Variation in the immune responses against Plasmodium falciparum merozoite surface protein-1 and apical membrane antigen-1 in children residing in the different epidemiological strata of malaria in Cameroon

Malaria constitutes a major health problem in Cameroon where it accounts for 48% of all hospital admission, 30% of morbidity and 67% of childhood mortality per year [1, 2]. Malaria in Cameroon is caused predominantly by Plasmodium falciparum but other species, including Plasmodium vivax, Plasmodium malariae and Plasmodium ovale have also been reported to cause malaria albeit at lower frequencies [3‐6]. The entire Cameroon’s population of over 22 million is at risk of infection [7]. The epidemiology of malaria in Cameroon is complex and has been described as “Africa in miniature” [8] because she has all the epidemiological strata of malaria present in Africa. Six epidemiological strata of malaria have been identified and mapped in Cameroon: the sudano-sahelian (SS) strata, high inland plateau (HIP) strata, savannah-forest transmission (SFT) strata, south Cameroonian equatorial forest (SCEF) strata, high western plateau altitude (HWP) strata, and the coastal (C) strata [9]. These epidemiological strata differ in terms of their geographical and ecological characteristics, transmission pattern and endemicity level, and in terms of the main vectors transmitting malaria parasites [9].

Children in Cameroon, as in most other endemic countries in Africa, are most at risk of clinical and severe malaria because of their low immunity [10‐12]. Immunity to malaria is known to be acquired slowly and in an age-dependent manner, after repeated exposure [10]. At a later stage, the capability of controlling parasitaemia in the blood is developed. The mechanism underlying development of anti-disease immunity and factors governing effective protection are still largely unknown as findings from different correlates of antibody-mediated immunity studies are often conflicting in their conclusions [13, 14]. Presently there is no single immunological correlate of protection to clinical malaria, moreover those described do not sufficiently account for the overall variation in susceptibility observed in a population [14].

Several blood-stage antigens of the malaria parasite with different structure and location have been evaluated for their role in inducing protective antibodies against clinical malaria, including the merozoite surface proteins (MSP-1, MSP-2, MSP-3, etc.), the apical membrane antigen-1 (AMA-1), erythrocytes binding antigen (EBA-175 RII) and the glutamate-rich protein (GLURP) [15‐20]. In this study, the immune responses against MSP-1 and AMA-1 was evaluated because they have been shown to have a strong association with protection against clinical malaria [21, 22] and there is a need to collect more information on natural immune responses in children living in a high malaria endemic area, such as Cameroon. The MSP-1 is one of the best characterized proteins in several Plasmodium spp. MSP-1 is the most abundant merozoite surface protein, and is thought to be involved in the initial attachment of the merozoite to the erythrocyte surface [22]. The 19 kDa C-terminal fragment of MSP-1 (MSP-1₁₉) has been recognized as the target of immunoglobulin G (IgG)-based protective immunity [23] and is a promising vaccine candidate [24]. MSP-1₁₉ was selected for this study over the other MSP-1 molecules because of the fine specificity of MSP-1₁₉ specific antibodies [25, 26] coupled to its role in protecting against clinical malaria. The recombinant 62 kDa apical membrane antigen-1 (AMA-1), on the other hand, is present in both the pre-erythrocytic and asexual blood-stage forms of the Plasmodium parasite. AMA-1 is involved in the re-orientation of merozoites prior to invasion of the erythrocyte and thereby plays a central role in erythrocyte invasion by Plasmodium species [22, 27]. Antibodies against this molecule display inhibitory activities against sporozoite invasion of hepatocytes [28] and against merozoite invasion of erythrocytes [29, 30]. Sero-epidemiological studies performed in other malaria-endemic areas have shown some evidence of antibodies against these antigens in protecting against malaria [20]. However, studies designed to assess the immune responses to malaria parasites in children in Cameroon are limited, with a few studies published thus far describing only the immune responses in neonates and infants [31, 32].

Previous studies in Cameroon, have shown that malaria transmission decreases steadily northward, from the C strata in the south to the SS strata in the north; meanwhile the rate of severe malaria attack and mortality rate appears to increase in the same direction [33, 34]. It was hypothesized that the observed trend could be attributed to the decreasing immunity against P. falciparum as a result of the decrease in transmission intensity as one move towards the north. In order to test this hypothesis, the humoral immune responses (total IgG) against the P. falciparum MSP-1 and AMA-1 antigens was measured and compared in children in five epidemiological strata of malaria in Cameroon. This will generate baseline data that may be useful for designing studies for future testing of malaria vaccine candidates.

The proportion of IgG responders observed in this study was high (72.1%). These findings are similar to studies performed on different populations in Cameroon [3, 32]. The high proportion of IgG responders in this study could be due to the constant exposure to infective bites of mosquitoes or the participants sustained an immune response after active infection [3]. MSP-1 was recognized more often than AMA-1 (58.5 vs 40.2%) in the serum of the study participants. Studies have shown that the immunogenicity of the large repertoire of antigens often targeted as malaria vaccine candidates are not the same [13, 43]. However, these data should be interpreted with caution bearing in mind that the dilutions of the two antigens were not the same (1/100 for MSP-1 and 1/1000 for AMA-1). Furthermore, the proportion of responders was higher among females compared to males (p = 0.002). In addition, the mean IgG level was observed to be higher in females compared to males for AMA-1 (p = 0.010) but not with MSP-1 (p = 0.055). These findings suggest that in the study area females were more prone to infection with malaria parasites than males, which is evident from the finding of a higher malaria prevalence in females in this study, although it was not significant. The overall mean IgG antibody level in the current study was observed to increase with increasing age (p < 0.0001). These findings are in line with the numerous studies that show that immunity to malaria increases with age as the frequency of exposure also increases [10, 13, 40, 44].

