Parasite Immunology, 1992, 14,95-109
Determination of the immunization schedule for field trials with the synthetic malaria vaccine SPf 66 C.L.ROCHA, L.A.MURILL0, A.L.MORA, M.ROJAS, L. FRANCO, J.COTE, M.V.VALER0, A . M O R E N 0 , R.AMADOR, F.NUREZ*, C.CORONELL & M.E.PATARROY0 Instituto de Inmunologia. Hospital San Juan de Dibs, Bogota, Universidad Nacional de Colombia, Bogota. Colombia and *Fuerzas Armadas de Colombia, Tumaco Accepted for publication 1 1 September 1991 SummaryThe synthetic malaria vaccine SPf 66 has been shown to be safe, immunogenic and effective in trials performed with controlled groups naturally and experimentally exposed to the disease. In order to continue the trials in open populations, it was necessary to standardize the vaccination characteristics. We have performed four field trials with soldier volunteers with the aim, among others, of defining the number of doses required, the intervals between applications, the protein concentration, and the adjuvant to be used. In these trials, the vaccinated individuals' immune responses were evaluated by assaying anti-SPf 66 antibody titres, in vitro growth inhibition of the P. falciparum parasite, and the vaccinees' capacity to recognize P.falciparum native proteins. From these results we conclude that the best vaccination schedule, for adults, is three doses administered subcutaneously on days 0,30 and 180, each containing 2 mg of the synthetic polymerized petide SPf 66 adsorbed to alum hydroxide. Keywords: synthetic malaria vaccine SPf 66, immunization schedule, antibody titres, parasite growth inhibition
Introduction
In previous studies with Aotus nancymai monkeys, we demonstrated the immunogenicity, safety and efficacy of the synthetic hybrid polymeric SPf 66 vaccine and its individual components against experimental challenge with. asexual blood stages of P . falciparum. (Patarroyo et al. 1987, Rodriguez et al. 1990). Later studies performed in humans demonstrated that this molecule is safe, non-toxic, immunogenic and able to generate protection in individuals immunized and challenged experimentally (Patarroyo et al. 1988). Because malaria is a disease that affects nearly 300 million persons annually, the Correspondence: Dr M.E.Patarroyo.
95
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C.L.Rocha et al.
development of new methodologies that permit the massive application of a vaccine is necessary. It is imperative to perform immunogenicity studies that might enable us to determine the optimal parameters for an adequate immunization schedule, such as vaccine concentration, adjuvant type and concentration, and the number of and intervals between doses. Standardizingthese parameters will enable us to begin studies on the SPf 66 vaccine’s efficacy, and its epidemiological impact in open populations. The main objective of this paper is to present, in a sequential form, the different immunogenicity studiesperformed on populations naturally exposed to malaria. We have now completed four field trials, denominated Tumaco A, B, C and D. The Tumaco A and B trials were performed to determine the safety of the SPf 66 malaria vaccine, its immunogenicity and ability to induce protective immunity. The analysis of the vaccinated individuals’ immune responses were performed with the objective of evaluating the boosting effect of a third dose of vaccine and the variations in immunogenicity when the interval between the second and third doses was varied. The Tumaco C and D trials were designed to give further information on the safety and immunogenicity of this vaccine. The Tumaco C trial compared the vaccinees’ immune responses when two different types of adjuvant, alum hydroxide (A1 (OH)3)and calcium phosphate (Ca3(PO&), were used to adsorb the vaccine. The purpose of the Tumaco D trial was to study the variations in the vaccinees’ responses when both vaccine and adjuvant concentrations were diminished simultaneously, maintaining the peptide/ adjuvant ratio (2/1) of previous trials. Subjects and methods VOLUNTEERS
