Anti-Anopheles darlingi saliva antibodies as marker of Plasmodium vivax infection and clinical immunity in the Brazilian Amazon
© Andrade et al; licensee BioMed Central Ltd. 2009
Received: 06 April 2009
Accepted: 05 June 2009
Published: 05 June 2009
Despite governmental and private efforts on providing malaria control, this disease continues to be a major health threat. Thus, innovative strategies are needed to reduce disease burden. The malaria vectors, through the injection of saliva into the host skin, play important role on disease transmission and may influence malaria morbidity. This study describes the humoral immune response against Anopheles (An.) darlingi saliva in volunteers from the Brazilian Amazon and addresses the association between levels of specific antibodies and clinical presentation of Plasmodium (P.) vivax infection.
Adult volunteers from communities in the Rondônia State, Brazil, were screened in order to assess the presence of P. vivax infection by light microscopy and nested PCR. Non-infected volunteers and individuals with symptomatic or symptomless infection were randomly selected and plasma collected. An. darlingi salivary gland sonicates (SGS) were prepared and used to measure anti-saliva antibody levels. Plasma interleukin (IL)-10 and interferon (IFN)-γ levels were also estimated and correlated to anti-SGS levels.
Individuals infected with P. vivax presented higher levels of anti-SGS than non-infected individuals and antibody levels could discriminate infection. Furthermore, anti-saliva antibody measurement was also useful to distinguish asymptomatic infection from non-infection, with a high likelihood ratio. Interestingly, individuals with asymptomatic parasitaemia presented higher titers of anti-SGS and lower IFN-γ/IL-10 ratio than symptomatic ones. In P. vivax-infected asymptomatic individuals, the IFN-γ/IL-10 ratio was inversely correlated to anti-SGS titers, although not for while in symptomatic volunteers.
The estimation of anti-An. darlingi antibody levels can indicate the probable P. vivax infection status and also could serve as a marker of disease severity in this region of Brazilian Amazon.
Malaria continues to be one of the most serious public health problems worldwide, exacting a huge impact on human wellbeing, mainly in tropical and subtropical countries. A better understanding of the interactions between the host, the vector and the parasite could be valuable to indicate future strategies. In endemic regions, residents are frequently bitten by both uninfected and infected mosquitoes. There is also a progressive acquisition of immunity, leading to a decreased number of malaria clinical attacks related to increasing age and time residing in the endemic area [1, 2]. Within the Brazilian Amazon, and mainly in riverine communities, the prevalence of asymptomatic malaria infection seems to be four to five times greater than the symptomatic infection [3–5]. Malaria clinical immunity has already been described in both Plasmodium (P.) falciparum  and Plasmodium (P.) vivax  infections and it seems to be related to higher titers of anti-Plasmodium antibodies . On the other hand, anti-parasite response might not be the unique determinant of the occurrence of symptomless malaria, as asymptomatic patients maintain parasitaemia at low levels in addition to controlling the clinical symptoms . Such asymptomatic carriers have developed just enough immunity to protect them from malarial illness but not from malarial infection. Regardless these facts, the specific mechanisms that underlie the occurrence of clinical immunity against the Plasmodium are not well understood.
In this scenario, the anopheline vector could play significant role in malaria clinical severity. Mosquito bites can induce immediate, delayed, and systemic hypersensitivity reactions in hosts . Moreover, pre-exposure to the vector saliva may create an inhospitable environment for the establishment of the parasites transmitted by these insects. Mice repeatedly exposed to bites from uninfected Anopheles (An.) stephensi increase a pro-inflammatory T helper 1 biased response that limits P. yoelii infection . In humans it has been shown that An. gambiae saliva is immunogenic for travelers transiently exposed to bites in African endemic areas , with the development of specific IgG and IgM antibodies. Specific anti-An. gambiae saliva IgG antibodies were also detected in young children from a seasonal malaria transmission region in Senegal, and antibody levels were higher in patients who developed clinical malaria episodes, suggesting that the estimation of humoral response to Anopheles salivary antigens can serve as potential marker for the risk of malaria . Moreover, anti-An. dirus salivary protein antibodies occur predominantly in patients with acute P. falciparum or P. vivax malaria, whereas people from non-malarious areas do not carry such antibodies . Little is known about anti-saliva humoral responses in other endemic areas, such as Latin America. In addition, the host response against the most widespread malaria vector in America, An. darlingi, is poorly explored. The objective of the present work was to measure the anti-saliva IgG responses against An. darlingi mosquitoes in the Brazilian Amazon and to evaluate the association of antibody levels with different clinical presentations of P. vivax infections.
