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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The challenge posed by developing an effective anti-Leishmania vaccine has led to innovative research on the ability of sand fly saliva from various species to enhance infectivity of Leishmania.6 For example, the saliva from Lu. longipalpis plays a role in establishment and exacerbation of L. major lesions.7 Furthermore MAX, identified only in Lu. longipalpis, is proving to be the most important immunomodulator in the saliva of this sand fly species complex.8–12 Both whole saliva and purified synthetic MAX equivalently increase the size of cutaneous lesions caused by L. major.13 Thus, Leishmania infection may be controlled by eliciting a host immune response against sand fly salivary proteins.
Although we have eluded to MAX thus far as one protein, Lanzaro and others described high levels of amino acid polymorphism in the mature MAX protein among Lu. longipalpis sibling species, after comparing eight DNA sequences from four laboratory fly colony populations originating from Brazil, Colombia, and Costa Rica.14 Some variants differed by as much as 23% in amino acid composition, with a mean ± SD variability of 15.7 ± 4.2%. They hypothesize that variability might represent antigenic polymorphism. The goal of this study is to test that hypothesis.
Since immunization with MAX elicits both a humoral and a cellular response in mice,13 our study addresses whether amino acid variants may elicit specific IgG responses from the host. The ability of mammals to make antibodies against arthropod saliva is well established in studies with laboratory animals.15–17 Even humans living with endemic populations of blood-sucking arthropods have serum antibodies specific against saliva of ticks, mosquitoes, triatomes18–20 and Lu. longipalpis.21 In the present study, we detected a host antibody response to sand fly-injected MAX using immunochemistry. Binding assays revealed antigenic specificity to the C terminal end of variant MAX proteins, suggesting the latter are recognized specifically by the host immune system.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sensitization of mice by sand fly bites. BALB/c mice (eight weeks old) obtained from Harlan Sprague Dawley (Indianapolis, IN) were used and maintained in compliance with National Institutes of Health guidelines. Mice were exposed to Lu. longipalpis females by a modified protocol.17 Briefly, prior to exposing them to sand fly feeding, mice were sedated with an intraperitoneal injection of 60 mg/kg of ketamine and 5 mg/kg of xylazine. Each mouse was exposed to at least 50 female sand flies twice a week, followed by a two-week interval for a total of greater than or equal to four exposures. Serum was collected two weeks after the last exposure, and antibody titers were measured by an indirect enzyme-linked immunosorbent assay (ELISA).
Collection of sand flies and preparation of salivary gland lysate (SGL). Lutzomyia longipalpis was collected via mouth aspirators from domestic animals and Centers for Disease Control (CDC, Atlanta, GA) light traps. In Nicaragua, the flies were collected in the communities of 1) Apumpua in the Department of Chontales and 2) Ojochal in the Department of Leon. Specimens were collected from the town of Baturité in the state of Ceará, Brazil. Field-collected sand flies were identified by species using standard morphologic characteristics.22 Lutzomyia longipalpis from Baturité were frozen in liquid nitrogen and were stored at -80°C. Field-collected female flies from Nicaragua were caged and provided a blood meal from hamsters. Gravid females were individually isolated in small containers and were transported to the laboratory after oviposition. The salivary glands of female progeny were used as an antigen in an indirect ELISA. Briefly, SGL was prepared from 2–8-day-old, laboratory-bred female flies, based on a previous protocol.5 The concentration of SGL protein was quantified by a Micro BCA (bicinchoninic acid) protein assay (Pierce Biotechnology, Rockford, IL) using bovine serum albumin as a standard.
Cloning and sequencing of the MAX gene.
Genomic DNA was isolated from individually dried Lu. longipalpis collected from Baturité, Brazil, Bucaramanga, Colombia and Liberia, Costa Rica14 by a previously described protocol.23 The MAX gene was amplified by a polymerase chain reaction (PCR) from DNA.24 The PCR products from individual flies were cloned using the TOPO TA Cloning system (Invitrogen, Carlsbad, CA) and INV
F’ competent Escherichia coli. Several white colonies were selected and plasmid DNA from individual clones was purified. The MAX gene was amplified from plasmid DNA using a BigDyeTM terminator cycle sequencing ready reaction (Applied Biosystems, Foster City, CA), cleaned on a Micro Bio-Spin P-30 Tris chromatography column (Bio-Rad Laboratories, Hercules, CA), and sequenced on an ABI-377 automated sequencer (Applied Bio-systems) according to the manufacturer’s instructions. DNA sequences were analyzed using DNASTAR Windows version 4.03 (DNASTAR, Inc., Madison, WI) for alignment and translation of DNA into protein sequence.
