INTRODUCTION
Plasmodium vivax is a human malaria parasite highly prevalent in Asia, Oceania, and the American continents, where it causes approximately 80–90% of the malaria cases.1 The increasing number of malaria cases worldwide that coincide with the failure of classic control measures has given new importance to the identification of additional effective and economically feasible methods that could interrupt transmission in malaria-endemic regions.2 Historically, vaccines have proven to be the most cost-effective measure to control infectious diseases, and during the last two decades several malaria vaccine candidates, mostly targeting pre-erythrocytic or erythrocytic asexual stages, have been tested in animal models and in human clinical trials. However, only a few are targeted at specifically interrupting parasite transmission to the mosquito. These transmission blocking vaccines (TBVs) are considered of greatest value for areas of low malaria transmission such as Colombia where they could prevent the spread of the disease at the household and neighborhood level, as well as preventing the spread of drug-resistant parasites and of escaped mutants from other vaccine candidates.3
Several P. falciparum antigens expressed either in gametocytes (Pfs48 and Pfs230) or in the mosquito parasite forms (Pfs25/28) have been identified and tested for their immunogenicity in animal models.3–6 Recombinant Pfs25/28 and Pf230 proteins formulated in different adjuvants have been shown to induce high specific antibody titers in mice and primates that blocked P. falciparum transmission in mosquito artificial membrane feeding assay (MFA).6–10 Recombinant Pfs48/45 protein was highly immunogenic in mice and rabbits, but expression of this protein in the correct conformation has proven difficult because the elicited antibodies failed to block parasite transmission to mosquitoes.11 Only two paralogous P. vivax antigens, Pvs25 and Pvs28, have been assessed as vaccine candidates.6 Both Pvs25 and Pvs28 produced in recombinant Saccharomyces cerevisiae and formulated with aluminum hydroxide induced strong transmission-blocking antibody responses in immunized mice.12,13 Moreover, Pvs25 co-administered with cholera toxin to mice by the intranasal route induced a strong specific IgG1 antibody response. These antibodies completely blocked oocyst development in mosquitoes fed with P. vivax obtained from infected human patients.14 The first phase I clinical trial using ookinete surface protein of P. vivax, Pvs25 as immunogen has recently been reported. This protein formulated in Alhydrogel was safe and generated transmission-blocking antibodies.15
Since P. vivax cannot be grown in culture, P. vivax gametocytes for the study of TBV must be obtained from infected human patients or susceptible chimpanzees. During the last decade, we have developed in Colombia an infective model with gametocytes from either human patients or experimentally infected Aotus lemurinus griseimembra monkeys and susceptible Anopheles albimanus mosquitoes.16,17 Using artificial MFA, mosquitoes can be fed with blood carrying mature gametocytes and the transmission-blocking activity of sera from human subjects from malaria-endemic areas, as well as from experimentally infected or immunized animals, can be assessed.
We have successfully used this model to address several critical questions relevant to the development of a P. vivax TBV for human use. We have determined whether a recombinant protein produced as a clinical grade product formulated in Montanide ISA-720 an adjuvant suitable for human vaccination trials is capable of inducing transmission-blocking antibodies in a non-human primate. We have also determined whether infection of immunized primates by P. vivax could boost a pre-existing immune response. Although Pvs25 is likely to be expressed primarily in insect stages, it is unclear under conditions of natural infection with circulating sexual stage parasites whether sufficient Pvs25 protein is expressed to boost the host immune response. Such boosting, if it occurs, will have an impact on the type of formulation required for TBV based on Pvs25.
MATERIALS AND METHODS
Plasmodium vivax Pvs25 recombinant protein.