A cross-sectional study investigating determinant factors for asymptomatic P. vivax malaria was performed during 2007 (June to August) in Buritis (10°12'43" S; 63°49'44" W), a recent urbanized municipality, and Demarcação (8°10'04.12" S; 62°46'52.33" W), a riverine community of the Rondônia State, in the south-western part of Brazilian Amazon. In general, Rondônia has a flat topography, with an average elevation of 300 m above sea level. The climate is tropical, with a long rainy season from January till May. It is argued that the environmental changes caused by deforestation have favored the main malaria vector in Brazil An. darlingi . Within the regions studied here, the malaria transmission is unstable, with increased number of cases being detected annually between April to September, and the risk of infection is moderate to high , with an Annual Parasite Incidence of 77.5 per 1,000 inhabitants in 2005 . In the Brazilian Amazon, P. vivax accounts for the majority of malaria cases, while P. falciparum infection prevalence is 23.7% . In addition, infection with P. malariae achieves 10% in Rondônia .
Baseline characteristics of the volunteers.
Plasmodium vivax current infection
(n = 80)
(n = 50)
(n = 74)
Age – years*
Malaria episodes referred*
Time residing in the area – years
Parasitaemia – parasites/μL
3 (6%) §
IgG anti-SGS – O.D.*
Plasma IL-10 – pg/mL*
Plasma IFN-γ – pg/mL*
Molecular malaria diagnosis
Primers used in Nested PCR reactions.
Oligonucleotide Sequence 5'-3'
Salivary Gland Sonicate (SGS) preparation
Salivary glands from field captured adult female An. darlingi mosquitoes were dissected and transferred to 20 μL of 10 mM HEPES pH 7.0, 0.15 mM NaCl in 1.5-mL polypropylene vials, usually in groups of 20 gland pairs. Salivary glands were kept at -70°C until needed, when they were disrupted by sonication using a Branson Sonifier 450 homogenizer (Branson, Danbury, CT). The homogenates were centrifuged at 10,000 × g for 4 min and the supernatants were used for the experiments. Protein concentrations were measured by the bicinchonic acid method (BCA, Pierce, Rockford, Illinois, USA). As the salivary glands used in this study were obtained from field captured mosquitoes, Plasmodium contamination needed to be checked by nested PCR. Briefly, it was performed the DNA extraction of a sample from the same SGS pool used in the serological experiments using the Qiagen Generation Capture Card Kit (Cat. No. 159982; Qiagen, Santa Clara, California, USA). Further, the nested PCR was performed as described above, in duplicate samples. There was no amplification of DNA in both duplicates (data not shown).
Anti-An. darlingi saliva serology
Volunteer's sera were collected and kept at -70°C. Serological tests of all samples were performed in a single experiment, with duplicate samples. ELISA was performed as described elsewhere . Briefly, plates were coated with An. darlingi salivary homogenate (SGS) equivalent to 1.5 μg/mL in carbonate buffer overnight at 4°C, then washed with PBS/0.05% Tween and blocked with PBS/0.1% Tween plus 0.05% BSA. Sera were diluted 1:100 with PBS/0.05% Tween and incubated overnight at 4°C. After further washings, the wells were incubated with alkaline phosphatase-conjugated anti-human IgG (Sigma-Aldrich, St. Louis, MO) at a 1:5,000 dilution. Following another washing cycle, the color was developed with p-nitrophenylphosphate. The reactions were blocked with NaOH and read at 405 nm using Soft Max-Pro Software v5 (Molecular Devices Corporation, Sunnyvale, California, USA) ELISA reader. The optical density (OD) values plotted represent the means between each sample duplicate, adjusted for the values from the blank wells.
