Clin. exp. Immunol. (1991) 84, 122-128

Studies

on

ADONIS

000991049100114M

the humoral immune response to a synthetic vaccine against Plasmodium falciparum malaria

M. SALCEDO, L. BARRETO, M. ROJAS, R. MOYA, J. COTE & M. E. PATARROYO Institute of Immunology, Hospital San Juan de Dios, Universidad Nacional de Colombia, Bogota, Colombia

(Acceptedfor publication 9 October 1990)

SUMMARY A synthetic vaccine against the asexual blood stages of P. falciparum, the SPf 66 synthetic hybrid polymer, composed of peptides derived from three merozoite membrane proteins as well as one peptide from the sporozoite CS protein, has been developed by our group and tested in different protection assays in Aotus monkeys as well as in human volunteers. This study evaluates the humoral immune response induced by the SPf 66 protein vaccination in adult human volunteers from the Colombian Pacific coast as follows: determination of specific IgG antibody levels against SPf 66 by FAST-ELISA after each immunization; analysis of antibody reactivity with P. falciparum schizont lysates by immunoblots; and determination of the in vitro parasite growth inhibition. A clear boosting effect, dependent on time and dose, was observed in the antibody production kinetics. These antibodies also specifically recognize three proteins of the P. falciparum schizont lysate corresponding to the molecular weights of the proteins from which the amino acid sequence was derived. These sera were also capable of markedly inhibiting in vitro parasite growth.

Keywords synthetic vaccines malaria vaccine human trial growth inhibition antibody production

block the erythrocyte invasion by merozoites or by affecting its subsequent development (Mitchele et al., 1976; Brown et al., 1982; Jepsen, 1983; Wahlin et al., 1984). Here we discuss the immunological aspects of these two large-scale human trials, in order to determine the relationship between the IgG antibody production kinetics and the immunization schedule. Other papers will discuss with the clinical aspects of the trials. The kinetics of antibody production and its duration, the specific recognition of the parasite native proteins by these antibodies as well as the in vitro P. falciparum growth inhibition were determined. The results of all these studies led us to design the most appropriate immunization schedule for the vaccine.

INTRODUCTION It is estimated that malaria caused by P. falciparum afflicts 234 million people per year, killing 2 3 million, mainly children (Sturchler, 1989). To control this disease, several studies have been carried out in an effort to find effective immunoprophylactic methods (Nussenzweig & Nussenzweig, 1984; Ballou et al., 1987; Herrington et al., 1987; Jendoubi & Pereira da Silva, 1987; Collins et al., 1988). Our group developed an effective synthetic vaccine against malarial parasites, the SPf 66 synthetic hybrid protein, directed mainly against the asexual blood stages of P. falciparum parasite, which in experimental trials has induced protective immunity in vaccinated human volunteers (Patarroyo et al., 1988). In order to expand the analysis of this vaccine we decided to perform two large-scale human trials to determine its safety, immunogenicity and protectivity and to define the best immunization schedule according to the kinetics of the immune response against the synthetic hybrid molecule, the parasite proteins and its growth inhibition. Quantitative determination of IgG-specific antibodies is important to determine the efficacy of a vaccine, since it has been suggested that these antibodies

SUBJECTS AND METHODS Human volunteers Three-hundred and ninety-nine healthy young male volunteers (age range 18-21 years) of the Colombian Military Forces, with at least 6 years of schooling were enrolled in the study; after a careful explanation of the project, written consent was obtained from all participants. All the volunteers were young soldiers recruited from the Colombian Pacific coast, who after 60 days of military induction stay in the malarial endemic area for approximately 300 days of military service. The study population was composed of two groups with similar characteristics,

Correspondence: Manuel Elkin Patarroyo, MD, Director, Instituto de Immunologia, Hospital San Juan de Dios, Carrera lOa, Calle la, Bogota D.E., Colombia.

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Serological studies in malaria vaccines who performed their military service patrolling the same areas. The group named Tumaco A was made up of 193 men, of whom 63 were randomly selected to be immunized with the synthetic vaccine and 130 were immunized with placebo, according to the same protocol, all on days 0, 20 and 220. The second group, Tumaco B, was enrolled 300 days later and included 206 people: 122 were immunized with the vaccine and 84 with placebo on days 0, 20 and 90. All the vaccinated volunteers received subcutaneously 2 mg of the hybrid synthetic protein SPf 66 dissolved in saline solution and adsorbed to AI(OH)3 in each of the three doses. The volunteers who received placebo were inoculated with Al(OH)3 in saline solution by the same route. None of the volunteers received prophylactic chemotherapy during the study. Serum samples Venous blood was drawn to perform all the serological test. The Tumaco A volunteers were bled on days 0, 19, 37, 52, 98, 200 and 240 after the first immunization. The Tumaco B group were bled on days 0, 14, 65, 110, 150 and 280 after the first immunization. The sera were separated, aliquoted, frozen immediately after collection and stored at - 20'C in polypropylene vials without preservative. On certain occasions it was not possible to obtain venous blood samples from the same individuals who were on guard duty or patrolling in places other than the sample collection site.

