Study of the safety and immunogenicity of the synthetic malaria SPf66 vaccine in children aged 1-14 years Gloria Patarroyo, Lina Franco, Roberto Amador, Luis Angel Murillo, Claudia Lucia Rocha, Mauricio Rojas and Manuel Elkin Patarroyo* Safety and immunogenicity tests of the SPf66 malaria vaccine have been carried out on a population o f children, aged 1 to 14 years, in the town of Tumaco, Colombia. Adverse reactions measured after each vaccination were local and minimal, and observed in only a small percentage of the vaccinated children. One year later, no delayed reaction was evident. The majority of the child population developed high antibody titres against SPf66 and the degree o f response did not vary with age. These induced antibodies recognize the native parasite proteins, in particular the molecules from which the amino acid sequence of this vaccine was deduced. These studies demonstrate that the SPf66 vaccine is safe and highly immunogenic for use in children > 1 year old. Keywords: Malaria; syntheticvaccine; safety; immunogenicity;children

INTRODUCTION

MATERIALS

It has been estimated that 230 million cases of Plasmodium falciparum malaria occur in the world each year. Of those infected, 10% die, mainly children under 5 years old. In Africa, in hyper- and holo-endemic areas, this age group is at the greatest risk 1-3. The SPf66 vaccine against the asexual blood stages has been studied experimentally in Aotus monkeys 4'5, and in a group of young adult human volunteers 6. In subsequent studies with young male adult volunteers, it was shown by clinical biochemical, haematological and auto-immune laboratory tests, that the vaccine was atoxic and safe. Furthermore, it induced specific antibodies against the SPf66 molecule% its individual peptides and the parasite's native proteins. These antibodies were able to inhibit the growth of P.falciparum

Study population

in vitro s. Reactions common to other vaccines, such as fever or collateral and delayed reactions, seen especially in live vaccines, have not yet been observed with this vaccine. Because SPf66 is the first chemically synthesized vaccine to be used in large field trials, the genetic make-up of the subjects vaccinated 9-11, the vaccine itself and its administration should be clearly defined 12. This paper reports the studies on the safety and immunogenicity of this vaccine on a population of children from Tumaco, Colombia, aged 1-14 years, who had been taken through the complete vaccination schedule with the synthetic SPf66 vaccine. Instituto de Inmunologia, Hospital San Juan de Dios, Universidad Nacional de Colombia, Carrera 10 Calle 1,

Bogot&, Colombia. "To whom correspondence should be addressed. (Received 29 May 1991; revised 24 September 1991; accepted 2 October 1991) 0264-410X/92/030175-04 © 1992 Butterworth-HeinemannLtd

AND

METHODS

A population of children of both sexes, aged 1-14 years, 92% of whom were non-white, was randomly selected from the town of Tumaco, on the Colombian Pacific coast. This area is holo-endemic for P.falciparum, with an Annual Parasitological Index of 12.2%, a parasite formula of 8 0 - 9 0 % for P. falciparum and a serological prevalence of 22% in adults. These indices were calculated as follows: Annual Parasitological Incidence (API) = (number of diagnosed cases of Plasmodium for 1 year/total population estimated for the same year) x 1000 ; Parasite formula = number of diagnosed cases of Plasmodium/number of diagnosed cases of P. falciparum ; Serological prevalence = (new or old cases in a given moment/total population sampled at the same moment) x I00. The vaccine was administered following the same procedures as in previous studies 6n. The schedule used was 0, 30 and 180 days with day 0 being defined as the day that the first dose was applied by subcutaneous administration of 1 mg of the vaccine adsorbed in alum hydroxide and dissolved in saline solution, for children < 5 years old, and 2 mg of the vaccine for children over this age. Before administering each dose of the vaccine, a thorough medical examination was carried out on each of the volunteers in order to detect the presence of any pathology. Written permission from the parents was obtained 13. For the purposes of vaccination, the population was organized into community groups, and a vaccination group was set up comprising a coordinating doctor from the Immunology Institute, three house doctors from the National University of Colombia, three paediatricians

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Safety and immunogenicity of malaria vaccine SPf66 in children:. G. Patarroyo et al. 1"able 1

Adverse reactions to SPf66 vaccine in children 30 minutes after vaccination Reaction (no. of cases ( % ) )

Dose

n

Local induration

Contralateral induration

Local pain

Erythema

Local pruritus

No reaction

First Second Third

292 287 223

0 0 4 (1.79)

