S I A L G I A R a b F c d e f g h i j k l R a A R R 1 A A K P H A 1 e s a r z s B G 0 d Acta Tropica 121 (2012) 152– 155 Contents lists available at SciVerse ScienceDirect Acta Tropica jo ur nal homep age : www.elsev ier .com/ locate /ac ta t ropica hort communication nfluence of HLA-DRB-1 alleles on the production of antibody against CSP, MSP-1, MA-1, and DBP in Brazilian individuals naturally infected with Plasmodium vivax uciane Moreno Storti-Meloa,b,∗, Daniela Reis da Costab, Wanessa Christina Souza-Neirasb,c, ustavo Capatti Cassianob,d, Vanja Suely Calvosa D’Almeida Coutoe, Marinete Marins Póvoaf, rene da Silva Soaresg, Luzia Helena de Carvalhoh, Myrian Arevalo-Herrera i, Sócrates Herrera i, ndrea Regina Baptista Rossit j, José Antonio Cordeirok, Luiz Carlos de Mattos l, icardo Luiz Dantas Machadob Departamento de Biociências, Campus Universitário Prof. Alberto Carvalho, Universidade Federal de Sergipe, Av. Vereador Olimpio Grande, s/n, 49500-000, Itabaiana, SE, Brazil Centro de Investigaç ão de Microrganismos, Departamento de Doenç as Dermatológicas, Infecciosas e Parasitárias, Faculdade de Medicina de São José do Rio Preto, Av. Brigadeiro aria Lima 5416, 15090-000, São José do Rio Preto, São Paulo, Brazil Instituto de Ciências Exatas, Naturais e Educaç ão, Universidade Federal do Triângulo Mineiro, Av. Frei Paulino, 30 - Bairro Abadia, 38025-180 Uberaba, MG, Brazil Pós-Graduaç ão em Genética, Universidade Estadual Paulista “Júlio Mesquita Filho”, Rua Cristóvão Colombo 2265, 15054-000, São José do Rio Preto, São Paulo, Brazil Ministério da Saúde, Núcleo Estadual do Amapá/CRDT, Rua Professor Tostes 2200, 68900-430, Macapá, Amapá, Brazil Laboratório de Pesquisas Básicas em Malária, Instituto Evandro Chagas, Secretaria de Vigilância em Saúde, BR316 Km 7, 67030-000 Ananindeua, Pará, Brazil Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Av. Professor Lineu Prestes, 580 - Bloco 17, 05508-900, São Paulo, São Paulo, Brazil Fundaç ão Oswaldo Cruz, Centro de Pesquisas René Rachou, Laboratório de Malária, Av. Augusto de Lima 1715, 30190-002, Belo Horizonte, Minas Gerais, Brazil Instituto de Inmunología, Facultad de Salud, Universidad del Valle, Cali, Colombia Departamento de Microbiologia e Parasitologia, Instituto Biomédico, Universidade Federal Fluminense, Rua Prof. Hernani de Melo, 101, 24210-130 Niterói, Rio de Janeiro, Brazil Departamento de Epidemiologia, Faculdade de Medicina de São José do Rio Preto, Av. Brigadeiro Faria Lima 5416, 15090-000, São José do Rio Preto, São Paulo, Brazil Laboratório de Imunohematologia, Departamento de Biologia Molecular, Faculdade de Medicina de São José do Rio Preto, Av. Brigadeiro Faria Lima 5416, 15090-000, São José do io Preto, São Paulo, Brazil r t i c l e i n f o rticle history: eceived 27 May 2010 eceived in revised form 9 September 2011 ccepted 12 October 2011 a b s t r a c t We evaluated the influence of allelic frequency of the human leukocyte antigen (HLA) -DRB1 on the acquisition of antibody response against malaria sporozoite and merozoite peptides in patients with Plasmodium vivax malaria acquired in endemic areas of Brazil. IgG antibodies were detected by enzyme- linked immunosorbent assay against four peptides of circumsporozoite protein (CSP) (amino, carboxyl, vailable online 4 November 2011 eywords: lasmodium vivax and VK210 and VK247 repeats) and peptides of merozoite surface protein 1 (MSP-1), apical membrane antigen 1 (AMA-1), and Duffy-binding protein (DBP). We found an association between HLA-DR3 and HLA-DR5 alleles and lack of antibody response to CSP amino terminal, as well as an association between HLA-DR3 and the highest antibody response to MSP1 (Pv200L). In conclusion, we suggest a potential -DRB n pop LA-DRB1 ntibody response regulatory role of the HLA CSP and MSP1 in Brazilia . Introduction Vaccines against malaria are viewed as a potentially cost- ffective measure for malaria control