SPf 66 antigen The SPf 66 polymeric synthetic hybrid protein used for immunization and for the FAST-ELISAs was produced at the Instituto de Immunologia by the Chemical Synthesis group following a published procedure (Merrifield, 1963; Barany & Merrifield, 1979), but using the multiple solid-phase synthesis method described by Houghten (1985). The amino acid sequence of this protein in three-letter code is: Cys-Gly-AspGlu-Leu-Glu-Ala-Glu-Thr-Gln-Asn-Val-Tyr-Ala-AlaPro-Asn-Ala-Asn-Pro-Tyr-Ser-Leu-Phe-Gln-Lys-GluLys-Met-Val-Leu-Pro-Asn-Ala-Asn-Pro-Pro-Ala-AsnLys-Lys-Asn-Ala-Gly-Cys. After chemical synthesis of 50 g of the vaccine and extensive dialysis against pyrogen-free doubly distilled water, the purity was checked by HPLC, SDS-PAGE, amino acid analysis and amino acid sequence. Toxicity, pyrogenicity, chemical composition and stability tests were performed and repeated before using this material as an immunogen in human volunteers. Falcon assay screening test ELISA (FAST-ELISA) The FAST-ELISA, briefly described here, is a modification of a detailed method reported by Campbell et al. (1987). For the assay, 10 pg of the peptide per ml diluted in phosphate-buffered saline (PBS), pH 7-2, were placed in microtitre plates and incubated with shaking at room temperature for 150 min. The antigen-coated lids were then washed by spraying with PBS0-5% Tween 20 and rinsed with de-ionized water. These lids were left to dry at room temperature and then stored in ajar with dessecant. After their thorough drying, the coated lids were immersed in microtitre wells containing the appropriate serum dilutions and incubated with shaking for 10 min at room temperature. The lids were then incubated with goat affinitypurified anti-human IgG peroxidase conjugate, shaking for I h, and finally incubated with the substrate for 5 min under the

123

same conditions. The lids were washed and rinsed after every step. When the lids were removed, the optical densities were determined at a wavelength of 620 nm, using an ELISA plate reader. The cut-off point for the final titre determination was the mean of the optical densities of the preimmune sera plus 3 s.d. Parasites

The FCB-2 P. falciparum strain from Bogota, Colombia, was cultured in vitro with human group 0 Rh+ erythrocytes using a modification (Zolg et al., 1982) of the Trager & Jensen (1976) culture method. Parasite lysate for Western blotting Late stage schizonts from continuous P. falciparum cultures, exhibiting 20% parasitaemia were collected, washed in sterile PBS and lysed in 0-2% saponine solution with vigorous vortexing for 20 sec. The pellet was washed twice with large volumes of PBS to remove hemoglobin and erythrocyte debris. Seven volumes of lysis buffer (5% SDS, 1 mm EDTA, I mM PMSF) were added to the pellet and then vortexing for 10 min; the supernatant was further centrifuged at 22 500 g for 30 min. This lysate was kept frozen in liquid nitrogen until use. The non-infected red blood cell lysate was prepared in the same way.