Pvs25 is a protein composed of four cysteine-rich epidermal growth factor–like domains expressed on the surface of zygotes and ookinetes of P. vivax. To produce a recombinant protein, Pvs25 was expressed in S. cerevisiae in a secreted form as described previously.13,14 Briefly, P. vivax genomic DNA from the Salvador I strain was used to amplify the gene fragment encoding the Pvs25 regions (Ala23-Leu195; GenBank accession no. BAC-66003), which was inserted into the yeast episomal plasmid YEpRPEU-3 that encodes a secretory α factor containing a 6-His tail.12 Supernatants of fermentation were recovered by tangential microfiltration, concentrated by ultrafiltration, and extensively dialyzed. The retentate was incubated overnight at 4°C with Ni-nitrilotriacetic acid agarose (Qiagen, Valencia, CA). The suspension was then transferred to a column, washed once with 2× PBS, pH 7.2, once with 2× PBS, pH 6.8, and once with 1× PBS, pH 6.4, and the protein was eluted from the resin by using 0.25 M sodium acetate, pH 4.5. Proteins were purified by size-exclusion chromatography and identity was confirmed by N-terminal sequencing and mass spectroscopy.13 This protein can be produced in S. cerevisiae as different conformers that migrate at different sizes in non-reduced sodium dodecyl sulfate–polycrylamide gel electrophoresis (19 and 29 kD). Recently, a large-scale production of a well-characterized, clinical grade Pvs25 product of 29 kD suitable for clinical use was achieved.13 Here we describe the results obtained with this product in pre-clinical trials conducted in Aotus monkeys.
Aotus monkeys and immunization. Aotus lemurinus griseimembra monkeys from the Primate Center of Universidad del Valle in Cali, Colombia were used. Male and female adult, malaria-naive animals with body weights of 0.8–1.0 kg were selected for the experiments. The animal experimental protocol was previously reviewed and approved by the Animal Ethical Committee of Universidad del Valle and was conducted in compliance with the U.S. National Institutes of Health guidelines. Monkeys were randomly allocated into two groups, an experimental group of six animals (group A) immunized with the recombinant Pvs25 vaccine and a control group of three animals (group B) immunized with adjuvant alone. Both groups were immunized on days 0, 60, and 120. Group A was inoculated with a total volume of 500 μL of vaccine formulated as 100 μg of the Pvs25 recombinant protein in Montanide ISA-720 in a 7:3 antigen:adjuvant ratio as recommended by the adjuvant producer (Seppic Inc., Paris, France). Group B was injected with distilled water containing no protein and mixed in the same adjuvant following the same procedure. The immunization was performed by the subcutaneous route distributed in five different sites of the thorax and abdomen of each animal.
Antibody response.
Plasma fractions were separated by centrifugation of the whole blood samples collected from immunized and control monkeys in tubes containing heparin as anticoagulant before each immunization and after challenge. Plasma samples were kept at −80°C until used for determination of specific antibodies to Pvs25 using an enzyme-linked immunosorbent assay (ELISA) as described elsewhere.13 Briefly, plates (Maxisorp; Nunc Roskilde, Denmark) were coated overnight at 4°C with 100 μL/well of Pvs25 at a concentration of 1 μg/mL in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). Plates were then blocked with 5% skim milk (Difco, Detroit, MI) in Tris-buffered saline (TBS; 60 mM Tris base, 0.15 M NaCl, pH 7.4) for two hours at room temperature. After washing with TBS-Tween (TBS, 0.05% tween -20) two-fold serial dilutions of the corresponding plasma prepared in diluting buffer (TBS, 1% bovine serum albumin, 0.5% Tween 20) were tested in triplicate. After incubation for two hours at room temperature, plates were washed with TBS-Tween (TBS, 0.05% tween -20) and incubated again for two hours with an alkaline phosphatase–conjugated goat anti-human polyvalent immunoglobulin (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) at a dilution of 1:2,000 in diluting buffer. After extensive washing with TBS-Tween (TBS, 0.05% tween -20) the plate was incubated for 30 minutes at room temperature with 100 μL of the substrate p-nitrophenyl phosphate in 1 mg/mL of 10% diethanolamine (Sigma, St. Louis, MO). Absorbance was read at 405 nm in a multi-channel spectrophotometer (SPECTRAmax 340PC; Molecular Device Co., Sunnyvale, CA). Serial dilutions of a standard rabbit anti-Pvs25 sera and rhesus monkey anti-Pvs25 serum were tested in each ELISA plate and assigned unit values as the reciprocal of the dilution giving an optical density at 450 nm of 1. An standard curve was designed and the absorbance of individual test sera was converted to antibody units (SOFTmax PRO version 3; Molecular Device Co.).