Volunteers were all young men, between 18 and 22 years of age, in excellent physical condition and with at least six years of schooling. All were soldiers belonging to the military forces of Colombia, living in low endemic areas for malaria. After immunization, they were sent to patrol the rural area of Tumaco on the Colombian Pacific coast, an endemicarea for P.falciparum malaria, where they were naturally exposed to the disease. Before being allowed to participate in the study, all individuals were clinically evaluated. Written consent was obtained from each individual (Amador et al. 1991). VACCINE
The polymeric synthetic hybrid protein SPf66 was synthesized in our laboratory by the solid phase peptide synthesis method described by Merrifield (Merrifield 1963, Barany & Memfield 1979) and modified by Houghten (Houghten 1985). The amino acid sequence of the synthetic peptide monomer in the three letter code is:
Cys-Gly-Asp-Glu-Leu-Glu-Ala-Glu-Thr-Gln-Asn-Val-Tyr-Ala-Ala-Pro-Asn-Ala-AsnPro-Tyr-Ser-Leu-Phe-Gln-Lys-Glu-Lys-Met-Val-~u-Pro-Asn-Ala-Asn-Pro-Pro-AlaAsn-Lys-Lys-Asn-Ala-GIy-Cys. For the different studies presented here, four different batches of the SPf 66 vaccine were synthesized: for the Tumaco A and B trials, batch no. 4 (1 g) was used; for the
Field trials with synthetic malaria vaccine
97
Tumaco A Immunizations
1st dose 2nd dose
r
I
I
20
0
Bleeding days
3rd dose
t
t
t
19
0
220
t
37
t
52
t
98
t
200
240
Tumaco B Immunizations
1st dose 2nd dose
I
I
I
90
20
0
Bleeding days
3rd dose
t t
0
t
t
110
65
14
t
150 280
Tumaco C Immunizations
1st dose
I
0
2nd dose
3rd dose
I
I
30
150
t
46
Bleeding days
t
68
t
150
t
180
Tumaco D Immunizations
1st dose
I
0
Bleeding days
0
2nd dose
3rd dose
I
I
30
180
t
43
t
65
t
202
Figure 1. Immunization schedule and bleeding days for the Tumaco A, B, C and D trials.
Tumaco C trial, batches nos. 6-7 (40 g) and 8 (150 g) were used; and finally, for the Tumaco D trial, vaccine from batch no. 8 was used. Each vaccine batch was tested after synthesis by HPLC, SDS-PAGE, amino acid content and sequence analysis. Sterility, cytotoxicity and pyrogenicity (in rabbits) tests were performed on the final product before its use in humans.
98
C.L.Rocha et al.
For all trials, three doses, each consistingof 2 mg of the synthetic polymerized peptide adsorbed onto 1 mg of alum hydroxide in a final volume of 0.5 ml, were administered subcutaneously. For the Tumaco D trial the dose was diminished, although maintaining the 2/1 ratio between peptide and adjuvant. TRIALS
The study groups were denominated Tumaco A, B, C and D. Their conformation, immunization schedule and specific objectives are described below: Tumaco A This study comprised 193 volunteers,divided between two groups. The first group had 63 individuals who were immunized on days 0,20 and 220 with the SPf 66 malaria vaccine; the second group of 130 individuals each received saline solution in alum hydroxide (Al(OH)3) (1 mg) as a placebo (Figure 1). Tumaco B In this trial, the interval between the second and third doses was diminished relative to the Tumaco A trial. Immunizations were performed on days 0, 20 and 90 (Figure 1). This group consisted of 206 volunteer soldiers, 122 of them receiving the vaccine and 84 saline solution in alum hyroxide as a placebo. Tumaco C I52 volunteers participated in this trial, separated into two groups, according to the adjuvant used, either alum hydroxide (Al(OH)3) (kindly provided by Instituto Nacional de Salud de Colombia) or calcium phosphate (Ca3(P0&) (Superfos Biosector a/s, Denmark). The vaccination schedule used was 0,30 and 150 days (Figure 1). Tumaco D In this trial, the 200 participating volunteers were randomly distributed into four groups (1, 2, 3 and 4). The peptide/adjuvant ratio of previous trials (2: 1) was maintained. However, total peptide concentration for each group was 2 , l and 0.5 mg, for groups 1,2 and 3, respectively. Group 4 received alum hydroxide in saline solution as placebo. The immunization schedule used in this trial was 0, 30 and 180 days (Figure 1). IMMUNOGENICITY STUDIES
Serum samples Venous blood was drawn to perform all the immunogenicity studies, according to a bleeding schedule in each trial (Figure 1). The serum was separated and aiiquots were frozen and stored at - 20°C without preservatives.
