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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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Shi and others59 reported on the construction of a multistage P. falciparum candidate vaccine believed to have potential for further development. The 41-kDa antigen originally referred to as CDC/NII MAL VAC-1 as modified here is referred to as FALVAC-1. Small animal studies indicated that the construct could induce a strong immune response; in vitro studies indicated that these antibodies inhibited the growth of blood-stage parasites in the presence of monocytes.
An immunization and challenge trial was designed to determine the protective immune response in A. nancymai monkeys immunized with the FALVAC-1 antigen in combination with various adjuvants; some of these adjuvants are believed to have potential for use in humans. At this preliminary stage in the developmental process, the standard for protection was those animals immunized with FALVAC-1 combined with Freund’s adjuvant. No attempts were made to include adjuvant control groups or to select immunization dosages or regimens. At the end of the immunization period, all animals were challenged with the FVO strain of P. falciparum to determine if one or more of the antigen/adjuvant combinations would provide protection comparable to that of animals immunized with FALVAC-1 combined with Freund’s adjuvant.
Here, we report the results of this preliminary study to test the ability of FALVAC-1 to induce protective immunity in A. nancymai monkeys. FALVAC-1 was combined with 1) QS-21 an adjuvant active saponin obtained from Quillaya saponaria, 2) ASO2a, 3) the nonionic block copolymer CRL1005 in saline, 4) CRL1005 in oil, 5) alum, and Freund’s complete (FCA) and Freund’s incomplete (FIA) adjuvants.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vaccine formulation. FALVAC-1 vaccine alone was prepared by diluting the antigen in PBS (pH 7.4; Gibco, Grand Island, NY) to a final concentration of 100 µg/dose and was administered in a final volume of 500 µL/monkey.
For vaccine containing Freund’s adjuvants (Sigma, St. Louis, MO), the antigen in PBS (pH 7.4) and the adjuvant were emulsified by connecting two separate syringes with a Luer fitting, and passing the contents back and forth for approximately 15 minutes. The stability of the emulsion was tested using the water-drop method. The first dose was given in Freund’s complete adjuvant (FCA); subsequent dose was given in Freund’s incomplete adjuvant (FIA).
The FALVAC-1 with alum formulation was prepared by the procedure developed by de Oliveira and others,62 with slight modifications. Briefly, aluminum hydroxide (2% rehsorptar, Intergen, Purchase, NY) was washed in acetate buffer (0.1 M, pH 6.2 with 0.15 M NaCl) and kept overnight at room temperature. The washed aluminum gel (1.2 mg) was then mixed with FALVAC-1 antigen (100 µg) at a ratio of 1:6 in acetate buffer (0.02 M, pH 6.2 with 0.15 M NaCl) and incubated at room temperature for three hours with gentle rotation. After the alum adsorption, the alum/FALVAC-1 mix was centrifuged at 3,500 rpm for 20 minutes and then resuspended in PBS (500 µL/monkey) for immunization. The supernatant was tested for the residual concentration using a spectrophotometer; it was found that all of the vaccine had been adsorbed to the alum.
FALVAC-1 with QS-21 (Antigenics, Inc., Lexington, MA) formulation was prepared by thoroughly mixing QS-21 with FALVAC-1 in PBS for a final QS-21 dose of 50 µg per monkey.
FALVAC-1 with ASO2a adjuvant formulation was prepared by mixing 287 µL of ASO2a (Glaxo Smith Kline, Rixensart, Belgium) with 426 µL of antigen (100 µg) in PBS. This preparation was thoroughly mixed by vortexing for 10 seconds and incubating for 10 minutes on a shaker at room temperature. Before injection, it was gently vortexed.
The copolymer formulation in PBS was prepared by thoroughly mixing copolymer CRL100563–65 (a gift from CytRx Corp., Norcross, GA) with FALVAC-1 in PBS to give a final dose of 5 mg of copolymer per monkey.