and elimination, and, ignificant effort has been directed toward the identification nd characterization of different Plasmodium antigens. Antibody esponse to pre-erythrocytic antigens, such as circumsporo- oite protein (CSP); and erythrocytic proteins, such as merozoite urface protein 1 (MSP-1), apical membrane antigen 1 (AMA-1), ∗ Corresponding author at: Universidade Federal de Sergipe, Departamento de iociências, Campus Universitário Prof. Alberto Carvalho. Avenida Vereador Olimpio rande, s/n, 49500-000, Itabaiana, SE, Brazil. Tel.: +55 79 34328222. E-mail address: stortilu@yahoo.com.br (L.M. Storti-Melo). 001-706X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. oi:10.1016/j.actatropica.2011.10.009 1 alleles in the production of antibodies to a conserved region of P. vivax ulation exposed to malaria. © 2011 Elsevier B.V. All rights reserved. and the Duffy-binding protein (DBP); have been systematically evaluated in the literature (Arévalo-Herrera et al., 2010). Various authors have considered the CSP as major target for the devel- opment of recombinant vaccines for Plasmodium vivax due to the high levels of antibodies elicited against synthetic peptides, which exhibit the same specificity generated in natural infections (Herrera et al., 2005; Rodrigues et al., 2005; Beeson and Crabb, 2007; Penny et al., 2008). Several molecules of asexual blood-stage parasites are being considered as vaccine candidates. Proteins expressed by merozoites play a critical role during the invasion of red blood cells (RBCs) and are responsible for perpetuating the parasite life cycle (Remarque et al., 2008). Human leukocyte antigen (HLA) class II genes were originally called immune response genes, since their alleles are known to influence antibody production (Germain, 1999). HLA class II dx.doi.org/10.1016/j.actatropica.2011.10.009 http://www.sciencedirect.com/science/journal/0001706X http://www.elsevier.com/locate/actatropica mailto:stortilu@yahoo.com.br dx.doi.org/10.1016/j.actatropica.2011.10.009 ta Tro m a f T t e o c m r a 2 a t o ( r r 2 B t r m 2 s a fi P c r s s d s r r ( m a F B t o t t ( o w R m m D m p t 2 6 a g b L.M. Storti-Melo et al. / Ac olecules bind antigenic peptides derived from exogenous as well s endogenous peptides, and the HLA class II-peptide complex ormed is exposed at the surface of antigen-presenting cells to the -cell receptor on CD4+ T lymphocytes, and the interaction with he B cell to activate a specific antigen–antibody response. How- ver, since HLA genes are the most polymorphic genetic groups n human population and allelic differences in these molecules an modulate their ability to bind and present antigenic deter- inants of proteins and, thereby, change the nature of T-cell ecognition. Various studies have investigated the influence of HLA lleles on malaria immunology (Banic et al., 2002; Johnson et al., 004; Oliveira-Ferreira et al., 2004). Some HLA-DR alleles have been ssociated with a better antibody response to Nt47 (p126 amino- erminal portion) (Banic et al., 2002), AMA-1 (Johnson et al., 2004) f Plasmodium falciparum, and VK247 CSP repetition of P. vivax Oliveira-Ferreira et al., 2004). Additionally, previous studies have eported an association between specific HLA alleles and immune esponse to malaria antigens in human vaccine trials (Nardin et al., 001; Zhang et al., 2009). Therefore, using plasma samples from razilian patients naturally infected with P. vivax, we evaluated he influence of the HLA-DRB1 allelic frequency on the antibody esponse against four different CSP peptides and against three erozoite antigens. . Materials and methods Patients enrolled in this study met the following criteria: pre- entation to medical care because of clinical symptoms of malaria, ge >18 years old, and a positive malaria diagnosis by thick blood lm for P. vivax. All patients signed a written informed consent. eripheral blood samples