Generation of synthetic MAX peptides and rabbit antisera.
Seven peptides based on MAX sequence data from individual, field-collected Lu. longipalpis (Figure 1
) were synthesized by SynPep Corp. (Dublin, CA). A Jameson-Wolf plot25 analysis of MAX predicted the relatively high antigenicity of the C terminal end. Six of the peptides were based on hyper-variable 19 amino-acid residues from the C terminal end 43–61 (vMAX), specifically, four vMAX peptides from Baturité, Brazil and one each from Liberia, Costa Rica and Bucaramanga, Colombia. One peptide, cMAX, was synthesized based on amino acid residues 23–40. The peptides were purified by high-performance liquid chromatography. As assessed by mass spectroscopy, the purity achieved was BZba1 = 93.6%, BZba9 = 79.8%, BZba15 = 88.3%, BZba17 = 80.8%, Liberia = 76.4%, BU12 = 84.2%, and cMAX = 61.7%. Proteins were cross-linked to a carrier protein, keyhole limpet hemocyanin, using maleimidobenzoic acid-N-hydroxysuccinimide ester.
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Indirect ELISA to measure anti-MAX, anti-SGL, and anti-peptide IgG. In general, antigen was coated (50 µL/well) on Immulon 4 microtiter plates (Dynex Technologies, Inc., Chantilly, VA). The ELISA was carried out using standard techniques26 with horseradish perioxidase–conjugated goat anti-mouse IgG, goat anti-rabbit IgG (Bio-Rad Laboratories), goat anti-human IgG, rabbit anti-pig IgG (Sigma), and ABTS (2,2'-azino-bis [3-ethylbenzothiazoline-6-sulfonic acid] diamonium salt) substrate (Pierce). Plates were read at 405 nm in a VERSAmaxTM microplate reader (Molecular Devices, Sunnyvale, CA). Relative titers of antibodies were measured in triplicate by determining the dilution factor at which 50% maximal binding occurred. The specific details of individual experiments are given below.
To measure levels of anti-SGL or synthetic MAX protein (SMAX), SGL equivalent to two pairs of salivary glands (
2.8 µg/mL) or SMAX (5 µg/mL) were used as coating antigens. SMAX was a generous gift of Dr. Richard Titus (Colorado State University, Fort Collins, CO) and was based on the published sequence of MAX from Lapinha Caves, Brazil.14 To establish a cut-off titer for each antibody assay, the IgG concentration of antibodies to saliva and antibodies to MAX was determined based upon pigs and humans living in an area where Lu. longipalpis are not found. To account for the possible cross-reactivity between IgG against another arthropod salivary protein and MAX, sera were analyzed from an area in Brazil endemic for triatomine bugs. The SGL coating antigen was derived from Lu. longipalpis from the same state. Negative control sera used in this ELISA originated from the United States. The optical density (OD) values (1:100) of control sera samples (mean + 3 SD) were used as the cut-off value for a positive response. The data obtained were verified by a minimum of three independent repeats.
To determine the binding capacity of rabbit antibodies to vMAX peptides (5 µg/mL) or to the SMAX protein (5 µg/mL), we compared binding of sera from immunized rabbits (n = 6) to that of pre-immune rabbit sera (n = 6). We then tested the binding capacity of six human and six pig sera samples to the vMAX peptides using six unexposed humans and pigs as controls. Data obtained were verified by a minmum of three independent repeats.
To address whether the vMAX portion elicits a stronger antibody reponse than the cMAX portion of MAX, we determined the binding capacity of sera from six Texan and six Nicaraguan pigs to the coating antigens cMAX (5 µg/mL) and vMAX (5µg of BZba17/mL). The OD readings from the Texas pigs were subtracted from the readings obtained for the Nicaraguan pigs. Data obtained were verified by two independent repeats.