The copolymer water-in-oil emulsion was prepared by mixing a 20% oil phase [10% sorbitan monooleate (Span 80) in squalene] and an 80% aqueous phase that contained the vaccine and the copolymer CRL1005 to give a final dose of 5 mg of polymer per monkey. The aqueous and oil phases were placed in separate syringes and emulsified using a Luer fitting where the contents were passed back and forth for 15 minutes or until a viscous white emulsion was formed.
PBS (pH 7.4; Gibco) was used to inject control monkeys (500 µL/monkey).
All vaccines were drawn into individual syringes for injection into monkeys.
Animals. Fifty-four A. nancymai (37 female and 17 male) monkeys were included in the vaccine trial. Twenty-two monkeys were laboratory-born (7 female and 15 male); 32 were feral animals imported from Peru (30 female and 2 male). All feral animals were quarantined upon arrival at the facility for at least 1 month, weighed, and tested by skin test to be free of tuberculosis. Parasitologic and serologic examination indicated that the animals were free of plasmodial infection. Protocols were reviewed and approved by the Centers for Disease Control and Prevention Institutional Animal Care and Use Committee, in accordance with procedures described in the U.S. Public Health Service Policy, 1986. Before immunization, and continuing throughout the trial, daily observations of the animals’ attitude, appetite, stool, and condition were recorded. Monkeys were weighed weekly. All were treated as medical conditions arose by the resident clinical veterinarian. All observations, parasite counts, and the results of laboratory tests were recorded on a daily basis and entered into a computerized database.
Animals were housed singly or doubly to avoid injuries caused by fighting with cage mates. Space recommendations for laboratory animals were followed as set forth in the Guide for the Care and Use of Laboratory Animals, NIH. All monkeys were fed a diet that has been shown to provide adequate nutrition and calories in captive Aotus used in malaria related research. Feed was freshly prepared. Animals were handled as little as possible. All procedures involving the animals were under the direction of the resident clinical veterinarian.
Recorded observations on local and/or systemic reactions (e.g., lymphadenopathy, cellulitis, abscesses, necrotizing lesions, arthritis, anorexia, and weight loss) to the candidate vaccines were made minimally once a week at the time of blood collection. In the event of a reaction, additional observations were made daily, and supportive treatment instituted.
Preparation of challenge inoculum. Before this trial, studies had shown that the Vietnam Oak Knoll (FVO) strain of P. falciparum would be expected to induce a high-density, life-threatening parasitemia following the inoculation of 10,000 parasitized erythrocytes. A frozen sample was used to induce a high-density parasitemia in a donor A. nancymai monkey. Blood was drawn via venipuncture, diluted in RPMI 1640 culture medium, and 10,000 parasitized erythrocytes passaged into each of the 53 monkeys remaining in the immunization trial. For rechallenge, a donor animal was again infected with parasites after being stored frozen; blood was drawn, diluted in RPMI 1640, and 50,000 parasitized erythrocytes injected intravenously into each of the remaining test animals.
Experimental protocol. A schematic of the trial: 1) immunization at 0 and 8 weeks, 2) first challenge at week 16, 3) treatment of all animals by week 24. Animals were assigned to 8 groups, taking into account sex and weight, using a table of random numbers. These groups received the following vaccinations. Group 1: 100 µg FALVAC-1 in PBS at weeks 0 and 8. Group 2: 100 µg FALVAC-1 combined with Freund’s complete adjuvant at week 0; a second immunization with 100 µg FALVAC-1 combined with Freund’s incomplete adjuvant at week 8. Group 3: 100 µg FALVAC-1 with alum at weeks 0 and 8. Group 4: 100 µg FALVAC-1 combined with QS-21 at weeks 0 and 8. Group 5: 100 µg FALVAC-1 combined with ASO2a at weeks 0 and 8. Group 6: 100 µg FALVAC-1 combined with CRL1005 in saline at weeks 0 and 8. Group 7: 100 µg FALVAC-1 combined with CRL1005-1 in oil at weeks 0 and 8. Group 8: phosphate-buffered saline (PBS) at week 0 and 8 (PBS control).