0 0 0

0 1 (0.34) 0

0 0 1 (0.44)

0 0 7 (3.13)

292 (100) 286 (99.65) 211 (94.61)

Table2

Follow-up of abnormal averages before and after each dose of SPf66 in children (1 14 years of age) Dose First (n = 43)

Test

Pre (%)

Post (%)

Haematocrits Haemoglobin White blood cell count Glutamyl oxaloacetic transferase (GOT) 7-Glutamyl pyruvic transminase (GPT) Glutamyl transferase (GGT) Creatinine Direct bilirubin Indirect bilirubin Total bilirubin

16 4 0 40 22 3 2 2.3 0 2.3

24 2 4 47 13 15 0 0 0 0

. pa 0.17

0.13 0.02

Second (n = 45) Pre (%)

Post (%)

20 14 2 14 5 0 3 6.6 6.6 4.4

5 14 0 37 5 0 0 4.4 2.2 2.2

p 0.02

0.007

Third (n = 85) Pre (%)

Post (%)

16 21 0 21 8 0 5 5.8 5.8 2.3

9 14 0 46 12 0 0 3.5 1.1 1.1

p

0.007

0.044

1 Year after (n = 62) Pre (%)

Post (%)

18 11 0 13 8 7 2 3.2 4.8 1.6

26 29 14 48 6 2 0 4.8 11.2 9.6

p

0.007 0.002 0.00003

0.02

"p Values were determined using Student's t test

trained in resuscitation techniques, and support personnel such as nurses and staff from the Malaria Eradication Service (SEM). The vaccination post was organized in such a way that the observation area was sufficiently ample and that a specialized treatment zone with resuscitation equipment and relevant medication was present.

Safety A total of 292 children participated in this study. Two types of adverse clinical reactions were looked for 30 min, and from the third to the fifth day, after each vaccination : minor reactions such as pain, erythema, local and contralateral indurations (LI and CI), local and contralateral pruritus (LP and C P ) ; and systemic reactions such as systemic pruritus (SP), laryngeal oedema (SL), bronchospasm (SB) and shock (SS) 14. One year after the first dose, a questionnaire was completed and a general medical examination was performed in order to detect any delayed adverse reactions, such as jaundice, palpebral oedema, joint inflammation or pain, bronchial hyperactivity, urticaria, dysuria or polyuria, fever, vomiting or diarrhoea, a n d / o r current illness. Clinical laboratory tests were also performed to measure haemoglobin, haematocrits, white blood cells, creatinine, glutamyl oxaloacetic transaminase (GOT), 7-glutamyl pyruvic transaminase ( G P T ) , glutamyl transferase ( G G T ) and bilirubin at the Shaio Clinic laboratory in Bogotfi, Colombia. These tests were performed before vaccination, after the second and third doses, and 1 year after the first dose. Normal values were defined as those established internationally for each test 15. Percentages of abnormal results were calculated, which were then tested for statistical significance. Using immunofluorescence and ELISA techniques, anti-nuclear, anti-DNA, anti-smooth

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muscle and anti-mitochondria antibodies were analysed in 84 post third dose sera. Titres > 1 : 20 were classified as positive (Immunology Laboratory, Cancer Institute, Bogotfi ).

Immunogenicity The IgG antibody titres against the synthetic SPf66 vaccine in the study population were measured using the FAST-ELISA technique (Falcon Assay Screening TestsELISA, Becton Dickinson Labware, Oxrand, CA, USA ), as previously described 8. For the purposes of this study, 401 pre-immune sera and 361 post third immunization sera were obtained from a randomly selected group from the total child population of the area. Of these, 99 were aged 1-4 years, 137 aged 5 - 9 years and 165 aged 10-14 years. The final titres were considered positive if the values were three standard deviations above the average pre-immune serum values. In order to determine the reactivity to the native proteins of the P.falciparum merozoite, the Western Blot technique described previously 8 was used for sera, diluted 1 : 100, from a group of vaccinated children. RESULTS

Safety After each dose of vaccine had been administered, there was a clinical observation period of 30 min for all the vaccinated children. No local reactions were observed in the 292 children who received the first dose (see Table 1 ). There was no local reaction in 94.61% of the children observed during the 30rain following the third dose. Three of the 12 children who experienced local reactions belonged t o the same family. Local induration persisted in < 10% of the population who received one, two or

Safety and immunogenicity of malaria vaccine SPf66 in children:. G. Patarroyo

et al.