Field trials with synthetic malaria vaccine
99
Determination of antibody titres
The IgG antibody levels of the vaccinated volunteers were determined by the FASTELISA technique (Falcon Assay Screening Test-ELISA, Becton Dickinson Labware Oxrand, CA) described by Campbell et al. (1987) and modified by Salcedo et al. (1991). The antibody titres were expressed as the maximum dilution of the sera in which the optical density value was higher than 3 standard deviations above the mean of values for the preimmune sera. All ELISA tests were calibrated with the same negative (preimmune serum) and positive (hyper-immune serum) controls. Western blotting P.falciparum schizont proteins were obtained from a continuous culture (Zolg et al. 1982, Trager & Jensen 1976, Salcedo et al. 1991), eiectrophoretically separated, transferred to nitrocellulose filter (Towbin, Staehelin & Gordon 1979) and subsequently incubated with either preimmune or immune serum at a 1/lo0 dilution. The reaction was developed with affinity-purifiedgoat anti-human IgG alkaline phosphatase conjugate (TAG0 Inc., Burlingame, CA, USA), using a mixture of NBT (nitroblue tetrazolium) (Sigma, St. Louis, MO, USA) and BCI (5-bromo, 4-chloro, 3-indolylphosphate) (Sigma, St. Louis MO, USA) as substrate, according to the method described by Blake et al. (1984). IN VITRO GROWTH INHIBITION ASSAY OF P . F A L C I P A R U M
For this assay, sera from the volunteers were obtained before vaccination, and 15 to 30 days after receiving the third dose, and for some studies, after the second dose as well. The assay was performed according to the method described by Salcedo et al. (1991). Growth inhibition over a 72 h period was determined by comparing the percentages of parasitaemia in the cultures containing immune sera with those of the cultures containing preimmune sera, according to the following formula (Hui & Siddiqui 1986). % Inhibition =
Parasitaemia (P-Ohr) - (T-Ohr) Parasitaemia (P-Ohr)
where: P = preimmune serum at 72 h; T =immune serum at 72 h; and Ohr = starting parasitaemia. Normal non-immune human sera obtained from individuals from non-endemic malaria areas were used in control cultures. The assays were performed for each sera in triplicate. For the Tumaco A and B trials the assays were repeated at least two times and the results were expressed as an average of the growth inhibition percentage obtained with each serum. STATISTICAL ANALYSIS
The statistical analysis of the differences between the antibody titres of each group was performed with the Friedman, Wilcoxon and Mann Whitney U-tests. A proportion differences test was applied to analyze the in vitro growth inhibition percentage (Siege1 1956).
100
C.L.Rochu et al.
Results TUMACO A AND B TRIALS
In these two first field trials the vaccine has proved to be safe and immunogenic (Amador et al. 1991). Although differences exist between the immunization schedules, the immune response pattern was similar in both studies. The most relevant findings were an increase in antibody titres for most individuals after the second dose, and the clear boosting effect of the third dose (P
Determination of the immunization schedule for field trials with the synthetic malaria vaccine SPf 66 C.L.ROCHA, L.A.MURILL0, A.L.MORA, M.ROJAS, L. FRANCO, J.COTE, M.V.VALER0, A . M O R E N 0 , R.AMADOR, F.NUREZ*, C.CORONELL & M.E.PATARROY0 Instituto de Inmunologia. Hospital San Juan de Dibs, Bogota, Universidad Nacional de Colombia, Bogota. Colombia and *Fuerzas Armadas de Colombia, Tumaco Accepted for publication 1 1 September 1991 SummaryThe synthetic malaria vaccine SPf 66 has been shown to be safe, immunogenic and effective in trials performed with controlled groups naturally and experimentally exposed to the disease. In order to continue the trials in open populations, it was necessary to standardize the vaccination characteristics. We have performed four field trials with soldier volunteers with the aim, among others, of defining the number of doses required, the intervals between applications, the protein concentration, and the adjuvant to be used. In these trials, the vaccinated individuals' immune responses were evaluated by assaying anti-SPf 66 antibody titres, in vitro growth inhibition of the P. falciparum parasite, and the vaccinees' capacity to recognize P.falciparum native proteins. From these results we conclude that the best vaccination schedule, for adults, is three doses administered subcutaneously on days 0,30 and 180, each containing 2 mg of the synthetic polymerized petide SPf 66 adsorbed to alum hydroxide. Keywords: synthetic malaria vaccine SPf 66, immunization schedule, antibody titres, parasite growth inhibition
Introduction
In previous studies with Aotus nancymai monkeys, we demonstrated the immunogenicity, safety and efficacy of the synthetic hybrid polymeric SPf 66 vaccine and its individual components against experimental challenge with. asexual blood stages of P . falciparum. (Patarroyo et al. 1987, Rodriguez et al. 1990). Later studies performed in humans demonstrated that this molecule is safe, non-toxic, immunogenic and able to generate protection in individuals immunized and challenged experimentally (Patarroyo et al. 1988). Because malaria is a disease that affects nearly 300 million persons annually, the Correspondence: Dr M.E.Patarroyo.
