Evaluation of naturally acquired immune responses against novel pre-erythrocytic P. vivax proteins in a low endemic malaria population located in the Peruvian Amazon Basin

doi:10.21203/rs.3.rs-3750232/v1

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Research Article

Evaluation of naturally acquired immune responses against novel pre-erythrocytic P. vivax proteins in a low endemic malaria population located in the Peruvian Amazon Basin

https://doi.org/10.21203/rs.3.rs-3750232/v1

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Background

Plasmodium vivax (Pv) represents the most geographically widespread human malaria parasite affecting civilian and military populations in endemic areas. Targeting the pre-erythrocytic (PE) stage of the parasite life cycle is especially appealing for developing Pv vaccines as it would prevent disease and transmission. Here, we explore naturally acquired immunity to a panel of Pv PE antigens which may facilitate vaccine development and lead to a better understanding of naturally acquired PE immunity.

Methods

Twelve Pv PE antigens orthologous to a panel of P. falciparum antigens previously identified as highly immunogenic in protected subjects after immunization with radiation attenuated sporozoites (RAS) were used for evaluation of humoral and cellular immunity by ELISA and ELISpot. We used samples from Pv infected individuals (n = 76) from a low endemic malaria region in the Peruvian Amazon Basin.

Results

In our clinical samples, all PE antigens evaluated showed positive IgG antibody reactivity with a variable prevalence of 58–99% in recently P. vivax diagnosed patients. The magnitude of the IgG antibody response against PE antigens was lower compared with blood stage antigens MSP1 and DBP-II, although titers persisted better for PE antigens (average decrease of 6% for PE antigens and 43% for MSP1, p < 0.05). A significant correlation between IgG antibodies and the number of previous malaria episodes was observed only for blood stage antigens (p < 0.001). High IgG responders across PE and blood stage antigens showed significantly lower parasitemia compared to low IgG responders (median 1,921 vs 4,663 par/µl, p < 0.05). We observed a positive T cell response in 35% vs 9–35% of total volunteers against blood stage MSP1 and PE antigens, respectively, but no correlation with IgG responses.

Conclusions

Our results demonstrate clear humoral and T cell responses against Pv PE antigens in individuals naturally infected with P. vivax. These data identify novel attractive PE antigens suitable for use in the potential development and selection of new malaria vaccine candidates which can be used as a part of malaria prevention strategies in civilian and military populations living in Pv endemic areas.

Plasmodium vivax

pre-erythrocytic stage

natural acquired immunity

Peruvian amazon Basin

Plasmodium spp. are the causative agent of malaria, one of the world’s deadliest infectious diseases. In 2022, about 247 million cases of malaria were reported by WHO with approximately 619,000 deaths, particularly in young children and pregnant women in sub-Saharan Africa (1). While Plasmodium falciparum (Pf) is the most prevalent malaria parasite on the African continent and responsible for most observed infections and deaths, P. vivax (Pv) represents the most widely distributed human malaria parasite worldwide. Outside of Africa, P. vivax is the dominant cause of malaria with over 3 billion people living within its transmission limits (1). Globally, several million clinical cases of P. vivax malaria are detected each year with a relevant portion in South East Asia and Latin America, where Pv is responsible for 50% and 70–85% of all malaria cases, respectively (1). Traditionally, Pv has been considered to cause a “benign” form of malaria but is now recognized as a significant cause of morbidity and mortality due to increasing evidence of severe cases with a possible fatal outcome (2).

Malaria elimination efforts have resulted in a substantial decline in the global malaria burden in the past two decades. It is estimated that these efforts have resulted in a reduction of global malaria infections by 29% and mortality caused by malaria by 60% between 2000 and 2018 (1). However, from 2014 to 2018 this downward trend has slowed considerably and even reversed in some regions (e.g., Venezuela) (1, 3). Pv represents a special challenge to control efforts due to its unique biological features, including low-density blood-stage infection, asymptomatic infections and the formation of hypnozoites—the dormant forms residing in hepatocytes that can cause relapses months or years after the initial infection. Hypnozoites are believed to be responsible for up to 80% of infections and there is currently no diagnostic tool available for their detection (4). Importantly, activation of hypnozoites can represents a big challenge for malaria elimination, especially in naïve civilian and military populations which travel to these Pv endemics zones. Previous studies showed relapse episodes in U.S. military population deployed to Afghanistan, where 38 Army Rangers reported activation of hypnozoites within an average of 233 days after return from P. vivax endemic areas, highlighting the risk to military personnel during deployments (5). Further progress towards malaria elimination will require additional tools including the development of an effective vaccine or intervention which can prevent or eliminate these dormant stages.

