The majority of clinical signs and complications of malaria result from the erythrocytic phase of malaria infection, and individuals with high-density parasitemia have a greater likelihood of experiencing malaria-related illness when results are adjusted for age (29
). Thus, a vaccine that confers protective immunity directed against the merozoite stage of Plasmodium falciparum
would have great benefit in preventing or mitigating the clinical burden of disease, particularly among infants and children who have not yet developed naturally acquired immunity and adults who experience waning immunity due to reduced exposure to malaria (15
). The merozoite protein and vaccine antigen MSP1 displays extensive polymorphisms between the two main naturally circulating alleles, 3D7 and FVO. Understanding the influence of circulating alleles on the stability or “boosting” of an allele-specific immune response in naturally exposed populations will help guide interpretation of immunogenicity outcome measurements in vaccine trials. In our study based on the DNA sequence of the region of msp1
, we found that both 3D7 and FVO alleles of P. falciparum
MSP1 are responsible for infections in western Kenya, with the majority being a single infection with 3D7. This is supported by other MSP1 genomic typing done in Kisumu, Kenya (C. Ockenhouse, personal communication). Due to technical limitations in sequencing the entire length of MSP142
we were not able to ascertain if other allelic variants of MSP1, i.e., CAMP/FUP (33-kDa 3D7 plus 19-kDa FVO) were present. It is also possible that detection of the FVO MSP133
allele was underestimated as a result of inadequate sensitivity of our PCR assay to detect low-level FVO parasitemia in the face of a coinfection with high-level parasitemia of the 3D7 allele. Using another PCR-based method to detect P. falciparum
allelic coinfections within this community in 2003, we found the FVO MSP119
variant to be dominant (11
), suggesting that the CAMP/FUP strain may be the dominant infection.
The genetic sequences of the block 15 to 16 msp1
, suggesting (MSP133
) isolated from 20 random samples from Kenyan adults and children demonstrated genetic sequences identical to those of the referent 3D7 and FVO vaccine strains, with no synonymous or nonsynonymous polymorphisms. This information provides optimism for time-consuming MSP1 vaccine development, since it suggests that although the 3D7 and FVO alleles differ, recent isolates do not reveal novel polymorphisms. Similar results were found in a study conducted in Thailand (22
), where sequencing of the region of msp1
from 19 individuals demonstrated only four nucleotide differences, all resulting in nonsynonymous changes. Taken together, these results suggest that MSP1 vaccines should not be hampered by emerging allelic variants within the 33-kDa region of individual MSP1 strains. In comparison, the extent of circulating polymorphisms of the MSP119
has been well documented from a vaccine trial site in Mali (43
). In that study, among 14 haplotypes identified, the prevalence of the 3D7 MSP119
allele was only 16%, with the CAMP/FUP and FVO alleles at 36 and 46%, respectively.
Previous work has supported the importance of MSP119
in inducing humoral immune responses and of MSP133
in providing necessary T-cell responses (8
). Udhayakumar et al. (44
) examined T-cell proliferation by residents of western Kenya to various 17-mer peptides from the 33-kDa region chosen by amphiphilic score. The peptide with the highest proliferation was PL147 (block 16). We used a peptide identical to PL147 (designated M41) that also generated one of the higher proportions of responders, reinforcing this peptide's immunodominance. An additional peptide from block 15, K29, was repeatedly immunogenic in our study. A recent study on the coast of Kenya used our overlapping MSP1 peptides to evaluate IFN-γ responses by cord blood T cells, reflecting prenatal exposure to malarial antigens (28
). Consistent with the findings presented here, the peptides generating IFN-γ responses by the greatest number of newborns' cord blood mononuclear cells were M41, M42, and M43, as well as K29, K30, and K31. The M41 peptide lies in a variable region with 10 of 18 amino acids being different between the FVO and 3D7 alleles, while the K29 peptide is in a semiconserved region with four amino acid differences between the two alleles, supporting the data from Udhayakumar et al. that cellular immune responses to 33 kDa are greatest in regions differing between these two allelic forms of MSP1.