a 25

D

20

15 c

"~ 10

0 20

100

200

400

800

1600

3200

6400 12 800 Figure 2 Western blots of pre-immune (PI) and the corresponding immune (I) sera taken from nine different patients and divided according to age

b

15

o 1o "o

R 0

20

100

200

400

800

1600

3200 6400 12800

C m

15

Immunogenleity :i~:i

o~O

c

three doses, being detected during the follow-up examination 3 5 days after vaccination. These reactions did not persist in subsequent check-ups, nor were there any delayed adverse reactions in the 62 children whom it was possible to evaluate 1 year after the first dose. On analysing the percentage of abnormalities of some haematological indicators and the blood chemistry (haemoglobin, haematocrits, leucocytes, bilirubins, GOT, GPT, creatinine and G G T ) no significant variation was detected in the percentage of abnormalities existing before and after each of the doses. Oflly for the G O T was there an increase in the percentage abnormality, although there was no association with the individual follow-up, and no correlation with changes in other simultaneous hepatic function tests (bilirubins, GPT, G G T and haemoglobin). This difference was significant for the groups studied after the second and third doses and for the group studied 1 year later (Table 2). No anti-nuclear or anti-DNA antibodies were detected in any of the samples; no anti-smooth muscle nor antimitochondria antibodies were found in the 23 samples analysed.

:::..::" 10

"13

0

I00

200

400

800 1600 Titres

~200 6400 12 800

Figure 1 Distribution of antibody titres against SPf66 in children. (a), Age group 1-4 years, n = 99; (b) age group 5-9 years, n = 137; (c) age group 10-14 years, n = 165

The sera obtained 20 days after the third dose showed that 93.7% of the population developed high titre levels, ranging from 1 : 100 to 1 : 12 800. Only 3.3% of the preimmune sera showed titres against protein SPf66, all of them with very low values ( < 1 : 100). A comparison by age group shows a clear serum conversion in all age groups: 93% of children aged 1-4 years, 93.4% aged 5 9 years and 94.5% aged 10-14 years present this seroconversion (Figure 1 ). Using the Western Blot method, the immune sera of the study children, taken 20 days after the third vaccination, showed a strong reactivity to proteins 135 kDa, 115 kDa (precursor of 55 kDa) and 83 kDa, and a mild reactivity to proteins 55 kDa and 35 kDa from the P.falciparum schizonts. The last three molecules were used in obtaining the aminoacid sequence included in the vaccine (Figure 2). This reactivity was found to

V a c c i n e , Vol. 10, I s s u e 3, 1992

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Safety and immunogenicity of malaria vaccine SPf66 in children:. G. Patarroyo et al.

be dependent on the anti-SPf66 antibody titres, since pre-immune sera as well as sera with low antibody titres to the vaccine are unable to recognize these proteins. No correlation was found between age group and serum reactivity.

grateful to Dr Oscar Sierra for his help in the design of this study, Dr Felipe Arboleda from Shaio Clinic for performing all the clinical laboratory tests, Luis Eduardo Sarmiento for statistical support, Edgar Cubillos for graphic design and Maria Teresa Gonzalez for secretarial assistance.

DISCUSSION Adverse reactions to the vaccines used in the Expanded Programme of Immunization (EPI) vary markedly according to factors associated with the vaccine itself, the subject vaccinated and the route of administration lz. The SPf66 synthetic vaccine is characterized by being chemically pure and free of contaminants and pyro.gens, which limits the likelihood of adverse reactions to hypersensitivity. First, the risk of immediate or delayed hypersensitivity was assessed, followed by an evaluation of the nature and severity of any local or general sign or symptom attributable to the vaccine. No significant differences were found between the laboratory parameters for haematology, hepatic or renal function prior to vaccination, after vaccination and 1 year later. Variations were only found in the G O T values, although no association was found with other tests of hepatic function on the same individual, carried out simultaneously or during follow-up. Malaria is one of many infections which has been associated with the production of autoantibodies. On the other hand, auto-immune disease is very rare in endemic malaria areas 16. In this study, vaccination with SPf66 did not lead to the formation of auto-antibodies 6 months or 1 year after the first vaccination in the population analysed. This demonstrates the safety of this vaccine, from the standpoint of auto-immunity. The distribution of antibody titres by age group against the synthetic protein and the parasite's native proteins shows a good response to the antigen, independent of age, and very similar to the response in adults. This suggests that the synthetic SPf66 vaccine may be used as a safe and highly immunogenic vaccine at this age, suitable to protect high risk populations such as children under 5 years of age resident in hyper- and holo-endemic areas, such as Africa or some regions of Latin America. This would have a high impact on the mortality resulting from this disease. Subsequent studies in such areas will enable the efficacy of the vaccine to be determined for these populations. ACKNOWLEDGEMENTS This study was supported by the Presidency of Colombia and the Ministry of Public Health. The authors are