Four criteria of protection were specified in the approved protocol: 1) decrease in the initial growth rate of parasites; 2) increase in the time to reach a level requiring treatment (> 200,000/µL); 3) an increase in the proportion not requiring treatment; 4) decrease in the maximum parasite count in the animals not requiring treatment. Note that these criteria applied to the experimental groups, not individual animals.
Immunization schedule. Animals were given 2 immunizations at 8-week intervals (also identified as Days 0 and 56). Day 112 (Week 16) was the day of the first challenge. Animals receiving FCA followed by FIA were immunized subcutaneously in 4 sites in the back to facilitate drainage if an ulcer developed. All other animals were immunized intramuscularly in the quadriceps muscles of the hind legs. The injection sites were shaved to allow visual monitoring for any adverse reactions. Animals were examined weekly, and reactions at the site of immunization were graded 1+ (erythema), 2+ (erythema or induration), 3+ (erythema and induration), or 4+ (ulceration).
The trial was conducted in a blind format. Persons responsible for the reading of blood smears, and making parasite count determinations, and the persons responsible for collection of specimens, and physical examinations did not know the experimental group to which the monkeys had been assigned. To achieve and maintain this level of masking, a person having none of the responsibilities listed above prepared a code book listing each animal by its tattooed identification number, its group assignment, and the corresponding treatment group to which each animal had been assigned. The same person was responsible for directing the administration of the immunizations.
Challenge. Eight weeks after the last immunization (Day 112–Week 16), each animal was injected with 10,000 parasitized erythrocytes of the FVO strain of P. falciparum taken from a donor animal, diluted in RPMI 1640 culture medium, and injected intravenously into the femoral vein of each animal.
Rechallenge. After termination of primary infection, animals were rechallenged with the homologous parasite to determine if there was persistence of immunity. For the rechallenge (Day 238–Week 34), each animal was injected with 50,000 erythrocytes parasitized with the same strain of P. falciparum from an infected donor animal.
Parasite monitoring. After challenge, thick and thin blood films were collected daily and stained with Giemsa by the method of Earle and Perez.66 Parasite counts were recorded per microliter of blood. After the parasitized erythrocytic challenge, blood films were made and examined from days 1 through 56, unless the animal had been previously cured of its infection. After the second challenge, blood films were examined from Days 239 to 280 (42 days after rechallenge).
When parasite counts exceeded 200,000/µL (approximately 5% of the erythrocytes infected), animals were treated orally with 20 mg mefloquine and 50 mg of quinine, which cured their infections. Fifty-six days after infection or 42 days after reinfection, all untreated infections in animals remaining in the trial were cured by treatment with 20 mg mefloquine. A parasite count of 
200,000/µL was selected for treatment of several reasons. Death of a primate can not be an end point in any of our vaccine trials. Our previous experience with infections with P. falciparum in New World monkeys has been that some animals may die if their parasitemia is allowed to exceed 10%. A parasite count of 200,000/µL (approximately 5% of erythrocytes infected) was considered an acceptable level that would allow most animals to survive if treated adequately.
Hematology. Biweekly collections of < 10% of each monkey’s estimated total blood volume (based on a total blood volume calculation of 40 mL blood per kilogram body weight), were made via venipuncture from the femoral vein. A complete blood count was done: 1) erythrocytes/µL; 2) leukocytes/µL; 3) hematocrit; 4) hemoglobin concentration; 5) platelets/µL; 6) mean corpuscular hemoglobin; 7) mean corpuscular volume; and 8) mean corpuscular hemoglobin concentration. The remaining blood was centrifuged and the plasma stored frozen for serologic studies.
Statistical analysis.
