• View in gallery

    Percentage of sera containing antibody to (PNAN)5 in each age group from three villages: A) Got Nyabondo, B) Raliew, and C) Oboch. Sera were tested at a dilution of 1:20. Absorbance values greater than 0.1 were considered positive.

  • View in gallery

    Frequency distribution of absorbance values of sera from three villages in Kenya: A) Got Nyabondo, B) Raliew, and C) Oboch. Sera were tested at a dilution of 1:20.

  • 1.

    Nardin E. H., Nussenzweig V., Nussenzweig R. S., Collins W. E., Harinasuta K. T., Tapchaisri P., Chomcharn Y., 1982. Circumsporozoite proteins of human malaria parasites Plasmodium falciparum and P. vivax. J. Exp. Med. 156: 2030.

    • Search Google Scholar
    • Export Citation
  • 2.

    Zavala F., Cochrane A. H., Nardin E. H., Nussenzweig R. S., Nussenzweig V., 1983. Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes. J. Exp. Med., 157: 19471957.

    • Search Google Scholar
    • Export Citation
  • 3.

    Ballou W. R., Rothbard J., Wirtz R. A., Gordon D. M., Williams J. S., Gore R. W., Schneider I., Hollingdale M. R., Beaudoin R. L., Maloy W. L., Miller L. H., Hockmeyer W. T., 1985. Immunogenicity of synthetic peptides from circumsporozoite protein of Plasmodium falciparum. Science, 228: 996999.

    • Search Google Scholar
    • Export Citation
  • 4.

    Dame J. B., Williams J. L., McCutchan T. F., Weba J. L., Wirtz R. A., Hockmeyer W. T., Maloy W. L., Haynes J. D., Schneider I., Roberts D., Sanders G. S., Reddy E. P., Diggs C. L., Miller L. H., 1984. Structure of the gene encoding the irnmunodorninant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum. Science, 225: 593599.

    • Search Google Scholar
    • Export Citation
  • 5.

    Enea V., Ellis J., Zavala F., Arnot D. E., Asavanich A., Masuda A., Quakyi I., Nussenzweig R. S., 1984. DNA cloning of Plasmodium falciparum circumsporozoite gene: Amino acid sequence of repetitive epitope. Science, 225: 628630.

    • Search Google Scholar
    • Export Citation
  • 6.

    Zavala F., Tam P., Hollingdale M. R., Cochrane A. H., Quakyi I., Nussenzweig R. S., Nussenzweig V., 1985. Rationale for development of a synthetic vaccine against Plasmodium falciparum malaria. Science, 228:14361440.

    • Search Google Scholar
    • Export Citation
  • 7.

    Campbell G. H., Brandling-Bennett A. D., Roberts J. M., Collins F. H., Barber A. M., Turner A., 1987. Detection of antibodies in human sera to the repeating epitope of the circumsporozoite protein of Plasmodium falciparum using the synthetic peptide, (NANP)3, in an enzyme-linked immunosorbent assay (ELISA). Am. J. Trop. Med. Hyg., 37: 1923.

    • Search Google Scholar
    • Export Citation
  • 8.

    Hoffman S. L., Wistar R., Ballou W. R., Hollingdale M. R., Wirtz R. A., Schneider I., Marwoto H. A., Hockmeyer W. T., 1986. Immunity to malaria and naturally acquired antibodies to the circumsporozoite protein of Plasmodium falciparum. N. Engl. J. Med., 315: 601606.

    • Search Google Scholar
    • Export Citation
  • 9.

    Druilhe P., Pradier O., Marc J-P., Miltgen F., Mazier D., Parent G., 1986. Levels of antibodies to Plasmodium falciparum sporozoite surface antigens reflect malaria transmission rates and are persistent in the absence of reinfection. Infect. Immun., 53: 393397.

    • Search Google Scholar
    • Export Citation
  • 10.

    Nardin E. H., Nussenzweig R., McGregor I., Bryan J., 1979. Antibodies to sporozoites: Their frequent occurrence in individuals living in an area of hyperendemic malaria. Science, 206: 597-599.

