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Am. J. Trop. Med. Hyg., 73(5 suppl), 2005, pp. 3-9
Copyright © 2005 by The American Society of Tropical Medicine and Hygiene

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SAFETY AND ELICITATION OF HUMORAL AND CELLULAR RESPONSES IN COLOMBIAN MALARIA-NAIVE VOLUNTEERS BY A PLASMODIUM VIVAX CIRCUMSPOROZOITE PROTEIN–DERIVED SYNTHETIC VACCINE

SÓCRATES HERRERA*, ANILZA BONELO, BLANCA LILIANA PERLAZA, OLGA LUCÍA FERNÁNDEZ, LEONARDO VICTORIA, ANA MILENA LENIS, LILIANA SOTO, HUGO HURTADO, LINA MARÍA ACUÑA, JUAN DIEGO VÉLEZ, RICARDO PALACIOS, MARIO CHEN-MOK, GIAMPIETRO CORRADIN, AND MYRIAM ARÉVALO-HERRERA
Instituto de Inmunología, Universidad del Valle, Cali, Colombia; Malaria Vaccine and Drug Development Center, Cali, Colombia; Fundación Clínica Valle del Lili, Cali, Colombia; Division of Infectious Diseases, Federal University of Sao Paulo, Sao Paulo, Brazil; Family Health International, Durham, North Caarolina; Biochemistry Institute, University of Lausanne, Lausanne, Switzerland


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Substantial experimental evidence indicates that the Plasmodium circumsporozoite (CS) protein has great potential as a vaccine candidate. We tested the safety and immunogenicity of vaccines composed of P. vivax CS-derived synthetic peptides. Sixty-nine healthy, malaria-naive volunteers were randomized to receive three injections of placebo or synthetic proteins N, R, or C (10, 30, or 100 µg/dose) in a double-blinded fashion. Vaccines were well tolerated and no serious adverse events were observed. Peptides N and R elicited humoral responses at all doses; peptide C elicicted these responses only at doses of 30 and 100 µg. The N peptide at a dose of 100 µg elicited the greatest antibody response. Antibodies to the three peptides recognized P. vivax sporozoites in an immunofluorescent antibody test. Peripheral blood mononuclear cells from most immunized volunteers also produced interferon-{gamma} upon peptide in vitro stimulation. These vaccines appear safe, well tolerated, and immunogenic in malaria-naive volunteers. Further optimization and development of this vaccine is being attempted to conduct phase II clinical trials.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An estimated 80 million cases of Plasmodium vivax malaria occur yearly worldwide. Although this parasite species does not produce significant mortality compared with P. falciparum, the disease negatively affects human health, economic productivity, and per capita income in malaria-endemic communities, as well as the health of travelers visiting areas not endemic for malaria.1 Since classic malaria control measures have failed to reduce the malaria burden, an effective vaccine is a priority to control this disease. Although the malaria parasite cycle provides at least three levels that may be targeted by immune responses, the pre-erythrocytic phase of the parasite cycle appears to be an ideal one because blocking hepatocyte invasion and further growth of the parasite would prevent subsequent clinical manifestations and relapse.

Vaccination using irradiated sporozoites has been shown to protect against human malaria parasites.2–4 Sera from individuals protected by this procedure recognize the circum-sporozoite (CS) protein, which is abundantly expressed on the surface of the sporozoite as well as during early liver parasite stages. Substantial experimental evidence has shown that the CS protein, which is involved in the process of hepatocyte invasion, has great potential as a vaccine candidate. Passive transfer of cytotoxic T cell clones or antibodies to CS protein to rodents and immunization of mice with irradiated sporozoites have been shown to protect animal models from sporozoite challenge.5–8 In addition, over the last few years, encouraging results have been obtained in clinical trials using a recombinant vaccine based on the carboxyl and repeat regions of P. falciparum CS protein and the hepatitis B surface antigen (RTS,S vaccine). This vaccine formulated in the adjuvant ASO-A2 (GlaxoSmithKline, Research Triangle Park, NC), has protected individuals from experimental inoculation with P. falciparum sporozoites,9 as well as semi-immune adults from natural infection in an endemic area.10 More recently, a trial conducted in young African children showed that after three RTS,S vaccine doses volunteers could be protected from clinical malaria in 29.9% of the cases.11