Human ethical clearance.
Ethical clearance to draw P. vivax–infected blood from human volunteers was obtained from the Institutional Review Board of Universidad del Valle and the Division of Microbiology and Infectious Diseases International Clinical Studies Review Committee of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. Twenty milliliters of whole blood was drawn from P. vivax–infected patients after written informed consent was provided, and blood was used for mosquito feeding using an artificial MFA. Immediately after blood donation, volunteers were provided with the anti-malarial treatment recommended by the Colombian Health Ministry (chloroquine/primaquine). Patients were informed that they would be treated even if they did not participate in the study.
Transmission-blocking assays.
Monkey plasmas were assayed for their capacity to inhibit oocyst development in artificial MFA using An. albimanus mosquitoes reared at the insectary of the Entomology Unit at the Malaria Vaccine and Drug Development Center in Buenaventura, Colombia. A total of 200 female mosquitoes per batch were fed in each assay using plasma samples with the highest antibody titer (115–4,900 units), collected on day 150 and tested in three independent assays. Monkey plasma samples were diluted 1:4 in non–heat-inactivated human AB serum, added to P. vivax–infected blood obtained from human volunteers, and used as a source of gametocytes. Seven to eight days after the blood meal, all surviving mosquitoes were dissected and their mid-guts were stained with 2% mercurochrome. A rabbit polyclonal antiserum and a mouse monoclonal antibody (1H10 at a concentration of 250 μg/mL) specific for Pvs25 were used as positive transmission-blocking controls. Plasma from monkeys obtained prior to immunization and a pool of AB normal human sera were used as negative controls.
Both the number of mosquitoes with oocysts and the number of oocysts per midgut were scored in all the mosquito lots. Results were expressed as the percentage of oocyst inhibition and were calculated using the formula (Xa/Xc) × 100, where X represents the proportions or the arithmetic means (total oocysts divided by total mosquitoes dissected) in mosquitoes fed with plasma from immunized monkeys (a) and those fed on naive monkey plasma (c). This result was subtracted from 100 to obtain the percentage of inhibition.
Isolation of DNA from clinical P. vivax isolates and Pvs25 gene sequence.
To obtain the sequences of the genes encoding Pvs25 in the clinical isolates used for transmission blocking assays, DNA was extracted from thin blood smears on glass slides using a QIAamp DNA mini-kit (Qiagen) with a slightly modified protocol. Briefly, 180 μL of pre-heated tissue lysis buffer was applied to each glass slide. After treatment with proteinase K solution, DNA was purified by a mini-spin column and 5 μL of resulting solution was used for polymerase chain reaction (PCR) amplification. Primers were designed to amplify the Pvs25 gene as follows: flanking primer Pvs25-F: 5′-CTGACTTTCGTTTCACAGC-3′ and Pvs25-R: 5′-TCGGTAAGTTCAGTAAAGAA-3′. The PCR products were directly sequenced by using BigDye terminator chemistry on an ABI3730XL sequencer (Applied Biosystems, Foster City, CA) as described previously.18
Boosting of anti-Pvs25 with infection.