were obtained from individuals from ities or towns in four malaria-endemic states of the Amazon egion of Brazil: Macapá, Amapá state; Novo Repartimento, Pará tate; Porto Velho, Rondônia state; and Plácido de Castro, Acre tate. The malaria epidemiology of these areas has been previously escribed (Storti-Melo et al., 2011). The samples were frozen, DNA amples were extracted using the Easy-DNATM extraction kit (Invit- ogen, Carlsbad, CA, USA), and a semi-nested polymerase chain eaction (PCR) using specific small-subunit (SSU) rDNA primers Kimura et al., 1997) was performed to confirm the single P. vivax alaria infection. The protocol for this study was reviewed and pproved (Process number 235/2006) by the Research Board of the aculty of Medicine from São José do Rio Preto, São Paulo state, razil. HLA-DRB1 alleles genotyping of the 55 DNA samples of he malaria patients was carried out. DNA concentrations were btained using a spectrophotometer at 260 and 280 nm, and con- ent measured of 100 ng/�L was used for low resolution typing of he HLA-DRB1 by PCR with sequence-specific primers (PCR-SSP) Micro SSPTM DNA Typing Trays, One Lambda, Inc., United States f America). Following manufacturer’s guidelines, 1 �L of sterile ater was added to the H1, H4, H7, and H10 wells of each plate. eactions in these wells served as controls for the assay. Taq poly- erase (7.5 U) was added to the DMiX® solution, and 9 �L of this ixture was then added to control wells. We then added 29 �L of NA (100 �g/mL) to the DMiX® solution, and 10 �L of this final ixture was placed in each reaction well. The reaction plate was laced in a thermal cycler (Applied Byosystems Gene Amp PCR Sys- em 9700) with the following settings: an initial phase of 96 ◦C for min and 63 ◦C for 1 min; followed by 9 cycles of 96 ◦C for 10 s and 3 ◦C for 1 min; followed by 20 cycles of 96 ◦C for 10 s, 59 ◦C for 50 s, nd 72 ◦C for 30 s. DNA fragments were visualized on 1.5% agarose els stained with ethidium bromide. IgG antibodies were detected by enzyme-linked immunosor- ent assay (ELISA) according to previously published guidelines pica 121 (2012) 152– 155 153 (Herrera et al., 2004; Valderrama-Aguirre et al., 2005; Rodrigues et al., 2005; Cerávolo et al., 2005) in the 55 plasma samples. We used four different CSP peptides; amino (N), carboxyl (C), a repetitive region corresponding to the VK210 (R) and a repetitive region corresponding to the VK247 (V) (Herrera et al., 2004); and three merozoite proteins N-terminal fragment of MSP-1 (Pv200L) (Valderrama-Aguirre et al., 2005), recombinant peptide of AMA- 1 (Rodrigues et al., 2005), and recombinant peptide of the DBP (Cerávolo et al., 2005). The ELISA IgG cutoff was defined as the mean optical density (OD) plus three standard deviations of the reaction from individuals (n = 30) who lived outside the Amazon region and who had never had malaria. The results were expressed as an index of reactivity (IR = OD405 values of tested sample divided by the value of the cutoff). IR values <1.0 were considered negative; IR values IR ≥1.0 were considered positive. Analyses were performed using R version 2.8.1 statistical soft- ware (The R Foundation for Statistical Computing, Vienna, Austria, available at http://www.r-project.org). Allele frequencies were cal- culated using the formula AF = a/N, in which a represents the number of positive samples for a specific allele and N represents the total number of alleles in the study population (Garavito, 2003). The heterogeneity of HLA allele frequencies between responder and non-responder groups was evaluated by multiple logistic regres- sion. Differences were considered significant when the p-value was <0.05. 