Background
As US armed forces continue to deploy to malaria endemic areas for both military and humanitarian missions, development of efficacious anti-malarial chemoprophylaxis with convenient regimens and minimal side effects remains a top priority for the Department of Defense [
1]. Weekly mefloquine administration is associated with well-described neuropsychiatric toxicity requiring mental health evaluation, and recently received a 2nd black box warning for this from the US Food and Drug Administration (FDA) [
2,
3]. Currently favoured options include daily doxycycline, which may have lower compliance than mefloquine and considerable side effects, or atovaquone/proguanil, which is both expensive and susceptible to resistance [
4].
Historically, chemoprophylaxis agents have been licensed based on the results of placebo-controlled studies in semi-immune volunteers. However, this approach raises two ethical dilemmas—justification for the use of placebo and post-trial access for the study population [
5]. Non-inferiority studies between active comparator drugs is an alternative approach hampered by the logistical challenges and costs associated with very large sample sizes needed to compare products with 95 % or better efficacy. This was underscored in a trial comparing weekly mefloquine with tafenoquine in Australian soldiers deployed to East Timor. While there were no cases of
Plasmodium falciparum malaria in either treatment arm, true efficacy could not be determined due to lack of a surrogate endpoint for malaria exposure [
6], and required an estimate based on attack rates in nearby indigenous personnel [
7]. Another key challenge is extrapolation of chemoprophylactic efficacy results from semi-immune populations living in endemic areas to non-immune travelers, a population who may be at great risk for more severe illness.
A surrogate biomarker for malaria exposure would ensure future active comparator trials are interpretable. Antibodies to blood stage malaria antigens, such a merozoite surface protein 1 (MSP1), have been evaluated in sero-epidemiological surveys as estimates of malaria exposure [
8,
9]. The serologic stability of MSP1 make it an attractive candidate as a surrogate endpoint of exposure for chemoprophylaxis trials, but unfortunately, in a proof-of-concept
P. falciparum controlled human malaria infection (CHMI) study by Moon et al. [
10], antibodies to PfMSP1
42 were not induced in malaria-naive, healthy volunteers taking mefloquine chemoprophylaxis with strict clinical and parasitologic monitoring; however, it may be possible that assessment MSP1
42 as a surrogate biomarker in endemic populations may be of more utility.
The US and Cambodian militaries have recently been working to develop new anti-malarial chemoprophylaxis agents [
11]. Malaria in Cambodia is characterized by a low incidence of
P. falciparum and
Plasmodium vivax infections in roughly equal proportion, focal transmission by forest-dwelling
Anopheles mosquitoes with sporadic infections, all occurring in the epicenter of anti-malarial resistance [
12‐
15]. Cambodian military personnel deploying from the non-endemic urban areas of Cambodia to forested areas along the border may be essentially malaria-naïve and at risk. This investigation assessed MSP1
42 titers in a cohort of healthy asymptomatic Cambodian soldiers in a malaria endemic area [
16] in order to evaluate its utility as a biomarker for pre-existing immunity as well as a surrogate endpoint of malaria exposure for future chemoprophylaxis studies.
Discussion
The US military continues to develop new anti-malarial drugs and vaccines as part of its mission to protect the war fighter. Historically, anti-malarial chemoprophylaxis studies for licensure in Asia and elsewhere have used placebo-controlled study designs in semi-immune populations, with recent examples including tafenoquine in soldiers of the Royal Thai Army [
22] and azithromycin in Indonesian soldiers [
23]. However, the declaration of Helsinki 2000 raised serious concerns regarding placebo-controlled study designs where alternative effective therapies exist [
5]. An established, validated biomarker for malaria infection would allow for calculation of protective efficacy in active-controlled malaria prevention studies. To this end, in 2009, Moon et al. [
10] performed a CHMI study administering
P. falciparum to healthy volunteers under mefloquine prophylaxis to assess seroconversion rates of
P. falciparum MSP1
42, defined as a fourfold rise in titers. None of the volunteers seroconverted, and only four of six controls, those who did not receiving mefloquine who did develop malaria, seroconverted.
This study aimed to characterize background and changes in humoral
P. falciparum and
P. vivax MSP1
42 responses during a single malaria season in order to gain insight to malaria exposure and immunity in a low transmission setting with multidrug resistant malaria in northern Cambodia and lay the groundwork for malaria prophylaxis studies. The predominantly resident military population was a mix of self-reported malaria-naïve and semi-immune individuals; some were newly arrived from non-transmission areas while other long-term veterans had likely had multiple exposures over several years while stationed in malarious areas. Transmission varied substantially between the two study locations, with significantly higher attack rates during the cohort study at the forested site B compared to semi-urban site A [
16]. Baseline titers to
P. falciparum and
P. vivax MSP1
42 proteins in asymptomatic, aparasitemic adults varied greatly by individual, with a 1–5 log difference not directly attributable to geographic site nor reported clinical history of prior malaria infection. Geomean titers were higher in those reporting a malaria infection in the past year compared to those with no history of disease, but significant overlap existed, underscoring the lack of utility of a single serological measurement as marker of immunity. Based on sero-positivity rates, prior exposure seemed quite evident, particularly for
P. vivax, even in those reporting no clinical history of malaria.
The wide variation in baseline serology is similar to prior sero-epidemiology studies in both high and low transmission settings. Fowkes et al. [
24] found that titers to merozoite surface antigens
P. falciparum and
P. vivax AMA1 could fluctuate widely in pregnant women living in Thailand even when measured biweekly. Another study in northern Thailand found that in individuals with documented falciparum or vivax malaria infection in the past 6 years, only 48 % were sero-positive to PfMSP
19 and 11 % to
P. vivax MSP1
19 [
25]. Even in a hyperendemic transmission area in western Kenya, approximately 60 % of semi-immune children and adults surveyed did not have a humoral response to any of the three main allelic variants (CAMP, FVO and 3D7/MAD20) of PfMSP1
42 [
26]. This lack of correlation with reported malaria exposure history raises significant doubts regarding the utility of MSP1 antibodies as an enrolment criterion for malaria prevention studies in even low transmission areas requiring malaria-naïve volunteers. At best, aggregate seroprevalence data could be used to identify locations to conduct prevention trials a priori and/or to interpret study results after the fact.
