adjuvanted with poly(I:C).
The lack of demonstrable differences in Th1 immunity with CSP compared to the αDEC-205 targeting approach and the failure to elicit CD8+ T cell responses differs from data obtained in mice [16
]. Several explanations may explain these findings. First, in this study we used a high dose (400 μg) of CSP to immunize NHP. Mouse studies reveal that very low amounts of αDEC-antigen are far more efficient than substantially higher amounts of protein [16
]. It is possible that a dose response of CSP and αDEC-CSP might have revealed a difference in their relative immunogenicity. Second, the lack of CD8+ T cell responses by the DEC targeting construct may reflect differences between mouse and NHP or a requirement of anti-CD40 antibody that was used in the mouse study [16
]. In addition, there may be a limited number of Pf CSP CD8+ T cell epitopes in NHP. Third, the avidity of the αDEC antibody may be critical for optimizing its effects. In this regard, clone MG38 used in this study has a much lower binding affinity for DEC-205 receptor as compared to the newer generation mAb clone 3G9 (data not shown). Indeed, in ongoing experiments in NHP using a higher affinity αDEC-205 (clone 3G9) with an HIV Gag protein administered with Poly-ICLC shows induction of CD8+ T cells that is not observed with HIV Gag protein and Poly-ICLC (manuscript in preparation). Thus, future studies using a higher affinity αDEC-205 antibody will assess whether DC targeting of CSP is advantageous for the development of a pre-erythrocytic vaccine against malaria.
An important aspect of any vaccine study is the durability of the responses. In this regard, low frequency CSP-specific CD4+ T cells were detected in the peripheral blood of both CSP or αDEC-CSP + poly(I:C) immunized groups at approximately 6 months post primary immunization. Moreover, there was also a decline in the titers of CSP-specific IgG antibodies over time. These results are similar to data obtained following immunization with adjuvanted RTS,S [15
]. Therefore, a CSP based vaccination regimen may require annual boosting to maintain a sufficient threshold of antibody to mediate protection. Accordingly, CSP-specific CD4+ T cell responses were boosted upon an additional immunization with a single dose of CSP + poly-ICLC at week-73 post primary immunization (data not shown). Finally, in terms of having a biologic effect in vivo
, we show that mice immunized with CSP + poly-ICLC had a decrease in the parasite burden in the liver upon i.v. challenge with a high dose of 10,000 chimeric Pb-Pf parasites two months following the last immunization. These data establish that a truncated Pf CSP with poly-ICLC as an adjuvant, has some protective effect by limiting parasite burden in the liver upon Pf infection. Ongoing studies using a full-length Pf CSP and poly-ICLC have shown more substantial reduction in parasite burden in the liver (manuscript in preparation). In comparing this study with other platforms, a prior report using QS-21 as an adjuvant with a multiple antigen peptide based vaccine conferred sterilizing immunity in mice using a mosquito challenge with Pb-Pf parasites. In this study, transfer of serum from immunized animals into naïve mice was sufficient to mediate sterile protection upon challenge with infected mosquito bite [27
]. Thus, it will be of interest to assess whether our vaccine regimen used here would have a greater effect on protection with this challenge model rather than with a high dose of parasites administered i.v..
The other major aim of this study was to assess whether a heterologous prime-boost immunization with CSP + poly(I:C) and irradiated PfSPZ could enhance immunity compared to either vaccine modality alone. Multiple immunizations with radiation attenuated or genetically modified Plasmodium
sporozoites generate sterilizing immunity across multiple species [39
]. Thus, establishing a combination vaccine regimen that could limit the number of immunizations with irradiated PfSPZ may have important clinical application. Here we sought to determine how boosting with a single immunization with irradiated PfSPZ influenced existing CSP-specific antibody and Th1 responses. CSP-specific CD4+ T cells and humoral immune responses generated following immunization with CSP/αDEC-CSP + poly(I:C) were not boosted by a single dose of 150,000 irradiated PfSPZ administered intravenously. Moreover, we failed to detect any CSP-specific antibody or T cell responses to a single immunization with irradiated PfSPZ. These data suggest that more than one boost with irradiated PfSPZ may be required to enhance the CSP-specific humoral and cellular responses. Indeed, data from human vaccine studies show that several immunizations with irradiated PfSPZ showed only a modest induction of CSP-specific IFN-γ producing cells [47
]. Similarly, we have shown that CSP-specific antibodies and malaria-specific T cell responses can be induced in NHP with irradiated PfSPZ but this required at least two immunizations given i.v. (manuscript in preparation). Additional possibilities to explain the lack of boosting by irradiated PfSPZ are that pre-existing CSP-specific antibodies from the primary immunization may bind to the irradiated PfSPZ thus effectively neutralizing any boosting effect. In summary, the inability of irradiated PfSPZ to boost CSP-specific CD4+ T cell and humoral immune responses generated in NHP following immunization with CSP or αDEC-CSP + poly(I:C) suggest that either these heterologous vaccine platforms do not optimize immunity, the order of prime-boosting needs to be changed or there needs to be a greater number of immunizations with irradiated PfSPZ.
In conclusion, this NHP immunogenicity study shows that CSP-specific antibody and CD4+ T cell responses can be elicited using an unformulated Pf CSP with poly(I:C). Thus, poly(I:C) offers a potential alternative to AS01B as an adjuvant with protein based vaccines. A critical question is whether the efficacy seen with the RTS,S/AS01B vaccine in humans is due to antibodies, CD4+ T cells or both. If poly(I:C) is a more effective adjuvant for inducing CD4+ T cell responses than AS01B and such responses have a direct effector role or lead to enhanced antibody responses, it offers the potential to improve upon the clinical efficacy of RTS,S/AS01B. In this regard, the data presented here in NHP with CSP and a non optimized formulation of poly(I:C) induced a significantly higher number of CSP-specific ELISPOT responses than detected in another NHP study following three immunizations with RTS,S/AS01B [48
]. The frequency of multi-functional CSP-specific CD4+ T cells secreting IFN-γ, IL-2 and TNF-α reported here is also higher than observed in NHP immunized with RTS,S/AS01B [14
]. While the role of multi-functional Th1 responses in malaria infection is unclear, a recent study reported a strong association of multi-functional CSP-specific CD4+ T cells with a better outcome upon malaria challenge following immunization with RTS,S/AS01B in a phase 2 clinical trial [11
]. In terms of humoral immunity, the antibody responses generated in this study are measured differently than the other NHP studies with RTS,S/AS01B. However, they are clearly robust and functional. An important caveat to making such comparisons amongst different studies is the variability associated with NHP studies and the assays used to measure adaptive immunity. Ultimately, a direct comparative study would be required to assess differences amongst RTS,S and CSP with poly(I:C) as adjuvant. Finally, since induction of CD8+ T cells has been shown to have a major role in controlling liver stage of malaria infection [6
] optimization of the protein and poly(I:C) formulation may be required to enhance cross-priming. As poly-ICLC used in the mouse studies is a more potent formulation than the poly(I:C) used in the NHP study, it is possible that it would enhance priming for CD8+ T cells. The induction of such responses may require additional formulation of the CSP, which is being evaluated. Overall, having a new non-live protein based platform that can induce potent antibody and Th1 responses combined with a recombinant live viral vector vaccine to induce CD8+ T cell responses could provide improved immunity and have a major impact upon the development of a pre-erythrocytic vaccine.