Western blot to measure anti-MAX IgG. Two pairs of salivary glands, or 3 µg of SMAX, were separated on a 4–20% gel by sodium dodecylsulfate–polyacrylamide gel electrophoresis (Bio-Rad Laboratories) under reducing conditions and transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories). After blocking non-specific sites, membrane strips were incubated overnight at 4°C with pig sera from Texas or Nicaragua. Subsequently, strips were incubated in horseradish peroxidase–conjugated rabbit anti-pig IgG for one hour at room temperature. Blots were then washed and developed with ECLTM substrate (Amersham Biosciences Corp., Piscataway, NJ).
Statistical analysis. An unpaired, two-sample t-test (two-tailed) was used to compare mean OD levels. Pearson’s correlation coefficient (R2) described associations between the OD levels achieved with binding to SMAX or SGL. A general linear model (GLM) gave statistical significance to the differences in OD units when more than two variables were compared. Exploratory data analyses were conducted to test for departure from normality, linearity, and independence, assumptions of the GLM, a model that also tests for analysis of variance (ANOVA). These descriptive analyses indicated no need to transform variables to meet assumptions. The ANOVA identified those variables responsible for observed differences in mean binding capacity between antisera and vMAX. A GLM was fitted in which the binding activity (OD level absorbance at 450 nm) was the dependent variable and independent variables (dilution factor, antibody x antigen interaction term, experimental replicate, population of origin of MAX peptide) were evaluated for their capacity to influence OD levels.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A positive correlation was found between levels of SMAX and SGL IgG (Figure 3A). Western blot analysis also confirmed detection of specific IgG to SGL and SMAX in Nicaraguan pig sera (Figure 3B). The location of the 7-kD band (Figure 3B, lane 1) corresponds with the mature form of MAX. The faint bands at approximately 131, 100, 65, and 33 kD are other proteins found in the salivary gland (Figure 3B, lane 2). When pig serum from the United States was used to probe the blots, the sera did not bind to SMAX or to the proteins in SGL. None of the people at risk of or infected with T. cruzi had measurable IgG levels against sand fly SGL or against SMAX by the indirect ELISA using serum from the United States to determine the cutoff point of the ELISA.
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). Variable sites in the mature, 61-amino acid protein are found predominantly in residues 9–22 and 43–61, with relatively little variability in other regions of the protein.
Specificity of antibodies against the C terminal variable region of MAX.
To examine whether variant MAX sequences generate a specific IgG response, peptide sequences were chosen to assess whether a positive correlation exists between amino acid sequence divergence and antigenic specificity. Sequences were chosen from within a single population from Baturité, Brazil, as well as from populations representing two other Lu. longipalpis sibling species from Bucaramanga, Colombia and Liberia, Costa Rica (Figure 1). These six peptides based on 19 amino acid residues differ from 5.2% to 36.8% in amino acid composition within the Baturité population and from 15.7% to 42.1% between populations of Lu. longipalpis sibling species from Brazil, Colombia, and Costa Rica. The sequences varied by approximately as much within populations as between populations.
Two interesting findings were obtained from anti-vMAX/vMAX binding experiments (Figure 4). First, the panel of anti-peptide sera for six vMAX peptides bound to the peptide- coating antigens with specificity (F = 10.5–24.5, degrees of freedom = 5, P = 0.0005, by ANOVA). The six peptide sequences were distinguished as though they were two different sequences. Peptides BZba17 and BZba1 bound in a nearly identical fashion with the panel of antisera (Figure 4A and B). In addition, peptides Liberia, BZba15, BU12, and BZba9 reacted with the panel of antisera indistinguishably (Figure 4C–F). Second, for the antigen/antibody combinations tested, an increase in amino acid differences produced a decrease in binding affinity. A difference in peptide residues 
26.3% caused the panel of anti-peptide sera to show statistically significant differences in binding.
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. The term antigen x antibody suggests that there is a synergy between antigen and antibody that produces changes in the OD level. Based on the GLM, differences among independent experiments (P = 0.975) or between populations (P = 0.223) did not contribute significantly to changes in the OD level. The population was defined as either those sequences from Baturité, Brazil or Bucaramanga, Colombia and Liberia, Costa Rica. The data suggest there is antigenic variability between populations as well as within a single population. If pressure from the host immune system produces variability in MAX, antigenic variability must be found within a single population.
Next, we determined that antibodies to vMAX specifically recognize full-length SMAX protein (Figure 5). Antibody to BZba15 showed the highest binding capacity to SMAX and antibody to BZba1/BZba17 showed the lowest binding capacity. This was an expected result since BZba15 was identical in amino acid sequence to the 19 residues at the C-terminal end of SMAX and BZba1/BZba17 peptide differed by greater than 26.3%. Antisera to the four remaining peptides bound with intermediate reactivity to SMAX, and their sequence divergence ranged from 10.5 to 15.7%.