    • Search Google Scholar
    • Export Citation
  • 11.

    Tapchaisri P., Chomcharn Y., Poonthong C., Asavanich A., Limsuwan S., Maleevan O., Tharavanij S., Harinasuta T., 1983. Antisporozoite antibodies induced by natural infection. Am. J. Trop. Med. Hyg., 32: 12031208.

    • Search Google Scholar
    • Export Citation
  • 12.

    Nardin E. H., Nussenzweig R. S., Bryan J. H., McGregor I. A., 1981. Congenital transfer of antibodies against malarial sporozoites detected in Gambian infants. Am. J. Trop. Med. Hyg., 30: 11591163.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

Age-Specific Prevalence of Antibody to a Synthetic Peptide of the Circumsporozoite Protein of Plasmodium Falciparum in Children from Three Villages in Kenya

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  • 1 *Malaria Branch, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333
  • 2 †Office of the Director, Division of Parasitic Diseases, Centers for Disease Control, Atlanta, Georgia 30333
  • 3 ‡Clinical Research Center, Kenya Medical Research Institute, Nairobi, Kenya

The presence of antibody to the repeating epitope of the circumsporozoite protein of Plasmodium falciparum was determined in children 1 month to 10 years old from three villages in western Kenya using the synthetic peptide (PNAN)5 in an enzyme-linked immunosorbent assay. The percentage of antibody-positive children increased with age and differed in the three villages. The village with the lowest percentage of antibody-positive children had the lowest percentage of infections as determined by detection of blood stage parasites. The villages also differed in the age at which antibody first appeared. In one village, only 12% of the children had antibody by the age of 5; while in the other two villages, 60% and 73% had antibody by 4 years of age.

Recent advances in monoclonal antibody production and recombinant DNA technology have led to the identification and production of specific antigens of the malaria parasite that are immunogenic and may be useful as target antigens for serologic assays. The most rapid progress has been made in characterizing surface antigens of the Plasmodium sporozoite. A major surface protein, the circumsporozoite (CS) protein, has been identified on all species of malaria parasites that have been studied.1 The protein is characterized by an immunodominant region of repeating amino acids that is species-specific.2, 3 The tandemly repeating regions (PNAN) of the CS protein of P. falciparum have been produced both by recombinant DNA technology and peptide synthesis and have been proposed as candidate vaccines.46 Both recombinant and synthetic peptides of the CS protein of P. falciparum have been used as antigens in serologic assays and appear to have potential for detecting antibodies in people living in areas endemic formalaria.7, 8

An enzyme-linked immunosorbent assay (ELISA) using a synthetic peptide of the repeating epitope of the CS protein of P. falciparum previously has been used to determine the presence of antibody in people living in Kenya.7 To further define the usefulness of such assays in describing the epidemiology of malaria, sera from children living in three villages in Kenya were tested in an ELISA for the presence of antibody to the synthetic peptide (PNAN)5.

The villages in this study were selected on the basis of topography that might support different mosquito populations, thus producing different parasite transmission patterns. Got Nyabondo is 10 km north of Kisumu in a hilly, very precipitous area at an elevation of 1,500 m. The surrounding terrain allows little pooling of water, although this village may receive more rainfall than the other villages. Oboch is about 10 km north of Lake Victoria at an elevation of 1,300 m. The terrain consists of rolling hills with small meadow areas that might be expected to retain some water. Raliew is within 1 km from Lake Victoria at an elevation of 1,140 m and is surrounded by fiat, broad meadows which flood and hold water during the rainy season. This paper describes the prevalence of antibody to sporozoites compared with the prevalence of detectable peripheral blood parasites in Kenyans in the three villages.

Table 1

Percentage of children infected with Plasmodium parasites from three villages in Kenya

VillageNo. slidesNo. parasitesPercentage of slides containmg:
F*FOFMFMOMO
Got Nyabondo147523817101
Oboch1421848328120
Raliew1962157513310

F = P. falciparum; FO = P. falciparum and P. Ovale; FMO = P. falciparum, P. malariae, and P. Ovale.