Due to its vaccine potential, the P. vivax CS protein has been the subject of significant immunologic characterization over the past decade.12–16 Such research has identified B and T cell epitopes that have been used to design and synthesize three polypeptides (N, R, and C) for assessment as vaccine candidates. Fragments N and C include functional domains (RI and RII plus) that bind to hepatocyte ligands and are involved in sporozoite invasion of the hepatocyte,17–19 whereas the central repetitive domain represented by peptide R has been shown to contain B cell epitopes16 able to induce invasion-blocking antibodies.20 Preclinical studies conducted by our group in mice and Aotus monkeys have indicated that the synthetic peptides described are safe and immunogenic in animal models.21,22

We report here the results of a randomized, double-blind, phase I clinical trial conducted in healthy, malaria-naive volunteers at the Malaria Vaccine and Drug Development Center in Cali, Colombia. Our study evaluated the safety, tolerability, and immunogenicity of three synthetic peptides (N, R, and C) derived from the P. vivax CS protein.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study participants. We recruited 69 healthy men and women volunteers 18–33 years of age with no history of malaria from universities in Cali, Colombia, a city not endemic for malaria. All participants provided written informed consent and were advised that they were free to withdraw from the study at any time. Volunteers were excluded if they had diseases or medical conditions that would alter the assessment of the vaccine, including human immunodeficiency virus, hepatitis B, or hepatitis C infection, cardiac, hepatic, renal, or autoimmune disease, anemia, allergies, pregnancy, immunodeficiency, existing antibodies to the P. vivax CS protein, splenectomy, or any other condition that could increase the risk of an adverse outcome.

Vaccines. To formulate the vaccine, we used three long synthetic peptides corresponding to the amino-flanking region (N-terminal), the repeat region (R), and the carboxyl-terminal region (C) of the P. vivax CS protein. The N polypeptide is amino acids 20–96 of the native CS protein and constitutes a 76-mer peptide. The R peptide is a hybrid 48-mer peptide that contains a B cell epitope in peptide p11 located at positions 96–104 of the protein and collinearly linked to a universal T helper cell epitope from the tetanus toxin protein.23,24 The 71-mer C peptide is composed of amino-acid residues 301–372 of the CS protein.

We synthesized the peptides under Good Laboratory Practices conditions at the Biochemistry Institute, University of Lausanne (Lausanne, Switzerland) using solid-phase fluorenylmethoxycarbonyl chemistry.25 Peptides were lyophilized under sterile conditions, packaged in individual doses of 36 µg and 120 µg per vial, and both sterility and pyrogenicity were tested. Additionally, mass and purity of the three peptides was assessed by high-performance liquid chromatography and mass spectrometry and had a purity ≥ 85%. Vials were shipped on dry ice from Switzerland to the Immunology Institute in Cali, Colombia and kept at –70°C until the vaccines were prepared. General safety tests were independently performed in animals by LCG Bioscience Laboratories (Turin, Italy).

We formulated the peptides in Montanide ISA 720 (Seppic, Inc., Paris, France) immediately before each immunization and stored the vaccine at 4°C according to manufacturer recommendations. Saline solution (Baxter, Deerfield, IL) appropriately formulated in Montanide ISA 720 was used as placebo. We mixed vaccine emulsions 20 times by up and down strokes using a 10-mL syringe with a 21-gauge needle, and without the rubber part of the piston. An independent monitor assessed peptide accountability at the beginning and the end of the study.

Study design. The study design was a randomized, double-blinded, phase I clinical trial. Participants and physicians in charge of immunization were kept blinded. The vaccine was prepared by an investigator who was blinded in regard to the recipient individual. Our protocol was reviewed and approved by the Institutional Review Boards of the Universidad del Valle and the Fundación Clínica Valle de Lili Hospital, and the study complied with Declaration of Helsinki principles, International Conference on Harmonization Good Clinical Practices guidelines, and all pertinent Colombian regulations.