To address the question whether P. vivax blood infection is able to induce boosting in the anti-Pvs25 antibody response, approximately 10 months after the last immunization (day 440) when specific antibodies to Pvs25 are no longer detected by ELISA, all monkeys were challenged with the P. vivax Salvador I strain by intravenous injection of 105 parasitized red blood cells. Parasites kindly provided by had been maintained in liquid nitrogen until they were used to infect an Aotus monkey that was further used as a parasite donor for the challenge. Total parasitemia and gametocytemia were followed every other day using thick and thin blood smears stained with Giemsa. Parasite concentrations were expressed as the number of gametocytes per microliter and the percentage of red blood cells parasitized by asexual parasite forms.19 Monkeys were bled post-challenge (days 447–503) to evaluate the presence of antibodies to Pvs25 by ELISA. In addition, the infectivity of circulating gametocytes was tested by feeding of An. albimanus mosquitoes with parasitized monkey red blood cells mixed with normal AB human sera using the MFA on days 460 as described earlier in this report. After challenge, if animals developed a hematocrit level below 25%, they were treated with a combination of sulfadoxine-pyrimethamine.20
RESULTS
Antibody responses to Pvs25 immunization.
Clinical grade Pvs25 was formulated with Montanide ISA-720 and used to vaccinate a group of six Aotus monkeys. Antigen-specific antibody responses to the Pvs25 protein as determined by ELISA were evident by day 30 after the first immunization at low levels (61–478 units of anti-Pvs25) (Table 1). By day 60, at the time of the first boosting dose, responses of most animals were similar and by day 90, antibodies were boosted in all but two animals (527*781 and 532*778). Only one monkey had an apparent boost with the third antigen injection given on day 120. All animals had maximum antibody levels by day 150. These levels started to decrease by day 180, but were still detectable 10 months after the first immunization (Table 1).
Outcome of parasite challenge on antibodies to Pv25 elicited by immunization.
To determine whether an active P. vivax infection could boost the pre-existing humoral immune responses to Pvs25, monkeys were challenged on day 440 of the study with P. vivax–infected erythrocytes. All monkeys developed patent parasitemia by day 453 of the study, approximately two weeks after intravenous challenge. The peak of parasitemia for most of the monkeys was observed between days 462 and 464 with parasitemias and ranged from 0.1% to 1.3% as determined by thin blood smear. All animals became negative on thick blood smears by day 470. Gametocytes were first evident between days 458 and 460 and remained at detectable levels in all animals until day 468. All monkeys were treated with curative doses of sulfadoxine-pyrimethamine on day 485 (Figure 1). Plasma samples obtained on days 447, 462, 482, and 503 after parasite challenge were negative for antibodies directed to the Pvs25 recombinant protein by ELISA. However, antibodies against parasite blood forms were evident by an immunofluorescent antibody test at low titers (1:400–1:800) on day 481 in both experimental and control animals. Gametocytes that developed in both groups were infectious to mosquitoes as determined in an MFA conducted with monkey blood drawn on day 460 in which plasma from AB human control sera was replaced by sera from infected monkeys (Table 2). This result supports the viability and functionality of the circulating gametocytes from both the Pvs25-immunized and the control animals.
Transmission-blocking activity of monkey plasma.
Monkey plasmas collected one month after the last immunization (day 150), were assessed for their ability to inhibit oocyst development (Table 3). Mosquitoes fed with P. vivax gametocyte-carrying human blood in the presence of either normal monkey plasma or normal AB human sera (negative controls) produced positive infections with an arithmetic mean of oocysts per midgut ranging between 0.3 and 3.8 and 0.2 and 1.0 oocysts, respectively. Within the range of plasma samples tested, transmission-blocking activity was independent of antibody titers to Pvs25 (115–4,900 units). Polyclonal rabbit anti-Pvs25 antisera completely blocked the development of infection in the mosquito midgut, except in one experiment in which 2 of 45 mosquitoes fed with rabbit anti-Pvs25 were positive (assay 3). Plasma from the Pvs25-immunized Aotus tested individually were highly inhibitory and completely blocked the development of oocysts, in all assays (reduction of the oocysts number > 98%) using three different P. vivax human isolates. Plasma from monkeys in the Montanide ISA-720 control group showed similar inhibition to the normal monkey plasma (negative control) (Table 3). The sequence of Pvs25 from all P. vivax isolated used was identical to the P. vivax Sal I sequence in the database (GenBank accession no. AF083502).