Materials and Methods

Human sera

Serum specimens were collected from children ranging in age from 2 months to 10 years who were present in the community in October 1985. Collections were made by visiting individual houses and by school surveys in these villages. Blood was collected by finger prick into heparinized capillary tubes from all children under 5 years of age and from children 5–10 years of age in the villages of Got Nyabondo and Oboch. Venipuncture was used to collect blood from children 5 years and over in the village of Raliew. Blood was kept cool until plasma was separated by centrifugation within 6 hr of collection. Plasma was stored frozen at − 20°C until it was transported to the Centers for Disease Control (CDC) on dry ice. After thawing and inactivating the sera at 56°C for 30 min, fibrin clots were removed by centrifuging the sera before use. At the time of serum collection, a thick and thin blood film slide was made. The presence and species of Plasmodium parasites were determined by microscopic examination of at least 200 fields of the thick film. The presence or absence of parasites of single or multiple species was noted.

Antibody assay

A modification of a previously described ELISA using synthetic peptide of the repeating epitope of the P. falciparum circumsporozoite protein was performed7 The 20-amino acid peptide, (PNAN)5 (Biosearch, Inc., San Rafael, California) had a purity of greater than 85% when analyzed by high pressure liquid chromatography and contained appropriate amino acid ratios. The peptide was conjugated 1:1 (w/w) to bovine serum albumin (BSA) at room temperature (RT) for 1 hr using a final concentration of 0.25% glutaraldehyde. Frozen stock of BSA-(NANP)5, 500 µg/ml, was diluted in phosphate buffered saline (PBS) to 3.12 µg/ml and added (50 µl) to each well of flat-bottom Immulon 2 microtiter plates (Dynatech, Springfield, Virginia) for 1 hr at 37°C. Control wells containing BSA and glutaraldehyde without the peptide were prepared identically to the peptide-BSA wells. Plates were washed with PBS before adding 200 µl of blocking buffer (PBS containing 1% BSA, 0.5 M 2-aminoethanol and 0.5% casein) for 18 hr at RT. Wells were washed three times with PBS-T (PBS with 0.5% Tween 20) before adding 50 µl of human sera diluted in blocking buffer with T. After incubation at 37°C for 1 hr, wells were washed and 50 µl of peroxidase-labeled (Ig fraction) rabbit anti-human IgG (provided by V. Tsang, CDC, Atlanta) was added for 1 hr at 37°C. Wells were again washed three times with PBS-T before adding the substrate o-phenylenediamine (Eastman Kodak Co., Rochester, New York). The reaction was stopped at 30 min by adding 25 µl of 8 N H2SO4. The absorbance at 492 nm of the solution in each well was recorded on a Titertek Multiskan MC ELISA reader (Flow Laboratories, McLean, Virginia).

Sera were tested at dilutions of 1:20 and 1:100 in wells containing peptide-BSA or control wells with BSA alone. Absorbance in control wells was subtracted from absorbance in peptide-containing wells as described previously.7 Absorbance difference values of greater than 0.1 at 1:20 or 0.05 at 1:100 dilution of sera were considered positive for antibody to (PNAN)5. The cut-off values were determined from the 95th percentile of a battery of 53 U.S. sera obtained from the CDC serum bank and previously determined to be nonreactive with blood stage P. falciparum parasites in an indirect fluorescent antibody (IFA) assay. The U.S. sera were tested in parallel with the Kenyan sera in the (PNAN)5 ELISA.

Table 2

Antibody to (PNAN)5 in sera from three villages in Kenya

VillageNo. seraPercent positive* when tested at dilutions of:
1:201:100
Got Nyabondo14712%5%
Oboch14248%25%
Raliew19644%26%
Total48535%20%

Antibody to (PNAN)5 determined in ELISA using an absorbance cut-off of 0.1 at a 1:20 dilution and 0.05 at at 1:100 dilution.

Figure 1.
Figure 1.

Percentage of sera containing antibody to (PNAN)5 in each age group from three villages: A) Got Nyabondo, B) Raliew, and C) Oboch. Sera were tested at a dilution of 1:20. Absorbance values greater than 0.1 were considered positive.