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REFERENCES 1 Struchler, D. How much malaria is there worldwide? Parasitol. Today, 1989, 5, 39-40 2 Wilier, D. Malaria resurgence, resistance and research. N. Engl. J. Med. 1983, 308 (15), 875-940 3 Wilier, D. Malaria resurgence, resistance and research I1. N. Engl. J. Med. 1983, 308 (16), 934-940 4 Patarroyo, M.E., Romero, P., Torres, M.L., Clavijo, P., Moreno, A., Martinez, A. et al. Induction of protective immunity against experimental infection with malaria using synthetic peptides. Nature 1987, 328, 629-632 5 Rodriguez, R., Moreno, A., Guzman, F., Calvo, M., Patarroyo, M.E. et al. Studies in Owl monkeys leading to the development of a synthetic vaccine against the asexual blood stages of Plasmodium falciparum. Am. J. Trop. Med. Hyg. 1990, 43, 339-354 6 Patarroyo, M.E., Amador, R., Clavijo, P., Moreno, A., Guzman, F., Romero, P. et al. A synthetic vaccine protects humans against challenge with asexual blood stages of Plasmodium falciparum malaria. Nature 1988, 332, 158 161 7 Amador, R., Moreno, A., Valero, V., Murillo, L.A., Mora, A.L., Rojas, M. et al. The first field trials of the chemically synthesized malaria vaccine SPf66: safety, immunogenicity and protectivity. Vaccine 1992, 10, 179-184 8 Salcedo, M., Barreto, L., Rojas, M., Moya, M, Cote, J. and y Patarroyo, M.E. Studies on the humeral immune response to a synthetic vaccine against Plasmodium falciparum malaria. Clin. Exp. Immunol. 1991, 84, 122-128 9 Patarroyo, M.E., Vinasco, J., Amador, R., Espejo, F., Silva, Y., Moreno, A. et al. Genetic control of the immune response to a synthetic vaccine against Plasmodium falciparum. Parasite Immunol. 1991, 13, 509-516 10 Murillo, L.A., Rocha, C.L., Mora, A.L., Kalil, J., Goldemberg, A.K. and Patarroyo, M.E. Molecular analysis of HLA-DR4-B1 gene in malaria vaccines. Typing and subtyping by PCR technique and oligonucleotides. Parasite Immunol. 1991, 13, 201-210 11 Murillo, L.A., Tenjo, F.A., Clavijo, P., Orozco, M, Sampaio, S., Kalil, J. and Patarroyo, M.E. A specific T-cell receptor genotype preference in the immune response to the synthetic Plasmodium falciparum malaria vaccine. Parasite Immunol. in press 12 Halsey, N.A. and Stetler, H.C., Reacci0nes adversas alas vacunas incluidas en los proyectos del Programa Ampliado de Inmuniazion. OPS 1983, 451, 93-101 13 Guterridge, F. Experimentacion con seres humanos y etica medica. Normas Internacionales para la Investigacion biomedica practicada en seres humanos. Cronica de la OMS 1981, 35, 233-236 14 Valentine, M.D. Allergy and related conditions. In: Principles of Ambulatory Medicine, 2nd Edn (Eds Barker, L.R. and Bur{on, J.R.) Williams & Wilkins, Baltimore, 1986, pp. 293-310 15 Vaughaun, V.C., McKay Jr, R.J. and Behrman, R.E. Textbook of Pediatrics 7th edn (Ed. Nelson, W.E.), W.B. Saunders, Philadelphia, 1982 16 Marsh, K. and Greenwood, B.M The immunopathology of malaria. Cfin. Trop. Med. Comm. Dis. 1986, 1, 91-125

Study of the safety and immunogenicity of the synthetic malaria SPf66 vaccine in children aged 1-14 years.

Safety and immunogenicity tests of the SPf66 malaria vaccine have been carried out on a population of children, aged 1 to 14 years, in the town of Tum...
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