For many years, the efforts to develop a PE malaria vaccine have mainly been focused on Pf while only two Pv vaccine candidates have reached clinical trials for Pv, targeting blood stage proteins DBPII and MSP-1 (6). The most advanced and recently approved vaccine by WHO is the Pf subunit vaccine RTS,S-AS01, which targets the Circumsporozoite Protein (CSP) via neutralizing antibodies and has shown consistent but short-lived efficacy of 25–36% in children in phase III and IV clinical trials (7, 8). In addition to CSP, other proteins have been under investigation as vaccine targets such as the PE antigen thrombospondin-related adhesive protein (TRAP) and the highly promising blood stage protein Reticulocyte-binding Protein Homolog 5 (Rh5) (9). Both candidates were chosen at least in part based on data showing protection in naturally infected persons, and Rh5 is progressing into phase I and II clinical trials after promising preclinical results in non-human primates (10, 11).

Only a few such studies of naturally acquired immunity to Pv have been conducted, largely with a focus on antibodies to erythrocytic antigens. In these studies, reticulocyte-binding proteins critical for merozoite invasion have been associated with protective immunity (e.g. PvDBP-II and PvRBP2b) (12–15). Large-scale screenings have identified antibodies to other blood-stage antigens associated with protection particularly when seen in combinations rather than with any single antigen alone (16, 17). However, even fewer studies have explored the natural immunogenicity of Pv PE antigens (18) despite the fact that the PE stages of Plasmodium can be targeted with both antibodies and T cells (19, 20). For Pf, a partially protective PE intervention that only results in a reduction of liver stage parasites will have no perceptible benefit to the person as even a small number of breakthrough parasites can yield fulminant blood stage disease. However, for Pv, preventing a portion liver stages would still not yield sterilizing protection against primary infection but would have proportional effects on elimination since the number of hypnozoites would be reduced, resulting in the reduced number of relapses that are the main driver of disease (4, 21)

Taken together, this suggests that antibodies and T cells targeting multiple PE Pv proteins could be an integral part of developing the first effective Pv vaccine. The almost exclusive focus on the blood stage has resulted in a general lack of Pv PE candidate antigens and an even more opaque understanding of their role in natural immunity or even simply immunogenicity. This paucity of data around PE immunity in Pv limits our ability to take the first steps towards novel vaccine candidates. Here, we explore naturally acquired immunity to a panel of Pv PE antigens as a key step to facilitate vaccine development and to better understand naturally-acquired PE Pv immunity.

Human plasma and lymphocyte samples

Human samples were obtained between 2012 and 2017 from three passive surveillance protocols approved by the Institutional Review Board of the U.S. Naval Medical Research Unit SOUTH (NAMRU SOUTH) in compliance with all applicable federal regulations governing the protection of human subjects (NMRCD.2007.0004, NMRCD.2010.0002 and NAMRU6.2012.0006).

Protocols NMRCD.2007.0004 and NAMRU6.2012.0006 were conducted in Iquitos, the largest city in the department of Loreto located in Peruvian Amazon Basin. Samples were collected from the Hospital Regional de Loreto (HRL, n = 41) and Hospital de Apoyo de Iquitos (HAI, n = 35). HRL is a specialized health center for complex diseases whereas HAI attends patients with less complex symptomatology, giving basic treatments and rapid diagnostics for transmissible and non-transmissible diseases. Subjects presenting malaria symptoms at both health centers were invited to participate in the study and blood samples were collected after providing their consent. All subjects enrolled (n = 76) were diagnosed by microscopy and confirmed by PCR (23) for Pv mono-infection. Loreto is described as hypoendemic malaria area with co-endemicity of Pv and Pf. Pv represents 80–90% of total malaria cases with an annual average incidence of 42,164 ± 7,779 cases during 2012–2017. In addition, Pv transmission is sustained and heterogeneous along the year (22), resulting in underestimated malaria prevalence due to low-parasitemia and/or asymptomatic cases.