Kaplan-Meier product moment estimators were used to estimate survivor functions describing the first day the parasite count was 100, initial treatment day, and first day negative. The Wilcoxon test was used to test for differences in survivor factor functions between each treatment group. Adjuvant differences in parasite counts were tested by Tukey’s HSD multiple comparison test. Geometric means were computed by taking the log of each parasite count, computing the mean of the log values, then expotentiating the log mean back to the original scale. Statistical significance was set at alpha = 0.05. SAS version 8.2 was used to analyze the data.
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RESULTS |
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; Figures 1 and 2). Prepatent periods were 7, 7, 6, 6, 6, 6, and 6 days. All monkeys had maximum parasite counts 200,000/µL and were treated on Days 14, 17, 12, 11, 12, 12, and 12, respectively.
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; Figures 1 and 2). Prepatent periods were 7, 7, 5, 7, 7, and 7 days. Three monkeys (T-0517, T-0767, and AI-1753) had maximum parasite counts 200,000/µL and were treated on Days 17, 19, and 14, respectively. Three monkeys (T-0708, T-0444, and AI-1740) had maximum parasite counts of 127,624, 1,530, and 108,000/µL, respectively. Monkey T-0708 died on Day 14; monkey AI-1740 was treated on Day 22, and T-0444 was treated on Day 54.
FALVAC-1 with alum adjuvant, Group 3: Seven monkeys (T-0811, AI-1763, T-0510, AI-1747, T-0483, AI-1738, and T-0815) were immunized and challenged (Table 1; Figures 1 and 2). Prepatent periods were 6, 7, 6, 6, 7, 7, and 6 days. All monkeys developed maximum parasite counts 200,000/µL and were treated on Days 14, 13, 12, 11, 12, 12, and 11, respectively.
FALVAC-1 with QS-21 adjuvant, Group 4: Seven monkeys (T-0822, T-0827, T-0828, T-0755, AI-1765, T-0818, and AI-1748) were immunized with FAL/VAC with QS-21 (Table 1; Figures 1 and 2). Prepatent periods were 6, 7, 6, 6, 6, and 7 days, respectively. Monkey T-0822 had a maximum parasite count of 380,000/µL on Day 12 and died. The remaining 6 monkeys had maximum parasite counts 200,000/µL on Days 12, 18, 12, 11, 11, and 12, and were treated.
FALVAC-1 with ASO2a adjuvant, Group 5: Seven monkeys (T-1004, T-1760, T-0207, T-0527, AI-1754, T-0826, and T-0543) were immunized and challenged. Prepatent periods were 6, 7, 7, 7, 7, 7, and 5 days, respectively (Table 1; Figures 1 and 2). All monkeys had maximum parasite counts 200,000/µL and were treated on Days 12, 15, 14, 12, 11, 13, and 11, respectively.
FALVAC-1 with CRL1005 adjuvant in saline, Group 6: Seven animals (AI-1752, T-0499, T-0800, AI-1741, T-0763, T-0533, and T-0768) were immunized and challenged (Table 1; Figures 1 and 2). Prepatent periods were 6, 6, 6, 9, 7, 7, and 6 days. All monkeys developed parasite counts 200,000/µL and were treated on Days 18, 12, 12, 12, 18, 11, and 12, respectively.
FALVAC-1 with CRL1005 adjuvant with oil, Group 7: Seven monkeys (T-0474, AI-1756, AI-1743, AI-1749, AI-1739, T-0694, and AI-1755) were immunized and challenged (Table 1; Figures 1 and 2). Prepatent periods were 5, 6, 6, 6, 7, 7, and 7 days. Monkey AI-1756 had a maximum parasite count of 136,000/µL on Day 14 and was treated on Day 56. The other animals all had maximum parasite counts 200,000/µL and were treated on Days 14, 12, 12, 12, 12, and 11, respectively.
PBS control, Group 8: Five monkeys (T-0805, T-1052, AI-1757, T-0762, and T-0698 were immunized with saline (PBS control) (Table 1; Figures 1 and 2). For some undetermined reason, the rise in parasitemia in monkey T-0805 was abnormally delayed. The parasite count eventually reached 244,000/µL and the animal was treated. Because of this, it was not considered in the analysis. Prepatent periods for the 4 remaining animals were 7, 6, 7, and 7 days, respectively. All of the animals developed parasite counts > 200,000/µL and were treated on Days 11, 12, 12, and 13, respectively, with quinine and mefloquine.