Eligible participants were enrolled to receive three doses of 10, 30, or 100 µg of vaccine. Each dose group included 23 volunteers who were assigned using block randomization to receive either N, R, or C peptide or placebo (n = 7 individuals/peptide or n = 2 individuals/placebo). Volunteers were recruited in successive groups to receive the different doses and no randomization was used in this step. Codes for volunteers and peptides were used to maintain blinding. We performed immunizations at months 0, 2, and 6 by intramuscular injection with a vaccine volume of 0.5 mL in the deltoid muscle, alternating arms between each injection. Participants who left the study before the second vaccine dose were replaced. Peptides were tested in a dose-escalating manner with the lowest-dose group vaccinated first. We maintained a two-month gap between the initiation of each dose/group and the higher one, so that an independent Safety Monitoring Committee could evaluate the occurrence and severity of adverse events (AEs) associated with the immunization, before each dose was escalated. According to the protocol, the occurrence of more than three AEs (severity grade ≥ 2 according to Common Toxicity Criteria version 2.0) or one serious adverse event (SAE) related to the vaccine in any of the groups would lead to study product discontinuation.

Safety and immunogenicity evaluation. Participants were observed for 60 minutes after vaccination and at 8 and 24 hours, as well as on day 7 after the procedure. To assess safety, we performed a complete physical examination as well as clinical laboratory tests on all study participants during scheduled visits at months 0, 1, 2, 3, 6, 7, and 9. Blood samples were collected for complete blood count, sedimentation rates, prothrombin time, partial thromboplastin time, glucose, blood urea nitrogen, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and bilirubin. Immediately before each vaccination, we tested female participants for pregnancy by a ß-human chorionic gonadotropin test. Adverse events were recorded throughout the study and were defined as any new or worsening sign or symptom of illness, or abnormal laboratory test during the protocol-specified follow-up period. Each AE was evaluated for its severity26 and relatedness with vaccination27 by the study clinician and SAEs were reported as required by the World Health Organization. Immunogenicity was assessed by an enzyme-linked immunosorbent assay (ELISA) performed at 0, 1, 2, 3, 6, 7, and 9 months; an ELIspot at 0, 1, 3, 6, 7, and 9 months; and an immunofluorescent antibody test (IFAT) at 0, 3, 7, and 9 months after enrollment. Additionally, tests to assess the induction of autoimmune antibodies (anti-neutrophil, anti-cytoplasmic, anti-nuclear, anti-cardiolipin, anti-DNA, and rheumatoid factor) were performed at 0 and 9 months. Follow-up was extended for three months after the last immunization (month 9).

Humoral response. Antibody response was measured by ELISA as previously described16 using N, R, or C peptide (1 µg/mL) as the antigen. The final reaction was read at 405 nm in a microplate reader (MRX; Dynex Technologies, Inc., Chantilly, VA). Cut-off points were calculated as three standard deviations above the mean absorbance value at 405 nm of sera from healthy volunteers who had never been exposed to malaria. A pool of sera from semi-immune donors from malaria-endemic areas was used as a positive control, and a pool of sera from malaria-naive volunteers from Cali was used as a negative control. The assay to assay variation was ≤ 20%. Parasite recognition by antibodies to CS protein was determined by IFAT using P. vivax sporozoites produced in Anopheles albimanus mosquitoes.15 Briefly, sporozoites were fixed to multispot microscope slides with phosphate-buffered saline (PBS) containing 2% bovine serum albumin and incubated with two-fold serial dilutions of sera starting at 1:100. This reaction was developed with fluorescein-conjugated goat antihuman IgG (heavy plus light chain) (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) diluted at 1:100. Slides were examined under an epifluorescence microscope and antibody titers were determined as the reciprocal of the end point dilution that showed positive fluorescence.