3. Results The HLA-DRB1 allele frequencies against the four different CSP peptides for responders and non-responders are shown in Table 1. A multivariate regression analysis showed a significant association between non-response to N peptide and the presence of HLA-DR3 (p = 0.016) and HLA-DR5 (p = 0.013) in individuals infected with P. vivax. No significant association, positive or negative, was observed between any HLA-DR molecules and antibody response to C, R, and V peptides. The HLA-DRB1 frequencies to MSP1, AMA-1, and DBP were sim- ilar between responders and non-responders; and no significant association between any HLA-DRB1 allele and antibody response against these proteins was found (Table 2). However, almost 90% of Brazilian malaria patients develop antibodies to the MSP-1 (Pv200L) antigen (Storti-Melo et al., 2011). In our study, only two samples were ELISA negative and, among positive samples we observed high antibody levels with value IR > 10. Thus, we analyzed the influence of HLA-DRB1 alleles on antibody levels to MSP-1, and a significant association between high levels of antibodies to MSP-1 and the presence of HLA-DR3 (p = 0.042) was observed by logis- tic regression analysis. In fact, among individuals with HLA-DR3, the frequency of high responders to MSP-1 (35.7%) was signifi- cantly (p = 0.040) higher than in patients with other alleles (14.4%). Because the prevalence and levels of anti-MSP-1 antibodies seem related to previous malaria infection (Storti-Melo et al., 2011), we examined the possibility that individuals with HLA-DR3 had been more exposed to malaria, but no difference was observed in the number of previous malaria episodes between these individuals and those without HLA-DR3 (p > 0.05). 4. Discussion HLA molecules show huge variability in humans and have an important role in immune response. HLA-DR is the most polymor- phic, with specific HLA-DR alleles influencing the acquisition and levels of antibodies to pre-erythrocytic and erythrocytic malaria antigens (Johnson et al., 2004). As previously suggested by Zhang et al. (2009) to improve the efficacy of statistical analysis, we pooled http://www.r-project.org/ 154 L.M. Storti-Melo et al. / Acta Tropica 121 (2012) 152– 155 Table 1 Frequencies of HLA-DRB1 alleles in responders and not responders to four peptides of circumsporozoite protein. HLA DRB1 alleles Amino (N) p-Value Carboxyl (C) p-Value VK210 (R) p-Value VK247 (V) p-Value Neg (N = 18) Pos (N = 92) Neg (N = 16) Pos (N = 94) Neg (N = 52) Pos (N = 58) Neg (N = 56) Pos (N = 54) DR1 (DRB1*01) 0.056 0.076 0.467 0.063 0.074 0.868 0.077 0.069 0.295 0.071 0.074 0.468 DR2 (DRB1*15, *16) 0.000 0.098 0.998 0.063 0.085 0.685 0.058 0.103 0.588 0.089 0.074 0.733 DR3 (DRB1*03) 0.222 0.065 0.016† 0.000 0.106 0.998 0.058 0.121 0.845 0.036 0.148 0.190 DR4 (DRB1*04) 0.278 0.185 0.605 0.250 0.191 0.776 0.212 0.190 0.806 0.196 0.204 0.880 DR5 (DRB1*11, *12) 0.167 0.043 0.013† 0.000 0.074 0.998 0.038 0.086 0.336 0.036 0.093 0.918 DR6 (DRB1*13, *14) 0.056 0.174 0.211 0.250 0.138 0.095 0.154 0.155 0.520 0.179 0.130 0.462 DR7 (DRB1*07) 0.056 0.174 0.280 0.125 0.160 0.887 0.173 0.138 0.675 0.143 0.167 0.605 DR8 (DRB1*08) 0.111 0.141 0.578 0.188 0.128 0.548 0.173 0.103 0.444 0.196 0.074 0.435 DR10 (DRB1*10) 0.056 0.043 0.753 0.063 0.043 0.787 0.058 0.034 0.853 0.054 0.037 0.814 N t a i t 2 w p n r M a i w l ( r t a a g a b i i a n a w n H b T F D N eg: non-responders (IR < 1); Pos: responders (IR ≥ 1); N: total number of alleles. † p < 0.05 by multiple logistic regression. he alleles identified in our study into a group representing equiv- lent serological antigens. The N-terminal region of CSP is highly mmunogenic and is recognized by T cell clones elicited by vaccina- ion of DR4 (DRB*0401 and *0403) individuals (Parra-López et al., 006). In our study, HLA-DR3 and HLA-DR5 alleles were associated ith failure to produce antibody response against the N-terminal eptide of CSP in naturally infected patients. To our knowledge, o previous associations between HLA-DR3 and malaria antigen esponse have been reported. In a phase I clinical trial of the ultiple Antigens