Comparison of different measures of efficacy. Initial growth rate. Four measures of initial growth rate were examined: 1) Prepatent period, day on which the parasitemia first exceeded 100/µL, 2) parasitemia on Day 11 (the day the first monkey was treated), 3) cumulative parasitemia up to and including Day 11, and 4) day to required treatment. The pre-patent period was relatively short with 3 of 53 monkeys patent on Day 5, 28 on Day 6, 20 on Day 7, and 1 on Day 9. The average time to > 100/µL for animals immunized with various combinations was Day 7.4 with 40 of the 52 being Day 7.
Inspection of the daily parasitemia figures indicated that there was some initial synchrony, with parasite density decreasing on every other day. Thus, 1) a plot of log(parasitemia) on the day when the first animals required treatment, and 2) the log(cumulative parasitemia) was used in the comparison of the different immunization groups. The groups of monkeys can only be compared by this method up to the time of the first treatment on Day 11. An examination of the parasite counts on Day 11 (Table 1; Figure 2) indicated that 15 of the 48 immunized monkeys had parasite counts < 50,000/µL: 2 of 7 in Group 1; 6 of 6 in Group 2; 2 of 7 in Group 3; 1 of 7 in Group 4; 3 of 7 in Group 5; 1 of 7 in Group 6; and 0 of 7 in Group 7.
We chose to examine log(cumulative parasitemia), as this is less affected by the 48 hour periodicity in some monkeys. Figure 2 shows the cumulative parasitemia to Day 11, plotted on a log scale for the monkeys in each group, together with the geometric mean cumulative parasitemia. Nineteen of the 48 immunized animals had accumulated parasitemias through Day 11 < 100,000/µL (Figure 2). There were 2 of 7 in Group 1; 6 of 6 in Group 2; 2 of 7 in Group 3; 2 of 7 in Group 4, 3 of 7 in Group 5, 3 of 7 in Group 6, and 1 of 7 in Group 7. It was readily apparent that those animals immunized with FALVAC-1 with the Freund’s adjuvants were better protected when compared with the other immunized groups of monkeys.
The results suggested that these groups contained responder and non-responder animals with respect to decreased initial parasite growth.
Proportion of animals requiring treatment. With the exception of Group 2 (FALVAC-1 plus Freund’s adjuvants), only monkey AI-1756 from Group 7 had a maximum parasite count < 200,000/µL after challenge and did not require treatment.
Parasitemia after rechallenge.
FALVAC-1 alone, Group 1: Six remaining monkeys (T-0683 had died) were rechallenged; prepatent periods ranged from 9 to 12 days (Table 2). Monkey T-0484 developed a maximum parasite count of 14,310/µL on Day 18 and died on Day 20. The remaining animals had maximum parasite counts from 4,454 to 180,000/µL and were treated 42 days after rechallenge.
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FALVAC-1 with alum adjuvant, Group 3: All 7 animals were rechallenged. Prepatent periods ranged from 6 to 14 days. Maximum parasite counts ranged from 0 to 140,000/µL. All infections were treated 42 days after rechallenge.
FALVAC-1 with QS-21 adjuvant, Group 4: Five monkeys were rechallenged with the FVO strain of P. falciparum. Pre-patent periods ranged from 9 to 14 days. Monkey AI-1848 had a maximum parasite count of 244,000/µL on Day 23 and was treated. The remaining monkeys had maximum parasite counts from 720 to 47,160/µL.
FALVAC-1 with ASO2a adjuvant, Group 5: All 7 monkeys were rechallenged. Monkey T-1004 failed to develop detectable parasitemia. Prepatent periods ranged from 9 to 14 days. Monkey T-0543 developed a maximum parasite count of 1,120,000/µL on Day 20 and died. The remaining animals had maximum parasite counts ranging from 150 to 38,160/µL, and were treated 42 days after reinfection.