In addition, we included a set of twenty human Pv negative control blood samples obtained from individuals living in the department of Piura, located in the North Coast of Peru (NMRCD.2010.0002), which is a very low incidence region for Pv (0.01 per 1000 inhabitants) during 2014 (Fig. 1).

Selection of Pv PE antigens for immunological assessments

The 12 Pv PE antigens are orthologs to a panel of Pf antigens previously identified as PE vaccine targets (24). These Pf antigens were selected from a larger set of 131 Pf recombinant proteins based on their reactivity against sera samples from sterilely protected subjects who underwent immunization with radiation attenuated sporozoites (RAS) followed by challenge with the bites of viable Pf-infected Anopheles mosquitoes (25, 26). This approach has been partially validated for three P. yoelii orthologs from this list of candidates: a putative cysteine protease inhibitor (Falstatin), the gamete egress and sporozoite traversal (GEST) and the early transcribed membrane protein (ETRAMP) which induced up to 60% protective efficacy when used in combination using the DNA and vaccinia virus prime-boost immunization approach (27). In addition, our subset of Pv proteins have been verified as expressed in the Pv proteome (28).

These proteins were successfully expressed by the cell-free wheat germ expression system (28) using the bi-layer method (small-scale) with a yield between ~ 300–1200µg of protein per reaction. In addition, we used the antigens MSP-1 (PVX_099980) and DBP-II (Sal-I) that are expressed at blood stage to compare immunogenicity levels with PE antigens.

ELISA - human samples

Briefly, thirteen Pv PE antigens and two blood stage antigens were used to determine antibody prevalence and relative immunogenicity. ELISA plates were coated with recombinant proteins at concentration of 2–4 µg/ml to high-binding 96-well microplates (Nunc Maxisorp) overnight at room temperature and then washed with T-PBS1X (PBS1X/0.05%Tween20) five times. Microplates were blocked for 1 hour with TBS1X-SM (TBS1X/Skim milk 5% buffer). After five washes with T-PBS1X, human plasma samples were added in duplicate at a 1:200 dilution in TBS1X-SM and incubated for 2 hours at room temperature. After five washes with T-PBS1X, antibodies were detected using peroxidase-conjugated anti-human IgG monoclonal antibodies (Jackson Immunoresearch Cat#: 309-035-033, 1:6,000 dilution) and incubated for one hour at room temperature. After five washes with T-PBS1X, we used o-phenylenediamine (Sigma Aldrich Cat#: P3804) with hydrogen peroxide as a substrate and the reaction was stopped after 1 hour with 50 µl of 3N HCl. Plates were read at 492 nm to determine optical density OD.

A two-fold plasma serial dilution of a positive pool of 30 individuals with more than 10 Pv confirmed events were used as a standard curve and included on each plate run. In addition, we used 20 plasma samples from individuals from malaria area with low incidence to calculate a positivity cut-off value for each protein using the average OD value plus 3 standard deviations.

ELISpot

A library of overlapping 15-mer synthetic peptides was obtained from Mimotopes Pty Ltd. The peptides overlapped by 9 aminoacids spanning the entire blood and PE proteins. Pv MSP-1 was used as a positive control of blood stage antigens, and seven pre-erythrocytic antigens were tested (CSP, CelTOS, Falstatin, ETRAMP, PVX_119755, GEST and HSP PVX_089585). These were resuspended according to the manufacturer’s instructions and each peptide pool used at 10 µg/ml. These peptides were used to determine the frequency of T-cell response producing IFN-γ to Pv PE proteins using Human IFN-γ ELISpot PRO (Cat.# 3420-2APW-10. Mabtech AB, Sweden) according to the manufacturer’s instructions.

Peripheral blood mononuclear cells (PBMCs) from P. vivax patients, who accepted large blood collection for immunological assays, were isolated from whole blood by density centrifugation using a Percoll gradient, counted and cryopreserved for storage in liquid nitrogen. Briefly, PBMCs were thawed in RPMI media with 10% FBS (SIGMA, Cat #: F4135), incubated overnight at 37°C and then plated at 0.2 x 10^6 cells/well in duplicates. Peptides were added at 10 µg/ml to stimulate T cell response for 18 hours with phorbol 12-myristate 13-acetate/ionomycin (PMA/ION) used as a positive stimulation control. Spots were counted using the CTL IMUNOSPOT Analyzer. Subjects were defined as positive when the number of spots was higher than the negative control by 20%.