Peptides (MAP) vaccine, higher anti-sporozoite ntibodies titers were restricted to three HLA class II alleles, includ- ng the HLA-DR5 (DRB1*1101) plus DRB1*0401 and DQB1*0603, hereas the HLA-DRB1*07 allele failed to elicit high antibodies evels (Nardin et al., 2000). In addition, Oliveira-Ferreira et al. 2004) implicated HLA-DR7 as a poor responder against the VK210 epetitive region of P. vivax CSP in Brazilian individuals. In con- rast, Zhang et al. (2009) observed an improved antibody response gainst a chimeric protein (PfCP-2.9) in the presence of HLA-DR7 lleles. P. vivax asexual blood stage antigens have been reported as tar- ets for the production of invasion-inhibitory or growth-inhibitory ntibodies. Among these, the MSP-1, the AMA-1, and the DBP have eing evaluated for their immune potential (Good et al., 2005). We nvestigated the possible influence of HLA-DRB1 polymorphisms n antibody production against these antigens but no significant ssociation between HLA-DRB1 allelic frequencies in responders or on-responders was found. In contrast, a higher antibody response gainst a recombinant AMA-1 of P. falciparum has been associated ith HLA-DR5 in Cameroon (Johnson et al., 2004). Nevertheless, o previous associations between antibody response to DBP and LA-DR alleles was reported. Since most Brazilian patients exposed to malaria produce anti- odies to Pv200L, there does not seem to be a limitation in the able 2 requencies of HLA-DRB1 alleles in responders and not responders to merozoite surfac uffy-binding protein (DBP). HLA DRB1 alleles Pv200L p-Value AMA-1 Neg (N = 2) Pos (N = 108) Neg (N = 12 DR1 (DRB1*01) 0 0.074 1.000 0.167 DR2 (DRB1*15, *16) 0 0.083 1.000 0.083 DR3 (DRB1*03) 0 0.093 1.000 0.167 DR4 (DRB1*04) 0 0.204 0.999 0.333 DR5 (DRB1*11, *12) 0 0.065 1.000 0 DR6 (DRB1*13, *14) 0.5 0.148 0.636 0.083 DR7 (DRB1*07) 0 0.157 0.999 0 DR8 (DRB1*08) 0.5 0.130 0.176 0.167 DR10 (DRB1*10) 0 0.046 1.000 0 eg: non-responders (IR < 1); Pos: responders (IR ≥ 1); N: total number of alleles. production of antibodies to this antigen that could be associated with HLA (Storti-Melo et al., 2011). However, a positive associa- tion was observed between the highest antibody levels to Pv200L and the presence of HLA-DR3 in this study. This antibody response did not seem related to the number of repeated exposures, since no difference was found between the number of previous malaria episodes and this allele presence. A similar study by Johnson et al. (2004), evaluating the HLA influence in the antibody response to P. falciparum asexual-stage antigens, found no association with any HLA-DRB1 or DQB1 alleles for antibody levels to MSP1-190l and to MSA2, but individuals positive for HLA-DR5 (*1201) had the high- est antibody levels to PfAMA-1. However, this evaluation was done in Cameroon, an African population, where the genetic compo- sition, the malaria transmission profile, and the Plasmodium spp. epidemiology are different from Brazil. The lack of association observed between HLA-DRB1 alleles and antibody response against some CSP peptides (C, R, and V) and against AMA-1 and DBP in our work may mean either that the HLA- DRB1 does not, in fact, modulate the antibody response against these P. vivax antigens, or that, because of the small sample size of our study, we were unable to detect such an association. This finding, therefore, needs to be further investigated. However, the genetic regulation of antigen-specific antibody responses does not seem to be caused only by HLA class II genes. A recent study has shown that genetic factors modulate different antibody isotype and subclass responses to malaria antigens, mainly due to undefined non-HLA-linked genes (Duah et al., 2009). In summary, the associ- ation of the HLA-DR3 and DR5 alleles with the absence of antibody response to the N terminal of CSP should be thoroughly investigated before a malaria susceptibility