Volunteers who did not get malaria during the course of the cohort study had essentially no change in titer. Mean PfMSP1 titers hovered just above the upper limit of seropositivity at both time points suggesting this is the natural ‘background’ titer of adults in rural Cambodia, although this would need to be validated during more than one transmission season. Ideally, sero-epidemiological studies conducted in areas with seasonal transmission would have humoral responses measured just before the onset of the transmission season. Since this study was nested in an active drug efficacy study, conducted several months after the rains had begun, baseline titer levels and rates of seropositivity may be overestimated. It is unclear if the high rate of sero-positivity signifies recent or remote infections, and it gives little indication of the number and species of prior infections, particularly for those with low titers. Regardless, the high rates did not appear to confer immunity.
Perhaps more importantly, there was little apparent ‘booster effect’ of baseline titers in response to infection, except for PfMSP1
42 for the four volunteers with three malaria infections during the study period. The PfMSP1 19-kDa haplotype for 10 of 11 infections for these four patients was EKNG; thus the higher titers do not represent a broadening of the immune responses by infection with different circulating haplotypes. With
P. vivax infections, titers appeared either to wane or remain remarkably stable over time. As reported elsewhere, it is likely that the majority of
P. vivax infections represented relapses [
27], and this would suggest that relapsing infection does not stimulate a significant rise in humoral response. A recent publication by Chuquiyauri et al. [
28] analysed antibody responses to
P. vivax antigens by microarray in Peruvian patients with
P. vivax mono-infection and found that both height and breadth of responses were not different in those who were determined to have relapse versus
P. vivax re-infection. Moreover, boosting may not be seen due to longer half-lives of MSP1 titers in low to medium transmission areas. Wipasa et al. [
25] used a mixed-effects regression model analysis to estimate the half-life of MSP1 in northern Thai volunteers to be 7.6 years. These evidences, combined with the data obtained here in northern Cambodia, strongly suggest MSP1 is unlikely to be a useful quantitative serologic marker for malaria exposure during a chemoprophylaxis study.
Alternative biomarkers to other antigens, such as antibodies to the circumsporozoite protein (CSP), which is located on the surface of the sporozoite, may be less affected by chemoprophylaxis. In an intervention-treatment-vaccination (ITV) study in healthy Dutch volunteers, those taking chloroquine prophylaxis during three successive mosquito-borne
P. falciparum infections were protected from subsequent
P. falciparum challenge and seroconverted to CSP but not AMA1 or GLURP [
29]. A study of Dutch soldiers under mefloquine prophylaxis while deployed to Zaire in 1994 showed 100 % efficacy while 11 % of soldiers had an increase in circumsporozoite antibodies, suggesting malaria exposure [
30]. Less is known about seroconversion with
P. vivax malaria, although one study of deployed Thai soldiers found almost three-quarters contracted
vivax malaria despite 62 % being PvCSP seropositive at deployment; those who were seronegative did seroconvert with acute illness though the rise in titers was modest and fell quickly [
31]. Thirty-eight percent of the Thai soldiers with documented, treated
P. vivax malaria did not mount any CSP antibody response. There were not successive increases in CSP titers with relapses, a finding also demonstrated in Cambodian adults in this study, suggesting that a biomarker for
P. vivax infection may be even more elusive. Whether pre-erythrocytic or erythrocytic, a single antigen may not suffice as a biomarker; thus, adding immunogenic antigens, such as MSP2 and schizont extract (SE) [
32], or assessing the breadth of responses [
33] plus newer bead arrays or microarrays [
34,
35] may prove to be more useful, as biomarkers but these alternate approaches will need to be validated in chemoprophylaxis field studies.
In design of chemoprophylaxis studies in endemic areas, it is possible that innate immunity in semi-immune subjects may attenuate infection, thereby inflating the apparent protective efficacy of prophylactic drugs and overestimate their potential benefit to malaria-naïve individuals. The previous evidence for the protective role of
P. falciparum and PvMSP1 in endemic areas has been mixed [
36‐
39]. In this study, those with higher MPS1 titers to either species at enrollment were more likely to develop malaria, and the proportion of volunteers remaining malaria-free was almost evenly split between those who were seropositive and seronegative, suggesting these higher titers were more reflective of exposure than semi-immunity. No 3D7 haplotypes to the C-terminus of MSP1 were detected, the region of MSP1 to which functional antibodies are thought to act, yet titers to this allelic haplotype were highly induced, perhaps merely reflecting cross-reactive immune response between three haplotypes 3D7, CAMP, FVO [
20,
36]. Cross-reactivity may not translate to cross-protection, although this may depend on haplotype, as it has been shown that vaccination with the FVO allele of PfMSP1
42 induces a better homologous and heterologous antibody response than the 3D7 allele of MSP1
42 [
40]. In addition, a traditional ELISA may not adequately capture function, as some antibodies have “blocking” activities and thus interfere with protective responses [
41].
Authors’ contributions
CL, DW and DS designed the study, with field site study coordination and execution by CL, SC, YS, SS, SP2 and CMC. Laboratory assays were performed by SP, PG, KY, UT, AL, CC. MS, SP, PG, CC, SC2, SD, EA, DS analysed and interpreted the data. All authors read and approved the final manuscript.