Plasma cytokine measurement
Interleukin (IL)-10 and interferon (IFN)-γ plasma levels were measured using de Cytometric Bead Array – CBA® (BD Biosciences Pharmingen, San Diego, California, USA) according to the manufacturer's protocol.
Data were analyzed using the GraphPad Prism 5.00® (GraphPad Software Inc.). For the ordinal variables (age, referred malaria episodes, IgG, IL-10 and IFN-γ serum levels), differences between groups were calculated using the non parametric Kruskal-Wallis test with Dunn's multiple comparison post test. Chi-square test was used to compare differences regarding categorized variables (Time residing in the area and parasitaemia). Mann-Whitney test was used to compare differences in IgG levels between non-infected individuals and those with symptomatic or asymptomatic P. vivax infection. This test was also used to estimate significance in IFN-γ/IL-10 ratios from volunteers with asymptomatic or symptomatic P. vivax infection. To evaluate the cut off value of IgG anti-SGS predicting malaria infection or asymptomatic infection, we performed Receiver-operator characteristic (ROC) curves, calculated the Area under curve (AUC), and then estimated the likelihood ratio for the discrimination between the conditions analyzed. Fine Lowess curves were plotted to evidence the trend of the data presented in correlation analyzes. Spearman test was used to verify the significance in the correlations between cytokine levels and anti-SGS levels. Differences were considered significant at P < 0.05.
Results and discussion
A ROC curve was built to assess the best anti-SGS OD value to discriminate P. vivax infection from the non-infected condition. A cut-off point of 0.0855 OD displayed a likelihood ratio to be infected of 2.11 indicating P. vivax infection (Figure 1B; AUC: 0.727; p < 0.0001). These data suggest that evaluation of anti-saliva antibodies could be a useful indicator to estimate exposure to P. vivax in this endemic area. High anti-SGS antibody levels were also proposed as putative biomarkers of exposure to bites of An. stetephensi or An. gambiae and also of risk of P. falciparum malaria . In this study, besides suggesting exposure to bites, high anti-An. darlingi saliva antibody levels could also indicate exposure to P. vivax.
This work is the first to evaluate human immune response against salivary components of An. darlingi, the most widespread specie of Anopheles mosquitoes and the major malaria vector in the Americas . In areas with unstable malaria transmission and moderate risk of infection, such as the Brazilian Amazon, adults, instead of children are largely affected by the disease. Hence, this study focused investigation on the adult population from a Brazilian endemic area.
Through the estimation of serum anti-An. darlingi saliva antibody levels, it is possible to infer the probable P. vivax infection status as marker of disease severity of an individual from the Amazon endemic area. Moreover, this study also suggests that the clinical immunity against P. vivax could be associated to a specific humoral response against the salivary components. As previously described to other vector-borne diseases, such as leishmaniasis, the detection of increased levels of anti-vector saliva could be pointed as an epidemiological marker of infection and also as a suitable indicator of clinical immunity in endemic regions.
BBA received a PhD fellowship and ARF a scientific initiation fellowship from the Brazilian National Research Council (CNPq). LAM, AB and MB-N are senior investigators from CNPq.
The authors would like to thank João Gambati, Sebastião Martins Neto, and Imbroinise Neto for technical support in field study area; Mr Jorge Tolentino, Ms Natali Alexandrino, and Mrs Adorielze Leite for logistic support. We are also grateful to Mr. Kiyoshi Fukutani for assistance with the molecular experiments and to Dr. Marcelo Jacobs Lorena for critical review of the manuscript. This work was supported by FINEP (010409605)/FNDCT-CT-Amazonia.
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