Statistical analysis

Variation of sample size per each analysis depended on the availability of samples, proteins quantity and data of epidemiological variables. Analysis was performed using STATA v16.0 statistical software (Stata Corp., College Station, TX, USA) and GraphPad Prism v9 (GraphPad Software, LLC). Differences among the frequency of epidemiological and immunological variables were analyzed using Chi-square test for categorical variables and Mann-Whitney or Kruskal-Wallis test for numerical continuous variables to compare the median values. Spearman rank correlation coefficient test was used to evaluate the correlation between epidemiological and immunological variables. To measure differences of antibodies level at different time points between groups (related samples) per each antigen we used paired Wilcoxon signed-rank test, the significance level for all statistical analysis performed was set at p < 0.05 or p < 0.001.

Epidemiological variables

Epidemiological variables by site of enrollment showed significant differences only for weight and number of previous Pv episodes (Table 1). Both variables were higher for patients enrolled at Hospital Regional de Loreto (HRL). Therefore, we chose to analyze samples from each location as a single group.

Table 1 Demographic information of Pv infected patients by site of sample collection. Differences of epidemiological variables between groups of sample collection were assessed using Mann-Whitney U test (°) and Chi-square test (") with significant values for. * p < 0.05, and ** p < 0.001. Hospital regional Loreto (HRL), Hospital Apoyo Iquitos (HAI)

Human plasma samples from Pv infected population and humoral response

In order to determine if antibodies recognizing Pv PE antigens are induced during naturally occurring Pv infections, we tested plasma samples from 76 Pv positive subjects enrolled at health centers of HRL and HAI during 2012–2017 by ELISA.

This group of 76 Pv subjects showed positive antibody reactivity with high prevalence for blood stage antigens MSP1 (92%), DBP-II (86%) and the PE stage antigen CSP (99%) (Fig. 2). Twelve other Pv PE antigens showed positive antibody reactivity with variable prevalence in the range of 58–99%. Prevalence for each antigen was: TRAP PVX_082735 (99%), HSP PVX_089585 (93%), GAP40 PVX_08046 (93%), CELTOS PVX_123510 (91%), GEST PVX_121950 (89%), FALSTATIN PVX_09903 (88%), Hypothetical protein PVX_119755 (82%), Hypothetical protein PVX_094725 (75%), Hypothetical protein PVX_111090 (75%), ETRAMP (UIS3 ortholog) PVX_121950 (72%), SPECT1 PVX_083025 (62%), and Hypothetical protein PVX_093660 (58%) (Fig. 1).

The magnitude of the antibody response as measured by OD values was higher for the blood stage MSP1 antigen with an OD average of 1.8 compared to the canonical PE antigen CSP with OD average of 0.8. The other 12 Pv PE antigens showed variable intensity with a mean OD range of 0.3–0.7. (Fig. 1). Overall, the antibody magnitude did not correlate with parasitemia levels at the time of sampling except for a modest negative correlation in four PE antigens (Spearman RHO: -0.23 to -0.28, p < 0.05) (Table 2). This indicates that variations in OD intensity for each antigen are likely a result of the intrinsic immunogenicity of each antigen and inter-individual differences in responses rather than differences due to the parasitemia at time of enrollment.

Table 2 Correlation of parasitemia levels with antibody response against blood and pre-erythrocytic and blood stage Pv antigens in Pv infected patients. Plasma samples from Pv patients (n = 76) were tested against 15 P. vivax antigens to determine correlation between antibodies levels vs parasitemia. Table shows Spearman Rho coefficient and p-values per each antigen.

To determine if antibody magnitude for each individual antigen correlated with previous malaria exposure, we used a subgroup of 59 patients from the total of 76 for which we had data of self-reported previous malaria episodes. Individuals were stratified by no previous (n = 26), one previous (n = 16) and two or more previous (n = 17) Pv episodes. There was a significant increase of IgG antibodies against only the blood stage antigens MSP1 and DBP in groups with one or more previous episodes as compared to the group with no previous P. vivax infection (Fig. 2). IgG antibodies against all Pv PE antigens showed similar IgG levels independent of the number of self-reported previous malaria episodes (Fig. 2). Together, these data reveal a broad and variable seropositivity to multiple Pv PE antigens during acute Pv infection that, unlike blood stage antigens, appear not to be boosted by multiple blood stage infections.

