We have developed viral vectored blood-malaria vaccine candidates that encode bi-allelic transgene inserts for the MSP1 and AMA1 antigens. These vaccines were developed with the aim of providing a broad immunological response that can protect against multiple strains of P. falciparum
. The use of a ChAd63 vector to prime responses followed by a boost with MVA eight weeks later has been shown to induce high levels of T cells and substantial antibody responses in both animal models and Phase I/IIa clinical trials in humans for both blood-stage vaccine candidate antigens (8
). However, previous studies have raised concerns that inclusion of multiple allelic variants in a vaccine may be detrimental to both the priming and in vivo
re-stimulation of antigen-experienced memory T cells owing to the immune evasion effects of antagonistic APLs present in polymorphic antigens. Here we explored the concepts of immune interference, antagonism and diversion of IFN-γ T cell responses in the context of human vaccination with viral vectored vaccines and subsequent malaria exposure following a Phase IIa CHMI study.
Previous studies have suggested that dimorphic malaria antigen variants arose and were maintained in parasite populations due to a survival advantage in the context of parasite co-habitation within the human host. MSP1 allelic T cell responses failed to correlate with differential antigen exposure in The Gambia (39
), and associated with this observation was the inference from in vitro
studies that co-habiting P. falciparum
parasites could interfere with the priming of T cell responses by the phenomenon of ‘immune interference’, and that the same would be true for re-stimulation of memory responses by ‘immune antagonism’ (38
). Such immune evasion mechanisms could be achieved by APL epitopes from both HLA class I and class II molecules present in dimorphic antigens leading to antagonism of responses (36
). Ultimately, concerns were raised about the ability to prime and boost cellular immune responses with vaccines encoding more than one allele of an antigen. Until now, these concerns could not be fully explored in the context of human vaccination due to the lack of bi-allelic malaria vaccine candidates that are capable of inducing strong cellular immune responses in human volunteers. Here we investigated these effects following human vaccination with the ChAd63-MVA viral vectored vaccine platform – one that has been optimized for the induction of both humoral and cellular immunity (22
Initially we investigated the priming and boosting of T cell responses using the ex-vivo
IFN-γ ELISPOT assay. T cell responses, as measured against the 3D7 and FVO alleles of AMA1, appeared to be primed and boosted consistently, and the ratio of the responses was roughly equal and also stable throughout the monitoring period. Similar results were obtained for the MSP119
region of MSP1. In agreement with this, the two alleles of AMA1 (as encoded in the vaccine) differ by 24 αα and the ectodomains share 95% sequence identity, and similarly, MSP119
shows 96% αα conservation between the 3D7 and Wellcome alleles. In contrast, the MSP133
fragment only shows 51% αα conservation between the two and in this case, responses towards the 3D7 allele were dominant over the Wellcome allele in most volunteers. This was apparent after priming and was maintained after boosting and throughout the memory phase. In the absence of a control GMP grade vaccine encoding a single allele that could be used for human vaccination, it remains impossible to establish whether responses against APL variants could display interference/antagonism at the priming and/or boosting phase, thus leading to reduced responses in the presence of both variants compared to the presence of one. However, the data clearly establish that strong and detectable IFN-γ T cell responses can be primed and boosted by immunization against both component alleles, and this was independently observed in two clinical trials of the same vaccines. The observation of strong cellular responses against the MSP133
region (in comparison to MSP119
) is in good agreement with many previous studies that have assessed T cell recognition within MSP142
in both animal models (20
) and humans (46
). It should also be noted that future studies could further assess such phenomena in the context of other T cell phenotype readouts, including different cytokine production.
We next assessed the potential effects of single strain blood-stage malaria parasite exposure on vaccine-induced T cells responses. In this case, 3D7 clone parasites were used to assess vaccine efficacy, following immunization with the bi-allelic vaccines (26
). In this experimental context, re-stimulation of heterologous epitope-specific T cell responses (Wellcome allele in the case of MSP142
or FVO allele in the case of AMA1) by antagonistic APLs from the 3D7 clone sequence could occur in conjunction with re-stimulation of the homologous 3D7 responses. No significant boosting was observed to either allele of either antigen during natural in vivo
infection by 3D7 clone blood-stage malaria parasites. In all cases, IFN-γ T cell responses post-malaria challenge continued to contract into the memory phase with similar kinetics to those observed in the Phase Ia trials where volunteers were not experimentally infected. It remains possible that the lack of boosting is due to a form of immune diversion, however in the absence of a single allele comparator clinical-grade vaccine, we cannot confirm responses were curtailed in such a manner. Interestingly, despite any apparent boosting of vaccine-induced T cell responses, de novo
T cell responses were observed against both MSP1 and AMA1 in control volunteers who had not received any vaccine. In the case of AMA1, responses were almost exclusively detected with peptides specific for the 3D7 allele, rather than those in common with the FVO allele. This is in contrast to vaccination where the common peptides are better recognized (25
), and may mean that immunization with two alleles can better focus T cell responses on conserved epitopes. In the case of MSP133
, responses that were cross-reactive with the Wellcome allele were noted in some volunteers. This is potentially due to conserved sequences between the two dimorphic MSP133
regions, or potentially represents T cells primed by 3D7 parasite exposure that cross-react with Wellcome allele sequence variants.