FALVAC-1 with CRL1005 adjuvant in saline, Group 6: Four of the 5 remaining animals were rechallenged. Maximum parasite counts ranged from 1,260 to 25,740/µL, and monkeys were treated 42 days after rechallenge.
FALVAC-1 with CRL1005 adjuvant with oil, Group 7: Six of the 7 remaining animals were rechallenged (monkey T-0474 was not because of poor health). Monkey T-1756 failed to develop detectable parasitemia. Prepatent periods ranged from 10 to 12 days. Maximum parasite counts ranged from 0 to 27,000/µL.
PBS control, Group 8: Three of the 5 monkeys were re-challenged with the homologous FVO strain of P. falciparum. Maximum parasite counts for AI-1757, T-0762, and T-0698 were 17,640, 25,200, and 65,240/µL, respectively.
Protection against rechallenge with homologous parasite.
Only 3 animals, AI-1748 from Group 4, T-0543 from Group 5, and AI-1753 from Group 2 were unable to control their infections after rechallenge (Table 2). Four monkeys (T-1004, AI-1740, AI-1763, and AI-1756), one each from Groups 5, 2, 3, and 7, failed to support detectable parasite counts during the 42 days of observation. Thirteen animals had maximum parasite counts < 10,000/µL; 5 of these had accumulated parasitemias < 10,000.
Adverse responses. Adverse responses at the immunization sites were recorded during the trial. Groups 1, 3, 4, 5, 6, and 8 had no discernable reactions. Of the 6 animals receiving FALVAC-1 with FCA/FIC, all animals had 3+ and/or 4+ reactions through the challenge period (3+ = erythema and induration; 4+ = ulceration). One monkey in Group 7 had 3+ responses after the second immunization; reactions in the other animals were of a low intensity and transient.
An unexpected observation during this trial was that some animals in all groups had marked dehydration that required treatment with subcutaneous fluids. Because this was seen in all groups, including the animals receiving PBS only, the exact cause of this was not determined. Such widespread dehydration has not been observed in previous trials or in another trial ongoing at the same time, although infected animals have occasionally required rehydration during infection.
Hematology. The hematocrits for the animals were determined every 2 weeks during the trial. At the beginning of the immunizations, the mean hematocrit value for all animals was 51.5%. At the end of the challenge period, the mean value was 50.3%. There was a marked drop during and immediately after infection. During the period after the second immunization, mean hematocrit values rose, reflective of the marked dehydration observed in the animals.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunization and challenge trials in Aotus and Saimiri monkeys have been used repeatedly in efforts to find an alternative to the primary testing of candidate antimalarial vaccines in human volunteers. The question of appropriate criteria of protection in nonhuman primates, and their influence on vaccine development continues to remain unclear. Is a strong serologic response in a nonhuman primate to the different components of a vaccine sufficient to proceed in the developmental process? If efficacy trials with monkeys are included in the critical pathway for vaccine development, what levels of protection are significant?
Few of the vaccine efficacy trials we have conducted in New World monkeys have provided evidence of solid protection after primary challenge.43–45,49 Nonetheless, New World monkeys are the only surrogate hosts that can be infected with P. falciparum to measure efficacy. Many of our studies have shown that these hosts are capable of developing high-level immune responses to infection and reinfection. Thus, we believe that demonstration of efficacy as well as immunogenicity would further support the continued development of candidate vaccines. A candidate vaccine that is highly immunogenic and highly protective in monkeys would appear to be a prime candidate for development in humans.