A study by Wipasa et al. (46
) used pools of overlapping peptides from the MSP133
region of Plasmodium yoelii
to examine the immune responses in mice. They found that two peptide pools induced responses by T cells that, when transferred to naïve immunodeficient (SCID) mice prior to challenge with blood stage P. yoelii
, generated partial protection against parasitemia and death. Our study, while not using peptide pools, found the number of participants with IFN-γ responses to MSP142
recombinant proteins to be much higher than the number of responders to individual peptides. These findings suggest that multiple epitopes in the C terminus of MSP1 generate effective T-cell immune responses. With the unavailability of recombinant MSP133
for use in the assays performed here, we were unable to determine if the increased IFN-γ responses to MSP142
were due to additional T-cell epitopes in MSP119
. However, in the study done by Udhayakumar et al. (44
) the peptides derived from MSP119
elicited weaker T-cell proliferation responses than peptides from MSP133
. Also, in a study by Lee et al. (24
), fewer than 10% of Kenyan adults demonstrated IFN-γ responses to individual 20-mer peptides from MSP119
. Lastly, in a study of five malaria-naïve adults vaccinated with the 3D7 allele of MSP142
, interleukin-5 (IL-5) levels from PBMCs stimulated with homologous MSP133
were both higher than in cells stimulated with MSP119
). Six additional volunteers were vaccinated with MSP142
FVO, and similarly IL-5 production with MSP142
was higher than with MSP119
. The aggregate of evidence from these studies would support our conclusion that the dominant T-cell epitopes lie in the 33-kDa fragment of MSP1, although we did not rule out contribution by N-terminal T-cell epitopes.
Over the 11-month observation period, IFN-γ responses to 3D7 recombinant MSP142 tended to be more prevalent, robust, and stable than those to FVO protein at all monthly time points and over two malaria seasons. This may reflect increased circulating prevalence of the 3D7 variant of MSP133 (which would include the 3D7 MSP142 variant plus the CAMP/FUP variant), the repeated exposure of adults in a region where malaria is holoendemic, or perhaps an increased immunogenicity of the 3D7 MSP133. We were not able to demonstrate any temporal relationship between IFN-γ responses and the prevalence of parasitemia in the same or preceding month as when the ELISPOTs were performed. As this study involved only 24 adults who were asymptomatic, it is unclear whether the IFN-γ responses were sufficient to be protective against malaria-related illness or partially responsible for the observed clearance of blood stage infections (in the absence of antimalarial drugs).
PBMC IFN-γ responses to the MSP133
18-mer peptides did not appear to be stable, due to the relatively low frequency of responders and low magnitude of IFN-γ responses measured by ELISPOT. There were only two individuals who consistently responded to a single peptide, suggesting that overcoming HLA haplotype restriction might improve responses; however, HLA matching a vaccine to individuals would be logistically impossible and prohibitively expensive due to the extreme HLA heterogeneity in African populations (25
). In this study, we were not able to determine the HLA class I and II genotypes of the study participants, results which would have allowed us to evaluate the influence of HLA restriction on the stability of IFN-γ-specific peptide responses. However, from our previously published studies we know that there is a high degree of HLA diversity with few dominant alleles and only rare instances of homozygosity (7
). Unlike other pathogens such as viruses and bacteria, eukaryotic parasites with complex life cycles and larger genomes display many more antigens and allelic variants of those antigens to the host immune system. This may thus diminish the impact of a single “immunodominant” T-cell epitope commonly used in recall assays, as there may be many such epitopes within and between epitope-variant malaria proteins. Accordingly, determining HLA genotype has less importance for studies using recombinant malaria proteins for vaccine or immunologic profiling studies. In addition, in this study we used overlapping peptides that should be able to be recognized by different HLA types, thereby supporting one of the main points of our study showing the lack of reproducible responses over time.
Other longitudinal studies examining cellular responses to the carboxyl-terminal region of the FVO allele (i.e., Wellcome strain) by individuals who had been mainly infected with parasites with 3D7 (i.e., MAD20) alleles were conducted in the Gambia by Riley et al. (37
). Consistent with our findings, lymphoproliferative and IFN-γ responses to MSP1 in adults fluctuated over time and were not associated with parasitemia or malaria transmission season. Their studies of children (3 to 8 years of age), however, showed that lymphoproliferative but not IFN-γ responses (by enzyme-linked immunosorbent assay [ELISA]) before a malaria season were significantly associated with asymptomatic malaria infections, although IFN-γ production did increase with age. Another study of children age 2 to 11 years in Uganda found only 9 of 117 had IFN-γ responses to MSP1. All of the responders were older than 4 years, with no relationship with prior episodes of parasitemia (40
). It thus appears that cellular immune responses to MSP133
increase with age and cumulative exposure, similar to humoral immunity to MSP119
). Studies examining antibody levels in adults, including one evaluating the same adult population in this paper, showed that titers remain high and stable over time (6
). The IFN-γ responses seen here may possibly reflect low-frequency memory T cells involved in protection in conjunction with antibodies. Longitudinal studies of effector and memory T-cell responses and cross-reactivity/cross-protection conferred by sequential allelic infections are ongoing to address the clinical relevance of IFN-γ-mediated responses.