ELIspot assay for single-cell release of interferon-{gamma} (IFN-{gamma}). Peripheral blood mononuclear cells (PBMCs) were separated from whole blood using Ficoll Histopaque density gradients, resuspended in RPMI 1640 medium, and used for enumerating IFN-{gamma}-producing cells by ELIspot assay. The latter was determined using a commercial kit for human IFN-{gamma} (ELIspot; Mabtech AB, Stockholm, Sweden). Briefly, microtiter plate wells (Multiscreen MAHAS 4510; Millipore, Bedford, MA) were coated overnight with 5 µg/mL of anti-human IFN-{gamma} monoclonal antibody (1-D1K; Mabtech AB) at 4°C. Plates were then blocked for two hours at room temperature in RPMI 1640 medium with 10% human AB serum. Fresh PBMCs (4 x 105/well were then mixed with 10 µg/mL of each synthetic peptide and plates were incubated for 40 hours at 37°C in an atmosphere of 5% CO2. After washing with 0.05% PBS-Tween 20, 1 µg/mL of biotinylated anti-IFN-{gamma} monoclonal antibody (7-B6-1; Mabtech AB) was added and incubated for an additional three hours at room temperature. Streptavidine-alkaline phosphatase (Boehringer Mannheim, Mannheim, Germany) diluted 1:1,000 was added and the reaction developed with the substrate 5-bromo-2-chloro-3-indolyl phosphatase/nitroblue tetrazolium) (Sigma, St Louis, MO), leading to the appearance dark blue spots. To determine the specificity of the response of PBMCs from individuals immunized with peptide R, cells collected at month 9 were stimulated with both ptt-30 and p11. The number of spots was counted with a spot-counting system (Scanalytics, Fairfax, VA) and the results were expressed as the mean number of IFN-{gamma} spot-forming cells (SFCs) per 106 PBMCs. Study participants were considered responders when the number of SFCs in their samples had increased from their own baseline level (before immunization on day 0).

Statistical methods. The main outcomes we evaluated were AE and SAE related to vaccination during the study period. An AE was considered related if it was determined to be possible, probable or most probable related to vaccination. Rates of related and other, common AEs for each peptide and dose compared with those seen in placebo groups were determined using Fisher’s test. P values < 0.05 were considered significant. Production of IFN-{gamma} was compared at months 0 and 9 for each dose group of peptide using the Wilcoxon signed-rank test. Antibody titers were compared among individuals from each group and a descriptive analysis was done to evaluate trends in humoral and cellular immune responses in each group of participants.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Study population. The mean age of the study participants was 23 years. Fifty-two (75%) were male and 17 (25%) were female. One participant was excluded before receiving the second immunization because of a visit to a malaria-endemic area; this participant was replaced with a new participant, according to the protocol. Another individual assigned to the 100-µg dose group left the study before receiving the third injection because of pregnancy. Thus, 68 participants completed the three-immunization regimen (Figure 1Go).



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  		    FIGURE 1. Recruitment process in the study.

 
Safety. Study participants experienced no SAE related to the vaccine. One participant developed an SAE not related to the study: a case of appendicitis reported during the safety follow-up period. All laboratory tests results remained within normal values during the study period. The three different doses were well tolerated by all participants. Low grade pain (mild or moderate) at injection site within 48 hours was the most frequent AE related to immunization, followed by local edema, and local erythema (Table 1Go). No differences in signs and symptoms related to the number of doses or the peptide used were observed. Autoimmune antibody test results remained negative after nine months of the first immunization.


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TABLE 1
Number (%) of adverse events
 
Humoral response. All participants immunized with 100 µg of peptide N or R and six volunteers immunized with 100 µg of peptide C seroconverted after the second vaccination, producing specific IgG antibodies to the corresponding peptides (Figure 2Go). At this dose, the response in the individuals immunized with the N peptide was faster and more consistent than the response in the groups immunized with the R or C peptides. At the 30-µg dose, the response started earlier for the N group, but three volunteers required all three doses to seroconvert, whereas in the R group, all participants seroconverted after the second dose. Participants immunized with peptide C at this dose were slower to respond and one of them never seroconverted. Among the groups that received the 10-µg dose, the N peptide group developed a very uniform response in six participants, but one did not respond at all. In contrast, with peptide R, two individuals failed to respond and the remaining participants responded irregularly. None of the individuals given 10 µg of peptide C seroconverted. Eighty-three percent of the 63 participants vaccinated developed antibody responses with higher titers for peptides N and R than for peptide C.



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  		    FIGURE 2. Humoral response to N, R, and C peptides of circumsporozoite protein of Plasmodiium vivax at doses of A, 10 µg, B, 30 µg, and C, 100 µg. Higher titers were obtained in participants immunized with the N peptide. Participants immunized with the N peptide at a dose of 100 µg seroconverted after day 30 post-immunization. Those immunized with peptide C showed a delayed onset and no response when immunized with a dose of 10 µg. Antibody titers were sustained until day 270 among all groups, except in the peptide C group at a dose of 10 µg.

 
Sera of groups displaying the highest ELISA antibody titers specifically recognized P. vivax sporozoites (Table 2Go). The specificity of the antibody response was ascertained by negative results obtained in ELISA and IFAT using sera obtained from the control group and from all volunteers prior to immunization.