Antibody breadth also showed substantial variability between volunteers, especially amongst pre-erythrocytic antibodies (Fig. 3). Some volunteers responded broadly to nearly every antigen while others appeared to have weak antibody responses in terms of both breadth and magnitude. Indeed, a common predictor of antibody response to one PE antigen was a response to another (Fig. 4) suggesting that some volunteers naturally respond to infection with a greater antibody response. Those “high responders”, defined simply as those in the top 50% of total IgG magnitude across all antigens were evaluated against epidemiological variables. We did not observe any significant differences between high and low responders by place of sample collection, sex, age, weight, temperature, and number of previous Pv episodes. However, high responders showed a significantly lower parasitemia compared to low responders (median par/µl 1921 vs 4663, p < 0.05) at time of enrollment (Table 3). Interestingly, high and low antibody responders did not differ in parasitemia levels after stratification by number of previous episodes (Fig. 5A and B).

Table 3. Demographic information of Pv infected patients by low and high antibody responders. Epidemiological variables were compared between low and high responder group. Categorical variables were tested by Chi2 and numerical variables by Mann-Whitney U test, significance was reported by p < 0.05, and p < 0.001. Hospital regional Loreto (HRL), Hospital Apoyo Iquitos (HAI)

Long-term analysis of a sub-group of 24 Pv patients for which we had samples six months after enrollment showed a significant decrease of OD values only for MSP1 (OD average decrease of -43%, Day 0: 1.71 vs Day 180: 0.97, p < 0.05) and CSP (OD average decrease of -28%, Day 0: 0.83 vs Day 180: 0.60, p < 0.001). Nine PE antigens showed more stable IgG levels with an average decrease of between antigens of 6% (range: -2 to -21 and SD ± 6%) (Fig. 6). These subjects did not experience a new malaria episode during six months of follow-up by monthly microscopy confirmation, indicating that some PE antibodies are maintained for a significant period after initial infection in the absence of boosting.

Human T cell responses

We next evaluated the T cell responses to eight antigens in a small set of individuals (n = 17) for which we had PMBCs. An ex vivo IFN-gamma response was detected in at least one subject for all the antigens tested. Positivity for IFN-gamma reactivity was the highest for the blood stage protein MSP1 and the pre-erythrocytic stage protein CSP, both at 35.3% (6/17 volunteers positive). From the remaining antigens tested, the highest percentage of positive responses was for ETRAMP at 33.3% (5/15), followed by Falstatin (25.0%, 4/16), CelTOS (23.5%, 4/17), the Hypothetical protein PVX_119755 (23.1%, 3/13) and HSP (18.2%, 2/11).

Across individuals, we observed broad and variable T cell responses to PE antigens (Fig. 7) with two subjects positive to CSP only, and six subjects positive to 2–4 PE antigens (in addition to or besides CSP). Variability between subjects in the number of SFU (spot forming units) per million cells was also seen, with two subjects highly reactive to multiple antigens. Additionally, two subjects were positive to only MSP-1 and no T cell response was found for seven subjects (41%).

We were able to assess the antibody response in seven of the subjects for which we had both plasma and PBMCs. In this limited set, there was no apparent correlation between positive IgG response and ex vivo IFN-gamma response (Table 4). Together, these data indicate that T cell responses to both blood stage and PE antigens are also present but variable during acute Pv infection.

Table 4 Comparative analysis of antibody and T cell responders against blood stage and PE antigens. Prevalence of ELISA and ELISPOT was measured by IgG and Interferon gamma positivity in Pv infected individuals, (n = 7).

Only a few Pv PE proteins such as CSP, TRAP and CelTOS have been thoroughly evaluated as vaccine candidates and still remain at the pre-clinical or early clinical stages. New Pv PE candidates and, in particular, discovery of new antigens will be necessary for a better understanding of Pv PE immunity and to provide insight into their role in the prevention of both liver and blood stage infection. In the absence of well-established animal models of P. vivax infection, studying the acquisition of PE antibodies during natural infection is a logical starting point with the long-term goal of studying the role these PE antibodies play in functional immunity in order to justify integrating functional targets into novel vaccine candidates. In addition, as more candidates move into clinical trials in Pv-endemic areas, the level and impact of pre-existing immunity will be important to measure. However, to date, few studies of this kind been done for Pv PE antibodies.