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). The human sera from the United States did not show specific binding to any of these peptides. These results followed the same trend as those from rabbits immunized with these peptides. The differential serologic response was more pronounced in humans than in pigs. The sera from Nicaraguan pigs, with greater exposure to Lu. longipalpis, responded to the peptides with an OD greater than 3 SD above the level given by sera from pigs from the United States. However, the Nicaraguan pig serologic response showed less difference between peptides (Figure 7). Pig NP34 responded less to peptides BZba17 and BZba1 compared with the other peptides. Individual serum responded differently to the peptides, depending on the degree of exposure of these animals to Lu. longipalpis, and therefore to MAX variants. No significant differences in the concentration of either total protein or IgG between the Nicaraguan and U.S. pig or human sera were found. Therefore, the differences are likely due to the presence or absence of specific IgG antibody to MAX.
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Binding capacity of Nicaraguan pig serum to the variable and conserved regions of MAX.
The MAX amino acid residues 23–40 (cMAX) are conserved relative to residues 43–61 (vMAX). We sought to determine whether antigenicity is associated with variability. However, cMAX was difficult to produce and purify from other slightly degenerate, synthetic forms. Despite the degeneracy of cMAX, sera from pigs bitten by sand flies in Nicaragua were found to have a greater specificity to residues 43–61 compared with residues 23–40 in an ELISA based on serum antibody capture (Figure 8). Texas pig serum did not bind with specificity to cMAX or to vMAX.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Extensive amino acid polymorphism in MAX from different sand fly populations, representing three sibling species from throughout Central and South America, was previously reported.14 In the present study, we found equally high levels of divergence among MAX variants from individuals collected from a single population in Baturité, Brazil. The variability of MAX is not explained largely by speciation because the Baturité population was a single gene pool with random mating among individuals.27 Polymorphism in MAX appears to be higher than the average degree of protein polymorphism in Lu. longipalpis. From analyses of enzyme-encoding loci, other Lu. longipalpis (Baturité Brazil) proteins do not have an unusual level of genetic polymorphism (37.5% polymorphic loci and 0.111 mean heterozygosity27) compared with values found in insects in general (38.0% polymorphic loci and 0.107 mean heterozygosity28).
Therefore, the salient question is why is MAX protein sequence so diverse? Maintenance of the large number of MAX variants observed in the Baturité population cannot be explained by genetic drift alone. If positive diversifying selection has operated on a gene, the number of non-synonymous substitutions causing adaptive amino acid changes is greater than the number of synonymous substitutions.29 Our finding that nucleotide substitutions in the MAX gene described here are primarily non-synonymous suggests that MAX variability may be due to selection rather than genetic drift. We are beginning to shed light on the nature of the selection pressure that produces polymorphism in MAX. Lanzaro and others14 demonstrated that a series of MAX variants all had equivalent vasodilatory potency. Therefore, polymorphism in MAX appears to be constrained by the necessity to conserve vasodilatory function.14
These results prompted us to find that MAX variants are specifically recognized by the host immune system. Although both human and pig sera recognized vMAX, individual human serum samples showed a more dramatic differential response to MAX than did individual pig serum. These results are probably due to the finding that Lu. longipalpis seem to preferentially bite pigs compared with other vertebrate hosts in Costa Rica30 and Colombia.31 For pigs bitten by a substantial number of flies every night over time, we predict that a given animal may have been exposed to a higher degree to all six of the peptide variants in our study. Humans have less exposure to sand flies compared with pigs or other domestic animals because Lu. longipalpis are not particularly anthropophilic.31
We also present evidence that epitopes in vMAX elicit a greater antigenic response than do epitopes in cMAX. It can be inferred from previous work32,33 that the variable regions of MAX are involved in binding to IgG because these regions are essential for MAX binding to the pituitary adenylate cyclase activation polypeptide type 1 (PAC1) receptor. In contrast, the conserved middle region of MAX appears essential for MAX vasodilatory activity, but not for binding to the PAC1 receptor. It appears that either residues 24–42 are conformationally unexposed to the environment, and thus are not involved in IgG receptor ligand binding, or the physical properties of these residues make them less antigenic. Regardless of the reason, our studies suggest that amino acid sequence variability in MAX is associated with antigenicity.