As the 3D7 MSP142
protein antigen is a leading blood stage vaccine candidate, several vaccine trials testing it have been completed (33
). In a phase 1 trial determining the safety and immunogenicity of MSP142
formulated in AS02A in malaria-naïve adults, moderate increases in IFN-γ ELISPOT responses were seen in most volunteers after the first but not the second or third immunization (33
). Preimmunization IFN-γ responses in malaria-experienced populations are important for establishing baseline values for subsequent vaccine trials, although as we have shown in this study that such responses vary greatly within and between individuals. Similar to our previous cross-sectional studies of pre-erythrocytic-stage malaria peptides (31
), while an individual's responses varied between time points, the overall proportions of responders to each allele were similar across each season. Given the large proportion of adults responding to the 3D7 allele in this study, outcome measurements for an MSP1 vaccine tested in areas in which the disease is endemic may have to incorporate a fold increase over baseline and prolonged duration of immune response in order to distinguish between a vaccine-attributable affect and normal variation as the result of natural exposure.
It has long been postulated that malaria may interfere with the generation of IFN-γ-producing memory T cells; thus, repeated natural infections may pose additional hurdles for vaccines in areas in which malaria is endemic. Evidence of this inherent difficulty was shown with another blood stage antigen, apical membrane antigen 1 (AMA1), when tested as a vaccine formulation with the adjuvant system AS02A in malaria-exposed adults in Mali (26
). Fifty-five percent of volunteers at baseline had measurable IFN-γ ELISPOT responses, and while there was an increase in IFN-γ production after three vaccinations, there was no statistical difference in outcomes between volunteers who received the AMA1 candidate vaccine and those who received the rabies control vaccine. This phase 1 study did not evaluate efficacy, but it is hoped that future efficacy trials conducted in areas of endemicity will have cellular assay endpoints to help elucidate the role of IFN-γ.
Humoral immune responses to MSP1 will play an essential role in vaccine-induced protection to disease, and with MSP1, antibodies generated to the conformational epitopes of the 19-kDa region are important in inhibition of erythrocyte invasion (3
). However, as has been shown in Aotus
monkey trials, better immunogenicity and homologous protection were achieved with immunization with recombinant MSP142
than with recombinant MSP119
), underscoring the importance of the 33-kDa region. Selecting the appropriate allele(s) of MSP142
to go forward as a vaccine candidate will be dependent upon the role the MSP133
plays in immunity. Our study has shown that the 3D7 MSP133
allele, the most prevalent circulating variant in western Kenya, generates a higher and more consistent IFN-γ response in asymptomatic adults with naturally acquired immunity to malaria. Such IFN-γ production has been shown to be protective in children in Papua New Guinea, with the underlying mechanism postulated to be increased cellular immunity and/or induction of antibody-dependent cell-mediated inhibition (ADCI) (12
). Alternatively, the MSP133
variant may, through T-cell help, be the determinant of effective MSP119
humoral responses. It is possible that the 3D7 allele in this regard may not be as desirable, as in the phase 2b vaccine trial of 400 Kenyan children where the 3D7 allele of MSP142
did not demonstrate any protection (35
). The ability of a vaccine to induce cross-reactive cellular responses to the multiple alleles of P. falciparum
encountered in the field may also be a determining factor in its success. In the study of malaria-naïve adults vaccinated with either the 3D7 or FVO allele of MSP142
, there was little IFN-γ produced by PBMCs when they were stimulated with the heterologous allele compared to when they were stimulated with the homologous allele (20
). A single blood stage antigen will not likely be sufficient as a stand-alone vaccine; thus, planned studies examining the nature and development of immune responses to MSP1 in Kenyan infants and children, and the involvement of these responses in protection, will be crucial to the development of a successful multistage, multiantigen vaccine candidate that can benefit children as well as adults, particularly as increased global efforts to reduce malaria transmission may reduce the strength and duration of naturally acquired immunity.