Given we had seen no apparent effects in terms of the overall IFN-γ T cell response following vaccination with bi-allelic antigens, we sought to confirm whether the APL antagonism previously reported by Lee et al
) could also be observed here in vitro
. One pair of previously reported peptides (M7/8) represented minimal class II epitopes that showed APL antagonism. These sequences were contained within our vaccine, and responses were detectable to either one epitope or both in about half of the volunteers tested. Interestingly, and in agreement with the previous data from naturally-exposed individuals, this epitope was shown to be convincingly antagonistic in 1/7 volunteers who possessed detectable IFN-γ responses. However, when 20mer peptides were used that contained the minimal epitopes, this antagonistic effect was no longer observed. Furthermore when minimal epitopes were combined with heterologous 20mers, antagonism was observed between M7* and M53 but not M8* and M92. The data suggested that the 20mer peptides could be processed into antagonistic APLs, but also that more optimal HLA binding sequences and/or other epitopes may be present within in the 20mers in comparison to the minimal peptides. Further testing with larger pools of 20mer peptides representing both alleles of MSP133
and AMA1 confirmed that there was no overall or obvious effect from antagonism, as measured using this ELISPOT assay readout for either the CD4+
T cell subsets. Neither antigen produced any antagonism in vitro
and responses to the combined allele peptide pools roughly equaled or were slightly greater than those seen to the strongest single allele. This observation was the same for volunteers who had a reduced or equivalent Wellcome MSP133
response as compared to 3D7, indicating that APL antagonism at the time of booster vaccination is unlikely to be contributing to the observed dominance of 3D7 responses.
Whilst performing the CD8+
T cell depletion assays, it became apparent that the 3D7 MSP133
T cell responses were dominant over the Wellcome allele in most volunteers, except those possessing HLA-B*1801. For two volunteers (#1, #14), who were heterozygous for HLA-B*1801, the 3D7:Wellcome ratio of response was roughly equal, whilst a third volunteer (#7) showed a dominant Wellcome response and was the only person homozygous for this HLA type. This observation led us to hypothesize that it was possibly a lack of HLA class I-specific epitopes in the Wellcome MSP133
sequence that may be responsible for the observed difference in immunogenicity between the alleles. We subsequently predicted twelve HLA-B*1801 epitopes, and of these, one showed dominant responses in 3 of the 14 volunteers screened, with four other epitopes also showing responses across the same volunteers. These were the three volunteers who possessed HLA-B*1801, and the broadest observed response was seen in the homozygous volunteer. These epitopes are all located close together toward the N-terminus of the Wellcome MSP133
fragment and are not present in the 3D7 sequence. Two previous studies (47
) have reported that T cell responses to MSP133
are directed to conserved epitopes, however, our data indicate this may not always be the case. Obvious differences exist between the studies, and most notably those related to study of class I versus class II epitopes, as well as host population genetics. Moreover, in the absence of single allele comparator clinical-grade vaccines, we cannot formally exclude the possibility that the observed differences between the 3D7 and Wellcome MSP133
responses are due to immune interference during T cell priming. It remains possible that the presence of the 3D7 MSP133
allele interferes with priming to the Wellcome allele in individuals, except in the case of certain HLA types such as HLA-B*1801.
It also remains of interest to assess such vaccine-induced responses in a malaria-endemic target population. A number of studies have characterized HLA types within and between West African, South African (50
) and East African populations (51
). Focusing on the HLA B locus, a number of typically African alleles such as HLA-B53 are seen across the populations at high frequencies, while other alleles vary in frequency depending on location. HLA-B*1801 was observed as the fifth highest allele frequency (6.21%) in a study in Kenya (51
), while lower frequencies have been described in West African populations such as The Gambia (5%), and Mali (2.4%), with Europeans around 10% (50
). Given that 3D7:Wellcome parasite ratios vary in different locations (52
), a lack of recognition by local HLA types as well as immune interference during co-infection would be of potential advantage to parasites. Similarly, lack of vaccine antigen recognition by local HLA types following vaccination may lead to different levels of vaccine efficacy, highlighting the importance of considering factors such as HLA types when undertaking vaccine field trials.
In summary, previous mechanistic studies have concluded that inclusion of multiple alleles of an antigen in a vaccine may be detrimental to both the priming and boosting of T cells. Our data, from human volunteers vaccinated with T cell-inducing viral vectored vaccines against two different but polymorphic blood-stage malaria antigens, suggest that T cell responses to bi-allelic antigens can be primed and boosted through vaccination. Following controlled infection with a single P. falciparum strain, vaccine-induced responses did not boost but neither were they apparently immune diverted to the heterologous strain, whilst strain-specific de novo responses could be measured in unvaccinated controls. Responses against the more conserved sequences of MSP1 and AMA1 are evenly distributed across the two alleles; however for the more divergent regions such as the MSP133 fragment, there does appear to be bias towards the 3D7 allele, most notably for CD8+ T cells. We cannot exclude the possibility that immune interference due to invariant APLs occurred at the time of priming, but we do report a paucity of HLA class I epitopes in the Wellcome allele sequence recognized by UK volunteers. We could also observe APL antagonism to minimal epitopes in vitro, however this effect did not appear to have a significant impact on the overall antigen-specific IFN-γ T cell response. Although the fine epitope specificity of responses is likely to be important in the context of vaccine-induced immunity, these data suggest host HLA-type may more significantly affect the magnitude or breadth of T cell responses. It will therefore remain of interest to further assess in target endemic populations the potential impact of circulating parasite strains, HLA type distributions and prior malaria exposure on vaccine immunogenicity.