A Pv cohort study (n = 31) in a low-transmission Pv area in western Thailand during 2013 showed prevalences of 76–86%, 18–21% and 0% for Pv CSP, CELTOS and TRAP antigens, respectively (30). In addition, a cross-sectional study (n = 299) of naturally acquired immunity performed in Brazil during 2016 against whole protein and linear synthetic peptides of PE TRAP showed prevalences of 49% and 25–32%, respectively (31). Our results showed naturally acquired immunity against all 13 PE antigens tested in n = 76 P. vivax patients from a low endemic malaria region located in the Peruvian Amazon Basin was generally higher, with some individuals responding to all PE antigens (range of 58–99%). This variation could be related to the inherent characteristics of different circulating Pv parasites, differences in transmission dynamics or simply differences in protein production platforms as these previous studies used HEK-293 cell lines for production compared to our wheat germ Cell-free system.

Compared to the magnitude of Pv blood stage responses, the IgG response to PE antigens was lower. This is likely due to the fact we did not see a boosting effect of anti-PE IgG with repeated infection. Rather, we found IgG responses to one antigen often correlated simply with a response to another antigen within individuals—leading to high and low antibody responders. Comparative analysis of high and low antibody responders against Pv PE and blood stage antigens vs. parasitemia showed that high responders had lower parasitemia levels. However, given the high correlation between antibody titers between both PE and blood stage antigens, we could not assign correlations that may indicate if antibodies to any stage or target might be leading to protection against higher parasitemia. These data suggest that high antibody titers may be a host-intrinsic factor, and this breadth of response—rather than a response to any single antigen—could be involved in the lower parasitemia we observed. This aligns with published data demonstrating protection from Pv is associated with the acquisition of a multi-antigen antibody response at the blood stage (17). However, we observed that the low parasitemia in high responders was seen irrespective of the number of previous Pv episodes, although these were not subsequent infections in the same individual. Thus, whether the lower parasitemia is the result of a recently-acquired diverse antibody response better controlling subsequent infections or that the antibodies are a correlate of some other immune phenomenon that results in lower parasitemia (33) will require investigation in well-designed longitudinal cohort studies with deeper immune phenotyping and active detection of new or relapse infections.

Previous studies have shown a correlation between antibody response and P. vivax parasitemia levels in cross-sectional and cohort studies. This includes a large (n = 466) survey of the antibody response against blood stage protein vaccine target DBP-II which showed a negative correlation between Pv parasitemia and antibodies in an area with hypo-endemic Pv transmission in Brazil (34). As such, PvDBP-II is the leading blood stage candidate for Pv blood stages. However, little is known about relationship between antibody response against PE antigens and parasitemia in cross-sectional or cohort studies. Here, in a cross-sectional study in hypoendemic malaria area we identified modest negative correlation between parasitemia and antibodies levels against 4 PE antigens (Spearman RHO: -0.23 to -0.28, p < 0.05), suggesting these antigens should be included in further studies as functional antibody targets.

We also observed that IgG antibodies six months after Pv infection had variable stability, with a significant decrease in the most naturally immunogenic antibodies MSP-1(-43%) and CSP (-28%) antigens. However, nine other PE antigens tested showed relative stability (− 6% STD ± 6) as did the blood stage DBP antigen. This characteristic could be useful in defining serological markers of recent vs. historical exposure to use in malaria endemic areas near to effective malaria control or on way for malaria elimination process.