The MAX protein induces vasodilation in the skin of a mammal that has been exposed to multitudes of bites from a population of sand flies over time (Milleron RS, unpublished data). This situation may occur because the host immune system may not neutralize the vasodilatory function of MAX when a population of sand flies injects numerous variants. In light of this observation, we hypothesize that the sand fly may have evolved diversity in MAX as a strategy to evade the host immune response. Although a potentially large B cell repertoire can be produced in any given vertebrate host, selective recognition of specific epitopes occurs due to the immunogen itself or to host factors involved in its selection.34 Studies of strains of human immunodeficiency virus type 1 and other animal lentiviruses show that these viruses have evolved immunodominant epitopes capable of undergoing antigenic polymorphism. Because of their antigenic diversity, these pathogens are able to limit or fix the humoral and cell-mediated responses to the initial resident pathogen; thus, the host seems unable to eliminate or control the agent that may be a closely related genetic variant.35 Whether the theory of deceptive imprinting or antigenic competition describes pressures encountered by MAX deserves further study.
The possibility that the host immune system exerts selective pressure on a salivary protein from a hematophagous insect has never been previously investigated or described. The data in the present study lend support to this hypothesis. We are completing studies to address whether selection is acting on MAX by examining blood feeding behavior and fecundity of flies feeding on mice immunized with MAX.
Our findings have implications for the general epidemiologic application of sand fly saliva. Antigenic diversity of MAX sequences is significant in light of the potential uses of salivary proteins in a diagnostic ELISA to test for sand fly or Leishmania exposure21 and a recombinant subunit vaccine.36,37 The observation that this antigen displays diversity could constrain these applications. Previously, a number of anti-parasite vaccines have failed based, in large part, on the high degree of amino acid diversity of a candidate vaccine antigen. Vaccinating mice against MAX elicits a protective Th1-type response against Leishmania, as well as a strong anti-MAX IgG response.13 However, the protective abilities of one MAX variant against exposure to another variant remain to be determined. Designing a successful vaccine may mean including all of the different immunogenic forms of MAX to give blanket protection. Alternatively, novel vaccination regimens will have to be developed to randomly reproduce the antigenic diversity displayed by these sand flies.
Received May 14, 2002. Accepted for publication March 3, 2003.
Acknowledgments: We thank Drs. Daniel Freeman and Frederic Tripet for discussing the statistical applications used in the manuscript, Dr. Dia Elnaiem for thoughtful comments, Dr. Richard Titus for his gift of synthetic MAX, Dr. Eva Harris for the human sera from Nicaragua, and Drs. José Ribeiro and Barbara Doughty for their critical reading of the manuscript and supply of essential reagents.
Financial support: This work was funded by National Institutes of Health (grants T32 AI-075261 and AI-39540).
* These authors contributed equally to this paper. 
Authors’ addresses: Rania S. Milleron, John-Paul Mutebi, Huaizhi Yin, and Lynn Soong, Department of Pathology and World Health Organization Center for Tropical Diseases, University of Texas Medical Branch, Galveston, TX 77555-0609. Sonia Valle and Alberto Montoya, Departamentos de Entomologia y Parasitologia, Centro Nacional de Diagnostico y Referencia, Ministerio de Salud, Managua, Nicaragua. Gregory C. Lanzaro, Department of Entomology, University of California, Davis, CA 95616-8579, Telephone: 530-752-5833, Fax: 530-752-1537, E-mail: gclanzaro{at}ucdavis.edu.
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REFERENCES |
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This article has been cited by other articles:
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  T. M. Brodie, M. C. Smith, R. V. Morris, and R. G. Titus Immunomodulatory Effects of the Lutzomyia longipalpis Salivary Gland Protein Maxadilan on Mouse Macrophages Infect. Immun., May 1, 2007; 75(5): 2359 - 2365. [Abstract] [Full Text] [PDF]
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 V. B. Reddy, A. O. Iuga, K. Kounga, and E. A. Lerner Functional Analysis of Recombinant Mutants of Maxadilan with a PAC1 Receptor-expressing Melanophore Cell Line J. Biol. Chem., June 16, 2006; 281(24): 16197 - 16201. [Abstract] [Full Text] [PDF]
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