profile can be established. Moreover, our finding that HLA-DR3 caused the highest antibody response to MSP1 needs to be confirmed by analyses using larger sample sizes, given the low frequency of this allele in Brazil. e protein 1 (MSP-1–Pv200L fragment), apical membrane antigen 1 (AMA-1), and p-Value DBP p-Value ) Pos (N = 98) Neg (N = 62) Pos (N = 48) 0.061 0.060 0.097 0.042 0.240 0.082 0.795 0.129 0.021 0.087 0.082 0.435 0.081 0.104 0.237 0.184 0.249 0.194 0.208 0.654 0.071 0.999 0.048 0.083 0.117 0.163 0.728 0.113 0.208 0.158 0.173 0.998 0.145 0.167 0.925 0.133 0.547 0.145 0.125 0.520 0.051 0.999 0.048 0.042 0.740 ta Tro F F S C ( C 1 ( I A s r d d W a l d d R A B B C D G G G L.M. Storti-Melo et al. / Ac unding Work reported in this manuscript was funded in part by undaç ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP), ão Paulo State, Brazil (02/09546-1; 06/09546-1) and in part by the onselho Nacional para o Desenvolvimento Científico e Tecnológico CNPq), Brasília, Brazil (302353/03-8; 410405/2006-0). Work in olombia was jointly sponsored by COLCIENCIAS (Grant 1106-04- 6489), the Ministry of Social Protection (Grant 2304-04-19524) Contract No.253-2005) and by the National Institute of Allergy and nfectious Diseases (NIAID Grant # 49486/TMRC). cknowledgments We are grateful to all the individuals who participated in this tudy. We would like to thank Carlos Eugênio Cavasini, Aline Bar- oso, Maria Cristina Figueredo, and Mauro Tada for their assistance uring the field work. We are also thankful to Alexandre Macedo e Oliveira and Beatie Divine for critical review of this manuscript. e are indebted to Juliana Cintra for her help in HLA genotyping nalysis, Kátia Sanches Franç oso for her assistance in AMA-1 sero- ogical analysis, and finally to Professor Luiz Hildebrando Pereira a Silva for permitting us to use his facilities at Centro de Pesquisas e Medicina Tropical (CEPEM). eferences révalo-Herrera, M., Chitnis, C., Herrera, S., 2010. Current status of Plasmodium vivax vaccine. Hum. Vaccin. 6, 124–132. anic, D.M., Goldberg, A.C., Pratt-Riccio, L.R., Oliveira-Ferreira, J., Santos, F., Gras- Masse, H., Camus, D., Kalil, J., Daniel-Ribeiro, C.T., 2002. Human leukocyte antigen class II control of the immune response to p126-derived amino terminal peptide from Plasmodium falciparum. Am. J. Trop. Med. Hyg. 66, 509–615. eeson, J.G., Crabb, B.S., 2007. Towards a vaccine against Plasmodium vivax malaria. PLoS Med. 4, e350. erávolo, I.P., Bruña-Romero, O., Braga, E.M., Fontes, C.J., Brito, C.F., Souza, J.M., Kret- tli, A.U., Adams, J.H., Carvalho, L.H., 2005. Anti-Plasmodium vivax duffy binding protein antibodies measure exposure to malaria in the Brazilian Amazon. Am. J. Trop. Med. Hyg. 72, 675–681. uah, N.O., Weiss, H.A., Jepson, A., Tetteh, K.K.A., Whittle, H.C., Conway, D.J., 2009. Heritability of antibody isotype and subclass responses to Plasmodium falci- parum antigens. PLoS One 4, e7381. aravito, G., 2003. Asociación HLA y Artritis Reumatoidea Juvenil: Em busca de las bases moleculares dependiente del MHC. Tesis Doctoral. Universidad del Norte, Colombia. ermain, R.N., 1999. Fundamental Immunology, 4th ed. Lippincott-Raven, Philadel- phia. ood, M.F., Xu, H., Wykes, M., Engwerda, C.R., 2005. Development and regulation of cell-mediated immune responses to the blood stages of malaria: implications for vaccine research. Annu. Rev. Immunol. 23, 69–99. pica 121 (2012) 152– 155 155 Herrera, S., Bonelo, A., Perlaza, B.L., Fernández, O.L., Victoria, L., Lenis, A.M., Soto, L., Hurtado, H., Acuña, L.M., Vélez, J.D., Palacios, R., Chen-Mok, M., Corradin, G., Arévalo-Herrera, M., 2005. Safety and elicitation of humoral and cellular responses in colombian malaria-naive volunteers by a Plasmodium vivax cir- cumsporozoite protein-derived synthetic vaccine. Am. J. Trop. Med. Hyg. 73 (S5), 3–9. Herrera, S., Bonelo, A., Perlaza, B.L., Valencia, A.Z., Cifuentes, C., Hurtado, S., Quintero, G., López, J.A., Corradin, G., Arevalo-Herrera, M., 2004. Use of long synthetic peptides to study the antigenicity and immunogenicity of the Plasmodium vivax circumsporozoite protein. Int. J. Parasitol. 