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TABLE 2
Recognition of Plasmodium vivax sporozoites by peptide-specific antibodies*
 
Production of IFN-{gamma}. Cells from 94% of the vaccinated (63) showed IFN-{gamma} responses at some point during the study, regardless of the peptide or dose (Table 3Go). All participants immunized with peptide N at any dose produced IFN-{gamma}, with the 100-µg dose group showing a significant difference at month 9 (P = 0.045), compared with participants immunized with the other two peptides (Figure 3Go). Analyses of IFN-{gamma} production at month 9 by individuals immunized with peptide R at the 100-µg dose showed that these individuals responded to both ptt-30 and to p11, with higher responses to the p11 peptide.


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TABLE 3
Frequency of interferon-{gamma} production
 


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  		    FIGURE 3. Comparison of interferon-{gamma} (IFN-{gamma}) production using fresh peripheral blood mononuclear cells (PBMCs) months 0 and 9. Results show the mean values of seven participants per group immunized with three synthetic peptides of the circumsporozoite protein of Plasmodium vivax at different doses. Error bars show the standard deviations (SD) of mean values. *A statistically significant difference was found only in the group immunized with N peptide at a dose of 100 µg. SFCs = spot-forming cells.

 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Vaccination of study participants with P. vivax–derived long synthetic peptides formulated with Montanide ISA 720 appears to be safe and well tolerated. Adverse events related to vaccination with any of the three peptides we studied were no different than those observed in the control groups. Local pain at injection site was the principal AE reported and was described by the participants as similar to that caused by commercially available vaccines. Previous studies with other vaccines formulated in Montanide ISA 720 adjuvant have reported minor effects.28–31 In addition, safety data from our study are comparable with those reported from a trial of P. falciparum CS protein-derived synthetic peptides.30

All three peptide formulations were immunogenic and induced both specific antibodies and IFN-{gamma} production. The antibody response pattern in this study against each one of the N, R or C peptides is consistent with responses of Aotus monkeys immunized with the same peptides.21 Vaccination with 100 µg of peptide was enough to induce seroconversion in all participants and consistently induced the strongest antibody responses. Even with peptide C, which induced only weak responses at 10 µg and 30 µg, immunization with 100 µg of the peptide induced high antibody titers that remained stable for up to three months after the last immunization. This was observed even in the participant who received only two 100-µg vaccine doses.

We found notable that antibody titers in the immunized participants were higher than those observed in people from malaria-endemic areas.16 Moreover, the patterns of antibody response to the three peptides were similar to those obtained in preclinical trials in Aotus monkeys vaccinated with the same peptides formulated in Montanide ISA 720. In these trials, antibody titers in immunized monkeys were significantly higher than those attained in immune donors.21 Peptides N and R produced a stronger specific antibody response than peptide C. The greater antibody production obtained in our study for peptides N and R correlates with the greater recognition of these protein fragments by sera from individuals exposed to natural infection in malaria-endemic areas. These two regions contain specific epitopes (P8 and P11, respectively) predominantly recognized by these individuals, as well as by monkeys immunized with the same peptides.16,21

The vaccine was able to induce a specific T cell-mediated response in most participants, as measured by IFN-{gamma} production. Although no significant differences were found among participants immunized with the different peptides, peptide N induced more uniform responses in all individuals in the different dose groups and it induced greater numbers of specific T cell responses at 100 µg than did peptides R or C. The higher levels of IFN-{gamma} production in cells from individuals vaccinated with peptides N or R could be explained by the presence of known epitopes32 or of T cell epitopes yet to be described. Additionally, in the case of individuals immunized with peptide R, an additive effect was observed between ptt-30 and p11 epitopes, which both contributed to IFN-{gamma} production. The findings described here are in contrast with those obtained in a clinical trial conducted with a P. vivax CS derived recombinant protein in which there was very limited immunogenicity.33

This study confirms that the use of long synthetic peptides represents a promising strategy in the development of new vaccine candidates. Such a strategy allows the inclusion of several relevant epitopes in a single peptide and the rapid assessment of malaria vaccine candidates in their early development phases. Synthetic vaccines may also allow a faster identification of the most accurate adjuvant to induce the strong immune responses required for malaria protection. These advances should contribute to P. vivax vaccine development and hasten the development of multispecies malaria subunit vaccine for use in malaria-endemic areas where both P. falciparum and P. vivax species are present and where parasite selection remains a problem. Given the results obtained with the peptides tested here, we are preparing a phase Ib clinical trial to assess the safety, tolerability, and immunogenicity of a combination of these peptides formulated in Montanide ISA 720, as well as in other adjuvants, before evaluation in a phase II clinical trial.

