Humoral immune response against Anopheles bites as a measure of exposure to Plasmodium falciparum in asymptomatic infections in a malaria endemic area of Colombia

Background The humoral immune response against Anopheles salivary glands proteins in the vertebrate host can reflect the intensity of exposure to Anopheles bites and the risk of Plasmodium infection. In Colombia, the identification of exposure biomarkers is necessary due to the several Anopheles species circulating. The purpose of this study was to evaluate human exposure to Anopheles bite by measuring antibody responses against salivary glands extracts from An. albimanus and An. darlingi and also against the gSG6-P1 peptide of An. gambiae in asymptomatic P. falciparum infections in the Colombian Pacific coast. Methods We eluted dried blood spots samples to measure the IgG antibodies against salivary gland extracts of An. albimanus and An. darlingi and the gSG6-P1 peptide by ELISA in uninfected people and microscopic and submicroscopic Plasmodium carriers from the Colombia Pacific Coast. A multiple linear mixed regression model, Spearman correlation, and Mann-Whitney U-test were used to analyze IgG data. Results Significant differences in specific IgG levels were detected between infected and uninfected groups for salivary glands extracts from An. albimanus and for gSG6-P1, also IgG response to CTG and gSG6-P1 peptide were positively associated with the IgG response to P. falciparum in the mixed model. Conclusion The CTG and STE An. albimanus salivary glands extracts are a potential source of new Anopheles salivary biomarkers to identify exposure to the main malaria vector in the Colombian Pacific coast. Also, the gSG6-P1 peptide has the potential to quantify human exposure to the subgenus Anopheles vectors in the same area. on the design and validation of specific peptides designed from immunogenic proteins of An. albimanus assess Central America. Our current study shows a high IgG response againstgSG6-P1 peptide in samples from infected compared to uninfected people. These findings agree with our previous study in Colombian and Chilean volunteers, where we found that the concentration of gSG6-P1 antibodies was significantly correlated with malaria infection status and that people with clinical malaria presented significantly higher levels of IgG anti-gSG6-P1 antibodies than healthy controls (11). Although, Anopheles species from the subgenus Nyssorhynchus are the main vectors of malaria in Colombia, at least six species from the sub-genus Anopheles have been described as potential malaria vectors in the region 24). Two of these species ( An. pseudopunctipennis and An. punctimacula) are present along the Pacific coast the main area where P. falciparum in Although Arcà et al. reported that gSG6 had no degree of identity with orthologous proteins from vectors in Central and South America, and therefore serological data published about the usefulness of this peptide in Colombia previously should be interpreted with caution previous work also showed that a deduced gSG6 from the New World species An. freeborni and An. quadrimaculatus (which belongs to the subgenus Anopheles) had between 67 and 71% of degree of identity with the gSG6 from Old World Anopheles species . In the same way, Pollard suggested that the antibodies to the gSG6-P1 peptide in the Colombian population may represent exposure to An. punctimacula, which is a member of the Anopheles


Introduction
Malaria is caused by the protozoan parasite Plasmodium and is transmitted by female Anopheles mosquitoes. Although significant advances have been made towards its elimination in several previously endemic countries, malaria remains a significant public health concern (1). The World Malaria Report in 2018 estimated that the global burden of malaria comprised around 219 million reported cases and 435,000 deaths worldwide (2). Specifically, in Colombia, there was a decrease in the estimated number of malaria cases by more than 20% between 2016 and 2017 (2).
Despite this, malaria remains one of the foremost public health concerns in some states in Colombia such as Nariño, which is located along the Pacific coast of the country. In 2017, 26% of malaria cases in Colombia came from Nariño where, unlike other regions, P. falciparum is the most common species (96.3%) (3).
More than 47 Anopheles species in five subgenera have been reported in Colombia (4). The majority of malaria vectors in Colombia belong to the subgenus Nyssorhynchus, with An. nuneztovari and An. darlingi as the most important malaria vectors in areas of high malaria transmission such as Urabá, Bajo Cauca and Alto Sinú regions (5). On the South Pacific coast, where comparatively little is known about the current spatial distribution of malaria vector species (5), it has been reported that An. albimanus is the main vector (6,7). However, the subgenus Anopheles, is the second most important group of mosquito vectors in the region, with An. pseudopunctipennis and An. punctimacula as the most representative (8).
Malaria is acquired when Plasmodium spp. sporozoites are injected into human skin through the bite of a female Anopheles mosquito along with the mosquito salivary proteins (9). Previous studies have shown that a significant number of mosquito salivary proteins are immunogenic and able to induce antibody responses, mainly IgG isotype. These antibodies can reflect the intensity of human exposure to mosquito bites and represent good indicators of the risk of infection with Plasmodium spp. (10)(11)(12)(13)(14). Significant higher IgG antibody levels against An.
albimanus and An. darlingi salivary proteins have been observed in people with active malaria infection in Central and South America when compared to uninfected people living in the same region (10,15). A similar pattern has been observed in areas where An. gambiae and An. stephensi are among the most important vectors. A significant number of these studies were performed evaluating IgG responses against the An. gambiae salivary protein gSG6, a highly conserved protein among Anopheles species from the Subgenus Cellia and Anopheles (16). The peptide, gSG6-P1, was designed from the original An. gambiae gSG6 sequence. IgG responses specific to this salivary peptide has been validated as a biomarker of human exposure not only in Africa but also in Asia and South America (11,14,17).
Although there are no known species of the subgenus Cellia in South America, the responses observed against the gSG6-P1 peptide can be attributed to the presence of An. pseudopunctipennis and An. punctimacula members of the subgenus Anopheles as described above (18).
Consequently, it is necessary to characterize a broader panel of biomarkers able to identify the risk of disease more closely in areas with a great diversity of Anopheles mosquitoes. Our research group plans to identify exposure markers that include not only the primary malaria vectors but also markers for the majority of the circulating species playing an important role in malaria transmission in Latin America, even when these vectors species are in a smaller proportion. Thus, the main objective of this work was to characterize Anopheles bite exposure in an area where low-density P. falciparum infections are frequent and where An. albimanus and An. darlingi (lacking the gSG6 protein) are present. Thus, we explored if human IgG responses to salivary gland extracts (SGE) from these two species are associated with lowdensity infections by P. falciparum. We also aimed to evaluate whether gSG6-P1 peptide continues as a useful marker to detect exposure to minor malaria vectors in Colombia.