95
96
C.L.Rocha et al.
development of new methodologies that permit the massive application of a vaccine is necessary. It is imperative to perform immunogenicity studies that might enable us to determine the optimal parameters for an adequate immunization schedule, such as vaccine concentration, adjuvant type and concentration, and the number of and intervals between doses. Standardizingthese parameters will enable us to begin studies on the SPf 66 vaccine’s efficacy, and its epidemiological impact in open populations. The main objective of this paper is to present, in a sequential form, the different immunogenicity studiesperformed on populations naturally exposed to malaria. We have now completed four field trials, denominated Tumaco A, B, C and D. The Tumaco A and B trials were performed to determine the safety of the SPf 66 malaria vaccine, its immunogenicity and ability to induce protective immunity. The analysis of the vaccinated individuals’ immune responses were performed with the objective of evaluating the boosting effect of a third dose of vaccine and the variations in immunogenicity when the interval between the second and third doses was varied. The Tumaco C and D trials were designed to give further information on the safety and immunogenicity of this vaccine. The Tumaco C trial compared the vaccinees’ immune responses when two different types of adjuvant, alum hydroxide (A1 (OH)3)and calcium phosphate (Ca3(PO&), were used to adsorb the vaccine. The purpose of the Tumaco D trial was to study the variations in the vaccinees’ responses when both vaccine and adjuvant concentrations were diminished simultaneously, maintaining the peptide/ adjuvant ratio (2/1) of previous trials. Subjects and methods VOLUNTEERS
Volunteers were all young men, between 18 and 22 years of age, in excellent physical condition and with at least six years of schooling. All were soldiers belonging to the military forces of Colombia, living in low endemic areas for malaria. After immunization, they were sent to patrol the rural area of Tumaco on the Colombian Pacific coast, an endemicarea for P.falciparum malaria, where they were naturally exposed to the disease. Before being allowed to participate in the study, all individuals were clinically evaluated. Written consent was obtained from each individual (Amador et al. 1991). VACCINE
The polymeric synthetic hybrid protein SPf66 was synthesized in our laboratory by the solid phase peptide synthesis method described by Merrifield (Merrifield 1963, Barany & Memfield 1979) and modified by Houghten (Houghten 1985). The amino acid sequence of the synthetic peptide monomer in the three letter code is:
Cys-Gly-Asp-Glu-Leu-Glu-Ala-Glu-Thr-Gln-Asn-Val-Tyr-Ala-Ala-Pro-Asn-Ala-AsnPro-Tyr-Ser-Leu-Phe-Gln-Lys-Glu-Lys-Met-Val-~u-Pro-Asn-Ala-Asn-Pro-Pro-AlaAsn-Lys-Lys-Asn-Ala-GIy-Cys. For the different studies presented here, four different batches of the SPf 66 vaccine were synthesized: for the Tumaco A and B trials, batch no. 4 (1 g) was used; for the
Field trials with synthetic malaria vaccine
97
Tumaco A Immunizations
1st dose 2nd dose
r
I
I
20
0
Bleeding days
3rd dose
t
t
t
19
0
220
t
37
t
52
t
98
t
200
240
Tumaco B Immunizations
1st dose 2nd dose
I
I
I
90
20
0
Bleeding days
3rd dose
t t
0
t
t
110
65
14
t
150 280
Tumaco C Immunizations
1st dose
I
0
2nd dose
3rd dose
I
I
30
150
t
46
Bleeding days
t
68
t
150
t
180
Tumaco D Immunizations
1st dose
I
0
Bleeding days
0
2nd dose
3rd dose
I
I
30
180
t
43
t
65
t
202
Figure 1. Immunization schedule and bleeding days for the Tumaco A, B, C and D trials.
Tumaco C trial, batches nos. 6-7 (40 g) and 8 (150 g) were used; and finally, for the Tumaco D trial, vaccine from batch no. 8 was used. Each vaccine batch was tested after synthesis by HPLC, SDS-PAGE, amino acid content and sequence analysis. Sterility, cytotoxicity and pyrogenicity (in rabbits) tests were performed on the final product before its use in humans.
98
C.L.Rocha et al.