Immunoblotting The proteins were electrophoretically separated, transferred to nitrocellulose paper (Towbin, Staehelin & Gordon, 1979) and incubated with pre-immune or immune serum at a 1/100 dilution. The reaction was revealed with affinity-purified goat anti-human IgG alkaline phosphatase conjugate, the substrate was nitroblue tetrazolium (NBT) and BCI (5-bromo, 4-chloro, 3-indonilphosphate) according to the method of Blake et al. (1984). High, intermediate or low antibody response sera from different bleedings, obtained in 20 volunteers from Tumaco A, were tested. In vitro growth inhibition assay Thirty serum samples from the Tumaco A vaccinees, collected on days 37, 98 and 240 after the first immunization, were chosen to evaluate the in vitro growth inhibition. For this assay, cultures were synchronized with a single treatment with 5% sorbitol (Lambros & Vanderberg, 1979). The mature parasites (late trophozoites and schizonts) obtained after culturing for 72 h, were diluted with normal group 0 Rh+ erythrocytes to a starting parasitaemia of 0-2% and adjusted to a 2% haematocrit with RPMI 1640 medium supplemented with 25 mm HEPES, 1 mg/ml hypoxanthine, 40 pg/ml gentamycin, 5 U/ml penicillin, 2 g/l glucose and 5% NaHCO3. Aliquots of these synchronized cultures (240 p1) were placed in 96-well flat-bottomed microculture plates to which 60 p1 of immune or pre-immune sera were added to a final concentration of 20%. Different concentrations of immune sera were used, diluting with human non-immune sera, while maintaining the same final concentration. Sera, thoroughly dialysed against saline solution using 6000-8000 molecular weight exclusion cutoff dialysis membranes, were also assayed. Chloroquine was used at a concentration of 0 16 pg/pl per well as a positive control of in vitro growth inhibition. The plated cultures were

M. Salcedo et al.

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incubated at 370C in a 5% 02/5% C02/90% N2 gas mixture for 72 h in order to see the serum effect during two cycles of parasite growth. The cultures were resuspended at 24 h, and the medium was changed at 48 h using fresh medium with the corresponding test serum. At the end of the incubation period, triplicate wells of each sample were pooled, centrifugated, and thin blood smears stained with Giemsa were prepared. The percentage parasitaemia was microscopically assessed reading 1000 erythrocytes. Eighty per cent of the sera were assayed at least three times. Growth inhibition over the 72-h period was determined by comparing the percentage parasitaemia in the cultures containing immune sera with that of the cultures containing preimmune sera, according to the following formula (Hui & Siddiqui, 1986): % Inhibition=

Parasitaemia (P -0)- (T -0) X Parasitaemia (P -0)

Where P = pre-immune serum at 72 h; T = immune serum at 72 h; and o= starting of parasitaemia. Normal non-immune human sera obtained from individuals from non-endemic malaria areas, were used in control cultures. RESULTS Antibody production kinetics When the specific anti-SPf 66 IgG levels were determined by FAST-ELISA, we found almost similar antibody production kinetics in the sera of both groups according to the number of immunizations. However, the response was not the same in all individuals. Following the second immunization (Grumet, Mitchell & McDevitt, 1971; Grumet, 1972) three patterns of the IgG antibody response against the synthetic polymer were observed using FAST-ELISA: group I, high responders, who increased their antibody titres between 1/ 1600 and 1/25 600; group II, intermediate responders, who increased their antibody titres between 1/200 and 1/800; and group III, low responders, who did not respond to the synthetic peptide or in whom the antibody titres never increased above 1/100 (Fig. 1). The antibody production kinetics showed the following pattern: a relevant increase in the specific IgG antibody levels against SPf 66 was not observed after the first immunization in any of the vaccinees. However, 15-30 days after the second and third immunizations, a very significant antibody increase was observed, which was higher after the third dose. Antibody titres tend to plateau by day 30 after the second and third dose, followed by a mild decrease in titre similar to that seen for the classical IgG half-time response to a given antigen. Antibodies remained in the peripheral blood for long periods, up to day 180, indicating that these antibodies last for long periods of time in the serum of the individuals. The few volunteers who received two doses had lower antibody titres than the ones with three doses. Neither the pre-immune sera nor the sera from placebo controls exhibited antibodies against SPf 66. Recognition of native P. falciparum proteins When analysed by Western blotting, the sera from volunteers vaccinated two or three times SPf 66 had IgG antibodies reacting with the following proteins: 135 kD, I 15 kD and 83 kD,