Samples selection
The samples used in this study were collected as part of a longitudinal study in which the purpose was to evaluate the dynamic of submicroscopic Plasmodium infections in Colombia. The following two sites are characterized as rural areas, and the last one is classified as a peri-urban zone. In 2017, P. falciparum was reported as the predominant species (96%) in Tumaco with an annual parasite index (parasite incidence per 1,000 population) of 13.5 (19). The main malaria vector in the area is An. albimanus with a HBR of 2.6 in 2006 (20) To compare the vector exposure between infected and uninfected individuals, all positive P. falciparum samples were selected (n = 63) from the 958 people that were enrolled in the main study. All of these infections were afebrile (axillary temperature <37.5°C), and 48 (76.2%) were submicroscopic (detected by LAMP or PCR but not by light microscopy (LM)). Furthermore, 50 uninfected samples were randomly selected by age (5 years) and sex from the total of non-infected individuals by using an Excel random list. Mosquitoes were blood feed at day 3 or 4 after emergence. A pool of 100 salivary gland pairs from each strain was then frozen and thawed three times to prepare the SGE. The concentration of the SGE was determined using a NanoDrop™ (Thermo Scientific, Wilmington, DE, USA) and 50uL aliquots were stored at -80 o C until use.

ELISA antigens and SGE preparations
The An. gambiae gSG6-P1 peptide was synthesized by Genscript (Piscataway, NJ, USA) and the P. falciparum Pf-MSP peptide (Fitzgerald, USA) was used to evaluate exposure to malaria parasites.
Indirect ELISA ELISA conditions were standardized as described elsewhere (10,11). Also, DBS GraphPad Software V5. OD normalization and plate to plate variation was performed as described elsewhere (11). Briefly, antibody levels were expressed as the ΔOD value: ΔOD = ODx − ODb, where ODx represents the mean of individual OD in both antigen wells and ODb the mean of the blank wells. For each tested peptide, positive controls of each plate were averaged and divided by the average of the ODx of the positive control for each plate to obtain a normalization factor for each plate as previously described. Each plate normalization factor was multiplied by plate sample ΔOD to obtain normalized ΔOD that were used in statistical analyses.
We estimated the median of antibody level for each antigen in uninfected people

Study sample characteristics and exposure to mosquito bites
We studied exposure to mosquito bites in the area of Tumaco in Narino (Colombia) (Figure 1).. Table 1 shows the characteristics of participants according to the status of infections. The gender and age groups distribution seem to be equally We also tested whether the difference observed in antibody level between infected and uninfected will be influenced by the village where samples were collected. Independent of location (random intercept at village levels), IgG response to CTG and gSG6-P1 peptide were positively associated with the IgG response to P. falciparum (regression coefficient (RE) = 0.105; 95% CI 0.0223-0.189 and RE = 0.070; 95% CI 0.013-0.126 respectively). In contrast with the IgG Pf-MSP, a negative association for all IgG responses to Anopheles was found with age showing there is a decreasing of IgG immune response with increased age ( Table 2).. A similar situation occurred with the time of residence in an endemic area for malaria; IgG responses to gSG6-P1 peptide was 3.4% lower in samples from people who had lived in a malarial area for more than five years (RE = -0.035; 95% CI -0.070 to -0.003).
Finally, no significant variation of specific anti-Anopheles IgG was observed according to the status of infection ( Table 2)..