DISCUSSION
The present study of the immunogenicity of a P. vivax transmission-blocking vaccine candidate addresses several is- sues important to the development of Pvs25 as a vaccine for human use. To make this study as relevant as possible, well-characterized clinical grade protein was used as the immunogen, and an adjuvant (Montanide ISA-720) was selected that is currently being used in a number of human vaccine trials.21,22
An important finding is that antibodies to Pvs25 were rapidly produced in Aotus monkeys after the first vaccination with Pvs25 and were still detectable in these animals for 180 days after the last booster immunization. When plasma from the immunized animals was tested for its capacity to block parasite transmission in a mosquito artificial MFA, plasma showed complete blocking of transmission in all assays, establishing the functionality of the elicited immune response.
One of the most important questions addressed by our studies is the possibility of boosting of immune responses to mosquito-stage antigens such as Pvs25. The timing of parasite expression of the Pvs25 protein has not been studied in detail, but there have been several studies of the orthologous protein from other species of Plasmodium. Generally, the protein is only found after zygotes formation within the mosquito (i.e., 0.5–2 hours after the blood meal).13,23,24 However, there has been one report of low level synthesis of Pfs25 in immature P. falciparum gametocytes in culture,25 and at least in P. berghei, high levels of P25 mRNA are present in gametocytes.24,26 Thus, there is a potential for low levels of expression in the vertebrate host that could provide a source of antigen for boosting of B cell memory. Since Pvs25 and its homolog in P. falciparum Pfs25 are major candidates for malaria transmission blocking vaccines and are in or close to clinical trials, the issue of boosting of adaptive immune responses by natural infection has significant practical importance. In our studies, Aotus monkeys whose antibody level to Pvs25 had decreased to non-detectable levels were infected with erythrocytic stages of the P. vivax Sal I strain. These animals developed normal infection profiles with production of gametocytes that were infectious to mosquitoes. However, such exposure of the animals to blood-stage and sexual-stage parasites did not boost their anti-Pvs25 titers, suggesting that this protein is not expressed in sufficient quantity in circulating asexual or sexual parasites to boost memory B cells. Therefore, the vaccine itself must induce the transmission-blocking immunity that is needed for vaccine efficacy without relying on booster immunizations during infections.
The main conclusion from this study is that boosting of antibodies to Pvs25 is not caused by the parasite infection. Thus, TBV strategies with Pvs25 must induce persistent antibodies that have transmission-blocking activity. The water-in-oil adjuvant used in the present study is a first step in inducing such activity.