In the current study, site-specific analysis revealed that the proportion of IgG responders was highest in Limbe (in C strata) and lowest in Ngaoundere (in the HIP strata) (p < 0.0001). Similarly the IgG antibody titres were observed to decrease northwards from Limbe (in C strata) where it peaked to Ngaoundere (in HIP strata) where it was lowest (p = 0.001). These observed differences could be attributed to the differences in the transmission intensity of malaria observed in the different epidemiological strata; malaria has been described as hyperendemic in Limbe [45], holo-endemic in Yaounde [46, 47], meso-endemic in Bamenda and Ngaoundere, and hypo-endemic in Maroua [33, 34]. The transmission of malaria in Cameroon has been observed to decrease steadily from the south towards the north of the country [33, 34]. The association between immunity to malaria and transmission intensity has previously been reported [35, 48‐50]. The differences in immune responses could also be attributed to factors including ethnicity [45], red blood cell defects [51] or use of mosquito nets, which could greatly influence exposure to malaria parasites and modulate the levels of antibodies in different populations. The decreasing trend in the magnitude of antibody responses observed in the current study supports the hypothesis that increased risk of severe malaria and fatality towards the northern regions as earlier reported [33, 34] could be attributed to the decreasing immunity against malaria. Site-specific analysis revealed two major groups of malaria transmission based on the serological reaction of the children: the southern group (comprising C and SCEF strata) and the northern group (comprising the HWP, HIP and SS strata). The southern and northern groups differed remarkably in terms of their serological reaction (p < 0.0001), but the different strata that make up the two groups were similar to each other. These findings may have significant implications in the design of future trials to test malaria vaccine candidates in Cameroon. However, larger studies involving more study sites within the different epidemiological strata will be required to confirm these observations.

In the current study, the overall clinical malaria prevalence was 14.6%. This prevalence was lower than the national prevalence of 29% reported in 2012 [52] and could be attributed to the relentless effort of the Cameroon Government to control the disease through mass distribution of insecticide-treated bed nets to all households in the country and to the intense sensitization of the population through media [47, 53]. The prevalence of malaria was observed to decrease steadily northward from Limbe (C strata) where it peaked to Maroua (SS strata). A significant association was observed between prevalence of malaria and study site. This pattern in malaria prevalence could be related to differences in the level of transmission of malaria and explains the observed trend in the antibody responses. Plasmodium falciparum was identified as the only Plasmodium species causing malaria in the target population, which is in line with other studies [33, 34] but contradictory to studies that have reported other Plasmodium species causing malaria, including P. vivax [3, 4]. These discrepancies between this study and others could be attributed to differences in the study designs; this study targeted mainly children, meanwhile others targeted adults in addition to children.

The prevalence of malaria was significantly higher in non-responders compared to responders (p < 0.0001) in this study. Furthermore, non-responders were observed to be at a higher risk of clinical malaria compared to responders. These findings serves as confirmation of studies reporting that antibodies to MSP-1 and AMA-1 are associated with protection against clinical malaria [13, 31] and therefore reinforces their potential as promising vaccine candidates. In this study, the mean IgG antibody level was observed to be higher in children infected with malaria parasites compared to their non-infected counterparts (p < 0.0001). This suggests that anti-MSP-1 and anti-AMA-1 antibodies are highest at the presence of infection and decay shortly after treatment. This observation conforms to studies performed elsewhere [54‐57]. Similarly, the mean parasite density was observed to be higher in non-responders compared to responders (p = 0.035). The observation of higher parasite density in non-responders conforms to the study by Taylor et al. [31] and suggests the role of antibodies in the protection against clinical malaria.

This study, which has revealed the pattern of the immune responses against malaria in Cameroonian children, is limited in that children were recruited in major urban centers only, and the findings may not be generalizable to children in rural areas. Only two malaria vaccine candidate antigens were used in this study, which may not be a true reflection of the overall immune responses in the target population. In addition, only total IgG antibody was measured and not the IgG sub-types. Larger studies involving many more malaria vaccine candidate antigens as well as measuring the different IgG sub-types will be required to give a clearer picture of the immunity against malaria parasites in Cameroonian children. Furthermore, the seasonal variation of the immune responses against malaria parasites was not investigated in the current study as participants were recruited during the rainy season only during which malaria transmission is usually higher. Transmission of malaria in Cameroon especially in the northern regions is seasonal and generally lower during the dry season. Studies designed to recruit children during the rainy and dry seasons will therefore be required to provide a clearer picture.

Overall, a high proportion (72.1%) of children in the current study were IgG responders. IgG antibody responses against MSP-1 and AMA-1 recombinant antigens were observed to increase in magnitude with increasing age. The risk of clinical malaria in this study was observed to be higher in IgG non-responders compared to responders. Furthermore, malaria parasite density was significantly higher in non-responders compared to responders. The immune responses against MSP-1 and AMA-1 vaccine candidate antigens in the current study were observed to decrease steadily from Limbe (in C strata) to Ngaoundere (in HIP strata). Furthermore, based on the serological reaction of the children, malaria transmission in Cameroon could be categorized into two major groups: the southern (comprising C and SCEF strata) and the northern (comprising HWP, HIP and SS strata) groups. These findings may have significant implications in the design of future trials to test malaria vaccine candidates in the country.