Received April 12, 2005. Accepted for publication July 7, 2005.

Acknowledgments: We thank the study participants for their invaluable collaboration in the study; Mauricio León, Edna Galindo, Fabián Méndez, and Marisol Badiel for support; Luz Elena García for valuable assistance in the recruitment of volunteers; Antonio José Ramirez for assistance in the administrative management; Vincent Ganne, (Seppic Inc., Paris, France) for supplying the Montanide ISA 720 adjuvant; Luis Rodriguez for peptides synthesis and purification; Gloria Palma (COLCIENCIAS (Bogotá, Colombia) and William Rojas (Corporación de Investigaciones Biológicas, Medellín, Colombia) for participating in the Safety Monitoring Board; Elizabeth T. Robinson (Family Health International, (Research Triangle Park, NC) and Amy Burks for critically reading the manuscript.

Financial support: This work was supported by grants from the Instituto Colombiano Francisco Jose de Caldas para la Ciencia y la Tecnología (COLCIENCIAS), the Health Department of the Valle del Cauca, and the Tropical Medicine Research Center (TMRC-Cali) (National Institute of Allergy and Infectious Diseases contract no. 49486). The UNDP/World Bank/World Health Organization Special Program for Research, and Training in Tropical Diseases provided valuable advice and clinical monitoring. Ricardo Palacios is supported by a studentship grant from the Brazilian Conselho Nacional de Desenvolvimento Ceientifico e Tecnológico – CNPq. Mario Chen-Mok is supported by the National Institutes of Health (NIH contract no. NIH/NO1-AI-05403).

* Address correspondence to Sócrates Herrera, Malaria Vaccine and Drug Development Center, Carrera 35 No. 4A-53, A.A. 25574, Cali, Colombia. E-mail: sherrera{at}inmuno.org Back

Authors’ addresses: Sócrates Herrera, Anilza Bonelo, Blanca Liliana Perlaza, Olga Fernández, Leonardo Victoria, Ana Milena Lenis, Liliana Soto, Hugo Hurtado, Lina Maria Acuña, and Myriam Arévalo-Herrera, Instituto de Inmunología, Universidad del Valle, Calle 4B No. 36-00, Facultad de Salud, A.A. 25574, Cali, Colombia, Telephone: 57-2-558-1931, Fax: 57-2-557-0449, and Malaria Vaccine and Drug Development Center, Carrera 35 No. 4A-53, A.A. 25574, Cali, Colombia, Telephone: 57-2-5583937, Fax: 57-2-5560141, E-mail: sherrera{at}inmuno.org. Juan Diego Vélez, Fundación Clínica Valle del Lili, Autopista Simón Bolivar, Carrera 98 No. 18-48, A.A. 020338, Cali, Colombia, Telephone: 57-2-331-9090. Ricardo Palacios, Division of Infectious Diseases, Federal University of Sao Paulo, Rua Napoleao de Barros, 715, Sao Paulo, CEP 040024-002, Brazil, Telephone: 55-11-55764357, Fax: 55-11-557745071. Mario Chen-Mok, Family Health International, Durham, NC 27713. Giampietro Corradin, Biochemistry Institute, University of Lausanne, 155 Ch. des Boveresses, 1066 Epalinges, Lausanne, Switzerland, Telephone: 41-21-6925701, Fax: 41-21-6925705.

Reprint requests: Sócrates Herrera, Malaria Vaccine and Drug Development Center, Carrera 35 No 4A-53, Cali, Colombia.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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A. Yadava, J. Sattabongkot, M. A. Washington, L. A. Ware, V. Majam, H. Zheng, S. Kumar, and C. F. Ockenhouse
A Novel Chimeric Plasmodium vivax Circumsporozoite Protein Induces Biologically Functional Antibodies That Recognize both VK210 and VK247 Sporozoites
Infect. Immun., March 1, 2007; 75(3): 1177 - 1185.
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