Discussion
The intensity of malaria transmission has been traditionally evaluated using the entomological inoculation rate, which is defined by the number of infected bites received per human per unit of time (22); nevertheless, this strategy has shown limitations in low endemic settings for malaria (12,23), such as Colombia. As a result, alternative methods to estimate human exposure to Anopheles bites have been proposed, including the detection of IgG responses to Anopheles SGE and salivary peptides. The purpose of the present study was to explore the possibility of using whole SGE from different Anopheles species to estimate exposure to Anopheles bites in a malaria-endemic area in Colombia where there is an important proportion of asymptomatic infections. We used SGE from two An. albimanus strains to try to capture potential differences in immunogenicity of salivary proteins from mosquitoes maintained for long periods in colony. Specifically, we compared immunogenicity of the CTG strains, a recently colonized strains of An. albimanus isolated in Colombia that could potentially resemble more closely exposure to "wild mosquito populations) against the STECLA strains, isolated in Central America in 1974, that has been previously used in our studies. We also wanted to evaluate the use of the gSG6-P1 peptide to evaluate exposure to minor malaria vectors in the area belonging to the subgenus Anopheles.
Consistent with our previous studies, we observed that the An. albimanus SGEs (STE and CTG) were associated with the infectious status, where people with infection presented significantly higher antibody levels against the salivary proteins. These results agree with previous findings in Haiti were the IgG antibody levels against An.
albimanus SGE salivary proteins were higher in patients with clinical malaria than those in uninfected people living in the same region (10). However, the fact that previous studies suggest that the antibody response against An. albimanus SGE is associated with Plasmodium exposure, highlights the relevance of using whole salivary content in the form of SGE as potentially useful antigen to measure mosquito bite exposure and risk of infection in areas of low and seasonal transmission where An. albimanus is one of the main vectors. Interestingly, the relationship between parasitemia and exposure was significant when using the antigen from the CTG strain and not the STE. This suggest that the antigens contained on the SGE from the CTG may be more closely related to the one the study subjects are exposed in the field. Still, more studies are needed to evaluate the effect of mosquito colonization on immunogenicity changes of salivary proteins of vectors.
However, we did not find an association between antibodies against An. darlingi SGE and malaria infection. This could be explained due to the low or probably absence of An. darlingi mosquito in areas where samples were collected (6,7). Still, the observed antibody response against the An. darlingi SGE may be explained by a potential cross reactivity between salivary proteins present in mosquitoes from the subgenus Nyssorhynchus, which An. darlingi belongs to. We are currently working on the design and validation of specific peptides designed from immunogenic proteins of An. albimanus SGE to assess exposure to the most abundant malaria vectors in Central and South America.
Our current study shows a high IgG response againstgSG6-P1 peptide in samples from infected compared to uninfected people. These findings agree with our previous study in Colombian and Chilean volunteers, where we found that the concentration of gSG6-P1 antibodies was significantly correlated with malaria infection status and that people with clinical malaria presented significantly higher levels of IgG anti-gSG6-P1 antibodies than healthy controls (11). Although, Anopheles species from the subgenus Nyssorhynchus are the main vectors of malaria in Colombia, at least six species from the sub-genus Anopheles have been described as potential malaria vectors in the region (8,24). Two of these species suggested that the antibodies to the gSG6-P1 peptide in the Colombian population may represent exposure to An. punctimacula, which is a member of the Anopheles subgenus or could represent exposure to minor vectors in the country (27).
When comparing IgG levels against An. albimanus among villages, we observed that SGE was higher in infected than uninfected people in all villages except California.
However, antibodies against the gSG6-P1 were equally high in infected and uninfected people from California residents; this is interesting because California is an area with urban characteristics, unlike Tangareal which is a sub-urban area and Robles and Candelillas which are rural areas. The evaluation of both SGE and peptides could help to refine diagnostic tools regarding malaria risk (28), especially in endemic areas with a high proportion of low density infections.
As discussed previously, Colombia has a high Anopheles species biodiversity (4) This study has some limitations. First, because this study was cross-sectional, association with the anti-Anopheles IgG levels should be interpreted with caution as they do not imply causality. Second, due to the lack of a symptomatic group, we could not analyze risk factors for this kind of infection, and we could not explore the differences in the anti-Anopheles IgG levels between uninfected, asymptomatic (both, submicroscopic and microscopic infections) and symptomatic groups. Despite these limitations, these results are useful to identify new potential biomarkers of exposure to Anopheles in Colombia.

Conclusion
This study demonstrates that SGE from An. albimanus strains CTG and STE could be a potential source of new Anopheles salivary biomarkers for the primary vectors on the Colombian Pacific Coast, and that gSG6-P1 peptide has the potential to quantify human exposure to some malaria secondary vectors. All of them could be useful to estimate the risk of malaria transmission and could provide relevant tools to better understand malaria transmission dynamics and orient control strategies according to the specific characteristics in low-endemic settings.
Declarations support in this research. We also thank Dr.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.   Figure 1 Study sites in Tumaco, Nariño, Colombia Modified from: http://moe.org.co/home/doc/comunic   Correlation between anti-Anopheles IgG levels and anti-Plasmodium IgG levels in the whole