For all trials, three doses, each consistingof 2 mg of the synthetic polymerized peptide adsorbed onto 1 mg of alum hydroxide in a final volume of 0.5 ml, were administered subcutaneously. For the Tumaco D trial the dose was diminished, although maintaining the 2/1 ratio between peptide and adjuvant. TRIALS
The study groups were denominated Tumaco A, B, C and D. Their conformation, immunization schedule and specific objectives are described below: Tumaco A This study comprised 193 volunteers,divided between two groups. The first group had 63 individuals who were immunized on days 0,20 and 220 with the SPf 66 malaria vaccine; the second group of 130 individuals each received saline solution in alum hydroxide (Al(OH)3) (1 mg) as a placebo (Figure 1). Tumaco B In this trial, the interval between the second and third doses was diminished relative to the Tumaco A trial. Immunizations were performed on days 0, 20 and 90 (Figure 1). This group consisted of 206 volunteer soldiers, 122 of them receiving the vaccine and 84 saline solution in alum hyroxide as a placebo. Tumaco C I52 volunteers participated in this trial, separated into two groups, according to the adjuvant used, either alum hydroxide (Al(OH)3) (kindly provided by Instituto Nacional de Salud de Colombia) or calcium phosphate (Ca3(P0&) (Superfos Biosector a/s, Denmark). The vaccination schedule used was 0,30 and 150 days (Figure 1). Tumaco D In this trial, the 200 participating volunteers were randomly distributed into four groups (1, 2, 3 and 4). The peptide/adjuvant ratio of previous trials (2: 1) was maintained. However, total peptide concentration for each group was 2 , l and 0.5 mg, for groups 1,2 and 3, respectively. Group 4 received alum hydroxide in saline solution as placebo. The immunization schedule used in this trial was 0, 30 and 180 days (Figure 1). IMMUNOGENICITY STUDIES
Serum samples Venous blood was drawn to perform all the immunogenicity studies, according to a bleeding schedule in each trial (Figure 1). The serum was separated and aiiquots were frozen and stored at - 20°C without preservatives.
Field trials with synthetic malaria vaccine
99
Determination of antibody titres
The IgG antibody levels of the vaccinated volunteers were determined by the FASTELISA technique (Falcon Assay Screening Test-ELISA, Becton Dickinson Labware Oxrand, CA) described by Campbell et al. (1987) and modified by Salcedo et al. (1991). The antibody titres were expressed as the maximum dilution of the sera in which the optical density value was higher than 3 standard deviations above the mean of values for the preimmune sera. All ELISA tests were calibrated with the same negative (preimmune serum) and positive (hyper-immune serum) controls. Western blotting P.falciparum schizont proteins were obtained from a continuous culture (Zolg et al. 1982, Trager & Jensen 1976, Salcedo et al. 1991), eiectrophoretically separated, transferred to nitrocellulose filter (Towbin, Staehelin & Gordon 1979) and subsequently incubated with either preimmune or immune serum at a 1/lo0 dilution. The reaction was developed with affinity-purifiedgoat anti-human IgG alkaline phosphatase conjugate (TAG0 Inc., Burlingame, CA, USA), using a mixture of NBT (nitroblue tetrazolium) (Sigma, St. Louis, MO, USA) and BCI (5-bromo, 4-chloro, 3-indolylphosphate) (Sigma, St. Louis MO, USA) as substrate, according to the method described by Blake et al. (1984). IN VITRO GROWTH INHIBITION ASSAY OF P . F A L C I P A R U M
For this assay, sera from the volunteers were obtained before vaccination, and 15 to 30 days after receiving the third dose, and for some studies, after the second dose as well. The assay was performed according to the method described by Salcedo et al. (1991). Growth inhibition over a 72 h period was determined by comparing the percentages of parasitaemia in the cultures containing immune sera with those of the cultures containing preimmune sera, according to the following formula (Hui & Siddiqui 1986). % Inhibition =
Parasitaemia (P-Ohr) - (T-Ohr) Parasitaemia (P-Ohr)
where: P = preimmune serum at 72 h; T =immune serum at 72 h; and Ohr = starting parasitaemia. Normal non-immune human sera obtained from individuals from non-endemic malaria areas were used in control cultures. The assays were performed for each sera in triplicate. For the Tumaco A and B trials the assays were repeated at least two times and the results were expressed as an average of the growth inhibition percentage obtained with each serum. STATISTICAL ANALYSIS
The statistical analysis of the differences between the antibody titres of each group was performed with the Friedman, Wilcoxon and Mann Whitney U-tests. A proportion differences test was applied to analyze the in vitro growth inhibition percentage (Siege1 1956).
100
C.L.Rochu et al.
Results TUMACO A AND B TRIALS
In these two first field trials the vaccine has proved to be safe and immunogenic (Amador et al. 1991). Although differences exist between the immunization schedules, the immune response pattern was similar in both studies. The most relevant findings were an increase in antibody titres for most individuals after the second dose, and the clear boosting effect of the third dose (P