Enzyme-linked immunosorbent assay antibody titers to Pvs25 in Aotus monkeys immunized with rPvs25
| Day of follow-up* | Day of challenge† | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Monkey code | 0 | 30* | 60* | 90 | 120* | 150 | 180 | 300 | 400 | 440 |
| * Day of immunization. | ||||||||||
| † Day of infection with 105 Plasmodium vivax Sal I parasites. | ||||||||||
| 327*544 | 0 | 459 | 388 | 487 | 1,058 | 3,731 | 660 | 581 | 217 | 345 |
| 298*882 | 0 | 274 | 195 | 607 | 267 | 4,866 | 473 | 245 | 102 | 131 |
| 099*382 | 0 | 61 | 305 | 866 | 439 | 4,415 | 204 | 114 | 121 | 84 |
| 532*778 | 0 | 253 | 152 | 208 | 41 | 685 | 100 | 68 | 17 | 115 |
| 528*097 | 0 | 211 | 213 | 376 | 292 | 1,185 | 124 | 80 | 54 | 60 |
| 527*781 | 0 | 174 | 206 | 181 | 43 | 115 | 56 | 24 | 37 | 69 |
Infectivity of mosquitoes fed with gametocytes from monkeys infected with Plasmodium vivax Sal I by artificial membrane feeding assays
| Monkey code | No. positive (total dissected)* | Average number of oocysts† | Gametocytes (μL)‡ |
|---|---|---|---|
| * Number of mosquitoes infected (total number of mosquitoes dissected). | |||
| † Average calculated as the total number of oocysts/total number of mosquitoes dissected. | |||
| ‡ Gametocytes per microliter of blood at day 460. | |||
| § Immunized monkeys. | |||
| ¶ Control monkeys. | |||
| 298*882§ | 3 (30) | 0.17 | 93 |
| 099*328§ | 13 (70) | 0.58 | 140 |
| V-123¶ | 2 (47) | 0.04 | 467 |
| 360*308¶ | 22 (62) | 1.35 | 140 |
Transmission-blocking activity of monkey plasma samples from day 150 in mosquitoes fed with Plasmodium vivax human isolates*
| Immunized group | Monkey | Anti-Pvs25 antibody units† | Assay number‡ | No. positive (total dissected)§ | Average number of oocytes¶ | % oocyst inhibition# |
|---|---|---|---|---|---|---|
| * ND = not determined; NA = not applicable; ELISA = enzyme-linked immunosorbent assay. | ||||||
| † Anti-Pvs25 antibody unit was determined by an ELISA using a standard assay where 1 unit is an optical density of 1. | ||||||
| ‡ Number of assay per sample using different P. vivax–infected human blood. | ||||||
| § Number of mosquitoes infected (total number of mosquitoes dissected). | ||||||
| ¶ Average calculated as total number of oocysts/total number of mosquitoes dissected. | ||||||
| # Percent inhibition of mean oocyst compared to normal monkey plasma (negative control) in each independent assay (1 − [mean oocysts per mosquito in normal monkey plasma/mean oocysts per mosquito in immunized monkey plasma] × 100. | ||||||
| rPv25 + Montanide ISA-720 | 327*544 | 3,750 | 1 | 0 (45) | 0 | 100 |
| 2 | 0 (46) | 0 | 100 | |||
| 3 | 0 (26) | 0 | 100 | |||
| 298*882 | 4,900 | 1 | 0 (56) | 0 | 100 | |
| 2 | 0 (64) | 0 | 100 | |||
| 3 | 0 (58) | 0 | 100 | |||
| 099*382 | 4,415 | 1 | 1 (53) | 0.02 | 99 | |
| 2 | 0 (72) | 0 | 100 | |||
| 3 | 0 (50) | 0 | 100 | |||
| 532*778 | 685 | 1 | 0 (52) | 0 | 100 | |
| 2 | 0 (80) | 0 | 100 | |||
| 3 | 0 (32) | 0 | 100 | |||
| 528*097 | 1,196 | 1 | 2 (47) | 0.04 | 98 | |
| 2 | 1 (42) | 0.02 | 99 | |||
| 3 | 0 (70) | 0 | 100 | |||
| 527*781 | 115 | 1 | 0 (48) | 0 | 100 | |
| 2 | 0 (55) | 0 | 100 | |||
| 3 | 0 (46) | 0 | 100 | |||
| Montanide ISA-720 | V136 | 0 | 1 | ND | ND | ND |
| 2 | 2 (44) | 0.11 | 97 | |||
| 3 | 2 (28) | 0.18 | −100 | |||
| V123 | 0 | 1 | 1 (45) | 1.0 | 52 | |
| 2 | ND | ND | ND | |||
| 3 | 7 (23) | 2.0 | −567 | |||
| 360*308 | 0 | 1 | ND | ND | ND | |
| 2 | 3 (44) | 0.18 | 95 | |||
| 3 | 15 (22) | 0.04 | 87 | |||
| Negative control | Normal monkey plasma | 1 | 95 (235) | 2.1 | NA | |