cleavage product from the precursor to the major merozoite surface antigens (PMMSA), the 195-kD protein. Some reacted with the 35-kD and some weakly with the 55-kD protein. The amino acid sequence of the vaccine was derived from these molecules. Pre-immune or placebo sera did not react with these proteins, although a large number of P.falciparum proteins was recognized by sera from patients with a previous history of malaria (Fig. 2). A correlation was found between anti-SPf 66 IgG titres and the reactivity of the sera in the Western blot, and this reaction was stronger in those volunteers who had three vaccinations and high antibody titres. Absorption of the sera with the synthetic hybrid polymer SPf 66 completely abolished all reactivity of these sera with the proteins in the Western blot and the ELISA (data not shown). There was no reactivity between the sera and the red blood cell membrane proteins at any time during the immunizations (data not shown). In vitro P. falciparum growth inhibition Parasite growth in the presence of pre-immune sera was similar to that of normal nonimmune human sera obtained from individuals coming from non-endemic malaria areas. Parasitaemia increased approximately 45 times during the 72 h period. Chloroquine inhibited parasite growth by 89% and was included as a growth inhibition positive control. Table I shows in vitro parasite growth inhibition using sera collected from the Tumaco A group at different times during the immunization process. Inhibition levels found in sera obtained 37 and 98 days after the first immunization (17 and 78 days after the second dose) were relatively low. In contrast, the inhibition percentage of sera obtained 240 days after the first immunization (20 days after the third dose) increased dramatically for most of the sera from high and intermediate responders as well as in the sera of some low responders. Table 2 includes the analysis of parasite growth inhibition from a larger number of sera from the Tumaco A group collected 20 days after the third immunization and their respective antibody titres against SPf 66. Most of these sera show a parasite growth inhibition above 45%, while a few show low inhibition percentages. The antibody titres ranged from 0 to 1/25 600. Inhibition levels ranging from 45% to 100% were seen in sera with high antibody titres (numbers 380, 353, 437, 424, 460, 355 and 371), similar to those seen with intermediate ones (numbers 422,429,417,357 and 492), but they were also present in some sera with low antibody titres (numbers 326, 503 and 368). Opposed to that, some sera with high or intermediate antibody titres showed very little inhibition of the parasite growth in vitro (sera 375, 408, 477, 473, 378 and 431). Sera from individuals injected with saline solution and Al(OH)3 and bled on the same day did not have antibodies against SPf 66 and did not inhibit in vitro growth of P.falciparum. This fact rules out a non-specific effect of the adjuvant used (Table 2). It is important to note that parasite morphology was not affected throughout the test, in sharp contrast with changes induced by crisis form factor described by Jensen et al. (1984). No significant difference was seen between inhibition levels produced by nondialysed as compared to the dialysed sera (Table 3). Plasmodiumfalciparum growth inhibition was directly proportional to serum concentration, the inhibition decreasing proportionally to the concentration of immune serum added (Fig. 3), suggesting a concentration-dependent effect.

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DISCUSSION Previous work from our laboratory has shown that the SPf 66 polymeric hybrid synthetic protein induces protection in Aotus monkeys as well as in humans against experimental challenge with the asexual blood forms of the P. falciparum malaria parasite indicating that vaccination with this synthetic molecule can induce protective immunity against infection caused by this parasite (Patarroyo et al., 1987, 1988; Rodriquez et al., 1990). This was the first vaccine against the asexual blood stages of the P. falciparum parasite for human use. The present work was carried out to determine the kinetics of antibody production in accordance with the immunization schedule, the duration of these antibodies in the serum, antibody reactivity with native molecules, and the biological mechanisms of substances induced by vaccination with SPf 66 which could produce phenomena such as inhibition of erythrocyte invasion.

The IgG antibody production kinetics against the SPf 66 polymeric molecule were established. These antibodies increased their levels at 15-30 days after the second and third immunization, clearly showing a boosting effect after the third dose. This effect was observed not only in the increase of antiSPf 66 IgG antibodies, but also in the parasite growth inhibition which increased dramatically after the third dose. Reactivity with the parasite native proteins, assessed by Western blotting, was also seen. It can be thus stated that these phenomena are the result of the immunization with the synthetic vaccine since all the preimmune sera and the sera from the placebo platoon-mate volunteers did not show any antibodies against the vaccine. Furthermore, their sera did not inhibit the parasite growth aftK the second or third dose of the vaccine nor were thy r with the parasite native molecules. . - rf; bwr1i18dO It was found that from day 25 toO~f3qtf*%fW the third doses, levels of anti-SPf 66 antibodies have a tendqjsy

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Fig. 2. Western blotting showing the reactivity of IgG antibodies from serum (diluted 1/100) of six volunteers to P. falciparum lysate. Sera from individuals were taken before the immunization and 20 days after the third dose. The volunteer 338 had previous malaria history. PI, pre-immune; 3rd, after third immunization. to slowly decrease, following the kinetics of all the IgG halflives. However, they can be detected approximately 180 days after the second (Tumaco A) and third doses (Tumaco B) in relatively high titres. SPf 66 immunization also induces the recognition of parasite native proteins, once again in a close correlation with the antibody titres against SPf 66, as seen in the immunoblots. Sera from volunteers with high antibody titres specifically and strongly recognized the proteins with molecular weights of 135 kD, 115 kD, 83 kD (a cleavage product of the 195-kD protein), 55 kD and 35 kD (in some cases) on P. falciparum /yMkW This phenomenon was most prominent in the sera obtained after the third immunization. This reaction did not be-lfi§&a4i-batiibody titres or pre-immune or placebo i&tJt[i'JI fL D Zinl ?¶Jt()iibrl [an