Even less well-studied than PE Pv antibodies are T cell responses to natural Pv infection. A few studies have assessed vaccine candidates eliciting CD8 + and/or CD4 + T cells that correlate with protection from liver-stage infection in mice (35, 36) and one clinical trial (37), but the mechanism that leads to T cell protection remains to be elucidated. Here, in a limited sample of volunteers, we observed a positive T cell response in 35% vs 9–35% of total individuals against blood stage antigen MSP1 and PE antigens, respectively. From patients with a positive T cell response, we observed that 40% showed reactivity for both blood stage and PE antigens which did not appear to correlate with IgG responses. While the role of T cell-mediated protection against rodent malaria liver stages is robust (38, 39), the absence of appropriate animal models for Pv and, in particular, hypnozoites leaves this possibility unconfirmed. Interestingly, recent evidence demonstrated that CD8 + T cells can eliminate infected reticulocytes (40) opening the possibility of multi-stage CD8 + T cell vaccines to prevent or limit Pv infection. Our limited data demonstrate that natural PE T cell immunity does exist and will need to be included in future studies with a larger population investigating the relationship between T cells and protection from infection at multiple stages.

In conclusion, we present the most antigenically diverse survey of PE immunity acquired during natural infection using samples from people living in an endemic area of the Peruvian Amazon Basin. Our results demonstrate broad but variable reactivity across Pv PE antigens and between individuals. Together, these data clearly demonstrate a previously underappreciated prevalence of natural immunity to PE antigens which warrants specifically designed longitudinal cohorts where better correlations to transmission and protection from infection can be assessed. Such studies can add to a growing understanding of immunity to Pv infection in terms of both antigens and immune effector mechanisms with the goal of designing vaccines capable of preventing or reducing infection in civilian and deployed military populations.

Plasmodium vivax

pre-erythrocytic

ELISA

enzyme-linked immunosorbent assay

P falciparum

MSP1

merozoite surface protein-1

DBP

Duffy Binding Protein

CSP

Circumsporozoite protein

WHO

World Health Organization

TRAP

thrombospondin-related adhesive protein

RH5

reticulocyte-binding protein Homolog 5

RAS

radiation attenuated sporozoites

GEST

egress and sporozoite traversal

ETRAMP

early transcribed membrane protein

STD

standard deviation

PCR

Polymerase Chain Reaction.

Acknowledgments

The authors are grateful to Dr. Hugo Valdivia, Dr. Danielle Pannebaker, Dr. Danett Bishop, Dr. Christie Joya, and Dr. Lisa Sperling for their support and critical review during the development of this work at NAMRU SOUTH.

Disclaimers

“The views expressed in this article reflect the results of research conducted by the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the United States Government”

Authors’ contributions

GCB, JCA and BKW conceived, designed, and supervised the project studies. JVA, LLT, RP, AF and ML performed experiments of protein production and/or evaluation of immunogenicity by ELISA or ELISPOT. JVA, LLT and BKW performed the data and statistical analysis. JVA, LLT and BKW wrote the first draft of the manuscript. JVA, LLT and BKW wrote the final version of the paper. All authors reviewed, edited and approved the manuscript.

Funding

This work was supported by the Military Infectious Disease Research Program Grants # F0398_14_LI, F0521_17 and F0578_19 awarded to Dr. G. Christian Baldeviano.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate

The human study protocols NMRCD.2007.0004, NMRCD.2010.0002 and NAMRU6.2012.0006 were approved by the U.S. Naval Medical Research Unit SOUTH Institutional Review Board in compliance with all applicable federal regulations governing the protection of human subjects

Consent for publication

Not applicable. No personal identifiable data was used in the study. All authors have provided consent to publish the findings in this study.

Competing interests

The authors declare that they have no competing interests.

Copyright Statement

“Several of the authors are members of federal/contracted employee of the United States government. This work was prepared as part of my official duties. Title 17 U.S.C. 105 provides that copyright protection under this title is not available for any work of the United States Government as part of that person’s official duties”

Author details

¹Vysnova Partners Inc.²U.S. Naval Medical Research Unit SOUTH, Lima-Peru (NAMRU SOUTH), ³Instituto Nacional de Salud (INS) Lima-Peru,⁴Jhons Hopkins University, ⁵CAMRIS International, LLC, ⁶Oregon Health & Science University, ⁷Universidad Peruana Cayetano Heredia Lima-Peru

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Evaluation of naturally acquired immune responses against novel pre-erythrocytic P. vivax proteins in a low endemic malaria population located in the Peruvian Amazon Basin

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Abstract

Background

Methods

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Background

Methods

Human plasma and lymphocyte samples

ELISA - human samples

ELISpot

Statistical analysis

Results

Epidemiological variables

Human T cell responses

Discussion

Conclusion

Abbreviations

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References

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