The planning and conduct of vaccine trials in nonhuman primates involves commitment of animals and many months of time before decisions can be made for movement to the next step in the development of a candidate vaccine. Trials have involved the testing of candidate antigens, usually combined with Freund’s adjuvant or with alum. Here, a novel candidate vaccine was used to test the potential of different adjuvants for the development of protective immunity in A. nancymai monkeys against the FVO strain of P. falciparum. When compared with the monkeys immunized with FALVAC-1 alone, FALVAC-1 with FCA/FIA reduced the mean parasite count (to Day 11), reduced the mean accumulated parasitemia (through Day 11), and extended the number of days to treatment. None of the other antigen/adjuvant combinations included had such an effect on parasitemia after challenge with the FVO strain of P. falciparum. This may indicate that much, if not all of the protection observed was due to the Freund’s adjuvant rather than the antigen. Thus, in an attempt to gather as much information as possible on these potential adjuvants, the exclusion of adequate adjuvant control groups, particularly for Freund’s makes assessment of the contribution of FALVAC-1 difficult.
Obviously, we expected the FALVAC-1 construct to be more protective than was shown in this preliminary trial. Otherwise, fewer adjuvants and more controls would have been included. A new vaccine construct, FALVAC-1A has been produced and is in preliminary stages of testing. Further studies with adequate controls to further test the potential efficacy of these various adjuvants when combined with the candidate vaccine will be needed. After rechallenge indeed, some animals failed to demonstrate detectable parasite counts and others had very low levels of parasitemia. However, whether the immunization or the previous parasitemia resulted in the protection shown against the homologous strain of P. falciparum upon rechallenge is not clear. There were similar statistically significant parasitologic differences noted between the various adjuvant groups or control groups upon homologous rechallenge. Whether protection would be shown against heterologous strains of the parasite remains to be determined. It may be determined that a vaccine directed against the erythrocytic stage of the parasite may have its greatest efficacy and field application after rechallenge and that the Aotus monkey model system may be sufficient to test this.
As expected, immunization with FALVAC-1 combined with FCA/FIA resulted in serious adverse responses at the site of immunization. The appearance of dehydration, primarily after the second immunizations and primary parasitemia was baffling. Frequency of this condition had not been encountered previously in our vaccine trials. Only occasionally do monkeys in vaccine trials require rehydration. The exact cause of this has not been determined, but the fact that the condition was scattered across all the groups suggests some factor not associated with the recombinant product or adjuvant.
Received December 3, 2004. Accepted for publication June 10, 2005.
Acknowledgments: The authors thank the staff of the Animal Resources Branch, NCID, for the care of the animals and Ira Goldman for maintaining the masking. The authors thank the administrative and statistical staff of the Division of Parasitic Diseases for their assistance in the conduct of the study and preparation of the manuscript. Special thanks goes to Charlotte R. Kensil, Antigenics, Inc., and Craig Hacket, Protein Sciences Corporation, for provision of QS-21 and FLVAC-1 antigen, respectively.
Financial support: The work was supported in part by USAID IAA no. 936-6001 between the Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention.
* Address correspondence to William E. Collins, Division of Parasitic Diseases, Mail Stop F-12, Centers for Disease Control and Prevention, 4770 Buford Highway, Chamblee, GA 30341. E-mail: wec1{at}cdc.gov 
Authors’ addresses: William E. Collins, John W. Barnwell, Venkatachalam Udhayakumar, JoAnn S. Sullivan, Douglas Nace, and Ya Ping Shi, Division of Parasitic Diseases, Centers for Disease Control and Prevention, Mail Stop F-12, 4770 Buford Highway, Chamblee, GA 30341. G. Gale Galland, Scientific Resources Program, Centers for Disease Control and Prevention, Atlanta, GA 30333. Tyrone Williams, Atlanta Research & Education Foundation, Atlanta, GA, 30033. Jon Eric Tongren, London School of Tropical Medicine and Hygiene, Keppel Street, London WC1E 7HT, UK. Altaf A. Lal, U.S. Embassy, New Delhi, India.
Reprint requests: William E. Collins, Division of Parasitic Diseases, Mail Stop F-12, Centers for Disease Control and Prevention, 4770 Buford Highway, Chamblee, GA 30341.
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