| 2 | 56 (119) | 3.8 | NA | |||
| 3 | 24 (328) | 0.3 | NA | |||
| Negative control | AB human serum | 1 | 23 (102) | 0.5 | NA | |
| 2 | 18 (44) | 1.0 | NA | |||
| 3 | 7 (69) | 0.2 | NA | |||
| Positive control | Rabbit anti-Pvs25 | 1:400 | 1 | 0 (57) | 0 | 100 |
| 2 | ND | ND | ND | |||
| 3 | 2 (45) | 0.1 | 72 | |||
| Positive control | Monoclonal anti-Pvs25 | 250 μg/mL | 1 | 0 (45) | 0 | 100 |
| 2 | 0 (44) | 0 | 100 | |||
| 3 | 0 (22) | 0 | 100 | |||
Parasitemia and gametocytemia in monkeys infected on day 440 with the Plasmodium vivax Sal I strain. Arrows mark the day of infection (day 440) with 105 P. vivax Sal I parasites. Patent parasitemia appeared on day 453 after intravenous inoculation. Gametocytes first appeared on days 458–460. Animals were treated with sulfadoxine-pyrimethamine on day 485.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 5_suppl; 10.4269/ajtmh.2005.73.32
Parasitemia and gametocytemia in monkeys infected on day 440 with the Plasmodium vivax Sal I strain. Arrows mark the day of infection (day 440) with 105 P. vivax Sal I parasites. Patent parasitemia appeared on day 453 after intravenous inoculation. Gametocytes first appeared on days 458–460. Animals were treated with sulfadoxine-pyrimethamine on day 485.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 5_suppl; 10.4269/ajtmh.2005.73.32
Parasitemia and gametocytemia in monkeys infected on day 440 with the Plasmodium vivax Sal I strain. Arrows mark the day of infection (day 440) with 105 P. vivax Sal I parasites. Patent parasitemia appeared on day 453 after intravenous inoculation. Gametocytes first appeared on days 458–460. Animals were treated with sulfadoxine-pyrimethamine on day 485.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 5_suppl; 10.4269/ajtmh.2005.73.32
Address correspondence to Myriam Arévalo-Herrera, Malaria Vaccine and Drug Development Center, Carrera 35 No 4A-53, AA 26020, Cali, Colombia. E-mail: marevalo@inmuno.org
Authors’ addresses: Myriam Arévalo-Herrera, Yezid Solarte, María Fernanda Yasnot, Angélica Castellanos, Adriana Rincón and Sócrates Herrera, Instituto de Inmunología, Edificio de Microbiología, Tercer Piso, Facultad de Salud, Universidad del Valle, Sede San Fernando, AA 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, AA 26020, Cali, Colombia, Telephone: 57-2-558-3937, Fax: 57-2-556-0141, E-mail: marevalo@inmuno.org. Allan Saul, Carole Long, and Louis Miller, Malaria Vaccine Development Branch, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20850, Telephone: 301-594-2701, Fax: 301-435-6725. Jianbing Mu, Laboratory of Malaria and Vector Research, National Institutes of Health, Bethesda, MD 20850, Telephone: 301-594-2701 Fax: 301-435-6725.
Acknowledgments: We thank G. Quintero (Primate Center of Universidad del Valle) and Z. Castillo and C. Prieto (Entomology Unit, Malaria Vaccine and Drug Development Center) for technical assistance during these experiments. We are grateful to the patients that provided gametocyte-carrying samples and to Dr. W. Collins (Centers for Disease Control, Atlanta, GA) for providing the P. vivax Sal I strain. María Fernanda Yasnot was recipient of a fellowship from the Colombian Research Council.
Financial support: This work was supported by the National Institute of Allergy and Infectious Diseases through Tropical Medicine Research Centers grant no. 49486 and by the World Health/Tropical Diseases Research Special Program (RCS contract no. MVDC 991006).
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