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Some individuals did not produce or increase their antibodies titres against SPf 66 after the third dose. This could be related to an ineffective presentation of the synthetic peptide associated to certain HLA molecules or could indicate a defective recognition at the T cell level and both possibilities are being currently explored in our Institute. Although the majority of the sera with high antibody titres against SPf 66 showed a great parasite growth inhibition and some low antibody titres sera showed a low parasite growth inhibition, we did not find a direct correlation between these antibody titres and the in vitro growth inhibition of the P. falciparum parasite. As shown in Table 2, some sera with low antibody titres induced a high-growth inhibition, and some sera with high antibody titres occasionally exhibited poor parasite growth inhibition. This observation suggested that (i) variations

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Serological studies in malaria vaccines Table 3. Effect of dialysis of sera on in vitro growth inhibition of P. falciparum FCB-2

Table 1. Correlation of in vitro growth inhibition of P. falciparum and immunization schedule

Inhibition (%)

Inhibition (%)* Serum no.*

After After After Serum no. Classification 37 days 98 days 240 days 00 00 180 260 20 00 70 0.0

H H H I I I L P

380 460 424 492 431 420 503 305

77-0 98-0 570

380 355 421

770 670 440 73-0 120 98 450 00

N.D. N.D. 0.0 6-0 00 00 00 00

Before dialysis After dialysis

75-0 98 0 520

* Sera were collected 240 days after the first immunization.

100

* Days after the first immunization. H, high, I, intermediate, L, low responders; P. placebo.

r-

80

Table 2. In vitro growth inhibition of P.falciparum FCB-2 and antibody titres against SPf 66 in sera collected 240 days after the first immunization

60

1-

c 0

Inhibition (%)

Serum no.

c

Classification

Three doses 380 353 437 375 408 424 460 440 355 477 473 371 378 431 422 429 417 492 420 357 326 503 415 368 Placebos 383 313 305 344 416 434

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could exist in the idiotypic specificity of the different antibodies against the SPf 66 molecule and hence against the parasite; (ii) that different immunoglobulin classes and/or IgG subclasses not detected by us by FAST-ELISA assays could be involved in the inhibitory response; or (iii) that factors (different from crisisform factor), other than IgG, present in the sera could induce this phenomenon. Thus, the magnitude of the parasite growth inhibition could depend on many factors, but clearly all these factors are induced by the SPf 66 vaccine, since none of the sera of the volunteers inoculated with AI(OH)3 in saline solution obtained on the same days and tested by the same assays, showed any of the above described phenomena. Growth inhibition was also independent from serum dialysis, and these data support the idea that the factor or factors that induce this phenomena have a molecular weight higher than 8 kD, and suggest also the possibility that this inhibition was not due to anti-malarial drugs present in the serum.

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These results confirm that the SPf 66 malarial vaccine is highly immunogenic in humans, and induces anti-SPf 66 IgG antibody production that recognizes the P. falciparum specific proteins from which the amino acid sequence of the vaccine was derived. These IgG antibodies, independently or jointly with other serum factors induced by vaccination with SPf 66, inhibit in vitro growth of the parasite. SPf 66 is an effective chemically synthetized vaccine against malaria for human use. Its action of mechanism could be mediated totally or partially by the humoral mechanisms described here. We propose, based on the data shown above, that one possible schedule of vaccination for adequate antibody production against the parasite is to immunize on days 0, 20 and 90 or 220. In our hands this immunization schedule produces high levels of antibodies in the vaccinated individuals, and these antibody titres persist for at least 180 days. ACKNOWLEDGMENTS This research has been supported by the Ministry of Public Health of Colombia, the German Leprosy Relief Association and Occidental Petroleum Company of Colombia. We would like to express our gratitude to the volunteers of the Military Forces of Colombia, Colonel Julio Cesar Caceres, Liutenent Francisco Nufiez, MD, and Liutenent Clara Zambrano, BSc, for their special collaboration during these studies and for their patience. Mauricio Calvo, Jos& Luis Carvajal, Dr Oscar Noya and Dr Carlos Eduardo Tosta provided helpful suggestions during this study.

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