34, 1535–1546. Johnson, A.H., Leke, R.G., Mendell, N.R., Shon, D., Suh, Y.J., Bomba-Nkolo, D., Tchinda, V., Kouontchou, S., Thuita, L.W., Van der Wel, A.M., Thomas, A., Stowers, A., Saul, A., Zhou, A., Taylor, D.W., Quakyi, L.A., 2004. Human leukocyte antigen class II alleles influence levels of antibodies to the Plasmodium falciparum asexual-stage apical membrane antigen 1 but not to merozoite surface antigen 2 and merozoite surface protein 1. Infect. Immun. 72, 2762–2771. Kimura, M., Kneko, O., Liu, Q., Zhou, M., Kawamoto, F., Wataya, Y., Otani, S., Yam- aguchi, Y., Tanake, K., 1997. Identification of the four species of human malaria parasites by nested PCR that targets variant sequences in the small subunit rRNA gene. Parasitol. Int. 46, 91–95. Nardin, E.H., Calvo-Calle, J.M., Oliveira, G.A., Nussenzweig, R.S., Schneider, M., Tiercy, J.M., Loutan, L., Hochstrasser, D., Rose, K., 2001. A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune response in volunteers of diverse HLA types. J. Immunol. 166, 481–489. Nardin, E.H., Oliveira, G.A., Calvo-Calle, J.M., Castro, Z.R., Nussenzweig, R.S., Schmeck- peper, B., Hall, B.F., Diggs, C., Bodison, S., Edelman, R., 2000. Synthetic malaria peptide vaccine elicits high levels of antibodies in vaccines of defined HLA geno- types. J. Infect. Dis. 182, 1486–1496. Oliveira-Ferreira, J., Pratt-Riccio, L.R., Arruda, M., Santos, F., Ribeiro, C.T., Goldberg, A.C., Banic, D.M., 2004. HLA class II and antibody responses to circumsporo- zoite protein repeats of P. vivax (VK210 VK247 and P. vivax-like) in individuals naturally exposed to malaria. Acta Trop. 92, 63–69. Parra-López, C., Calvo-Calle, J.M., Cameron, T.O., Vargas, L.E., Salazar, L.M., Patar- royo, M.E., Nardin, E., Stern, L.J., 2006. Major histocompatibility complex and T cell interactions of a universal T cell epitope from Plasmodium falciparum circumsporozoite protein. J. Biol. Chem. 281, 14907–14917. Penny, M.A., Maire, N., Studer, A., Schapira, A., Smith, T.A., 2008. What should vaccine developers ask? Simulation of the effectiveness of malaria vaccines. PLoS One 11, e3193. Remarque, E.J., Faber, B.W., Kocken, C.H.M., Thomas, A.W., 2008. Apical membrane antigen 1: a malaria vaccine candidate in review. Trends Parasitol. 24, 74–84. Rodrigues, M.H., Rodrigues, K.M., Oliveira, T.R., Cômodo, A.N., Rodrigues, M.M., Kocken, C.H., Thomas, A.W., Soares, I.S., 2005. Antibody response of natu- rally infected individuals to recombinant Plasmodium vivax apical membrane antigen-1. Int. J. Parasitol. 35, 185–192. Storti-Melo, L.M., Souza-Neiras, W.C., Cassiano, G.C., Taveira, L.C., Cordeiro, J.A., Couto, V.S.C.D., Póvoa, M.M., Cunha, M.G., Echeverry, D.M., Rossit, A.R.B., Herrera, M.A., Herrera, S., Machado, R.L.D., 2011. Evaluation of the naturally-acquired antibody immune response to the Pv200L N-terminal fragment of Plasmodium vivax merozoite surface protein-1 in four areas of the Amazon Region of Brazil. Am. J. Trop. Med. Hyg. 84, 58–63. Valderrama-Aguirre, A., Quintero, G., Gómez, A., Castellanos, A., Pérez, Y., Méndez, F., Arévalo-Herrera, M., Herrera, S., 2005. Antigenicity, immunogenicity and pro- tective efficacy of Plasmodium vivax MSP1 PV200L: a potential malaria vaccine subunit. Am. J. Trop. Med. Hyg. 73, 16–24. Zhang, Q., Xue, X., Xu, X., Wang, C., Chang, W., Pan, W., 2009. Influence of HLA-DRB1 alleles on antibody responses to PfCP-2.9-immunized and naturally infected individuals. J. Clin. Immunol. 29, 454–460. Influence of HLA-DRB-1 alleles on the production of antibody against CSP, MSP-1, AMA-1, and DBP in Brazilian individuals n... 1 Introduction 2 Materials and methods 3 Results 4 Discussion Funding Acknowledgments References