Citation: The American Society of Tropical Medicine and Hygiene 37, 2; 10.4269/ajtmh.1987.37.220

Results

Table 1 shows the percentage of children in whom parasites were detected by microscopy at the time the serum specimen was taken. Forty-eight percent of the children in Got Nyabondo were infected with one or more species of Plasmodium. Eighty-two percent of the children in Oboch were infected compared with 79% in Raliew. In Oboch, 28% of the infections were combinations of both P. falciparum and P. malariae.

Figure 2.
Figure 2.

Frequency distribution of absorbance values of sera from three villages in Kenya: A) Got Nyabondo, B) Raliew, and C) Oboch. Sera were tested at a dilution of 1:20.

Citation: The American Society of Tropical Medicine and Hygiene 37, 2; 10.4269/ajtmh.1987.37.220

Of the 485 sera from the three villages, 35% were reactive at a dilution of 1:20 and 20% at 1:100 (Table 2). Got Nyabondo, the village with the lowest percentage of infections, also had a significantly lower percentage of positive sera (P < 0.001) than was found in Oboch and Raliew. In all villages, both the percentage of sera and the absorbance value were related to increasing age. No differences in the percentage of positive sera were observed in males vs. females (data not shown).

The differences among villages were also demonstrated by the age of first appearance of antibody to (PNAN)5 (Fig. 1). The consistent presence of antibody was not observed in children from Got Nyabondo until the age of 5 years (12% positive). High percentages of children in Oboch (60% positive) and Raliew (73% positive) had antibody by age 4. The difference in the ages of individuals having antibody in the three villages is also reflected in the absorbance values of the sera (Fig. 2).

Discussion

The sera used in this study were not obtained by a statistical sampling method and may not be precisely representative of the communities. They were obtained as preliminary samples to define the areas for further study. They should, however, provide a good indication of the age-specific prevalence of antibodies in the villages.

The difference between the childrens’ age at which antibody appeared in the village of Got Nyabondo compared to that in Oboch and Raliew was striking. The exposure to parasites as indicated by the presence of antibody to (PNAN)5 was also reflected in the percentage of children infected, with Got Nyabondo having the lowest. Both the serologic and parasitologic findings are consistent with the expected transmission rates judged by the terrain in which the villages were located. In addition, ongoing Anopheles gambiae and An. funestus mosquito collections indicate that at peak collection times, five- to ten-fold greater numbers of mosquitoes are obtained from Oboch and Raliew than from Got Nyabondo (A. D. Brandling-Bennett, personal communication). Determination of infected mosquito rates using ELISA methodology is underway. The relationship between antibody rates determined by reactivity to sporozoites in an IFA assay and transmission rates in villages in Senegal and Burkina-Faso has been established.9 Use of antibody assays as described here, when combined with entomological data, will more precisely define the relationship between antibodies to CS protein and transmission of malaria.

A previous study of sera from children 3–10 years of age living in the general area of these villages in terrain similar to Oboch and Raliew found 46% to have antibody to the repeated peptide of the CS protein.7 This agrees with the combined total of 46% in children in Oboch and Raliew found in this study to have antibody to (PNAN)5. Similar observations of the early age at which antibody occurred were reported in a study of Indonesians using CS recombinant protein of P. falciparum.8 The higher prevalence in young children than that reported in earlier studies is probably the result of a higher rate of transmission in this and the Indonesian study areas.10, 11

The overall percentage of specimens containing antibody was greater when sera were tested at dilutions of 1:20 than at 1:100, indicating a relatively low titer of antibody. Even at 1:100 dilution, however, 20% of the sera contained detectable antibody. Examination of positive sera from children less than I year of age showed that the number of sera containing antibody was not greater in children 1–3 months of age than in those 6–12 months of age (data not shown). This suggested a minimal influence of maternal passage of antibody on the results shown here. A previous study using sporozoite IFA has shown maternal passage of antibody.12 Further longitudinal studies will be necessary to clearly show the prevalence and duration of maternally transferred antibodies as detected by the ELISA.

An interesting finding in the current study was the presence of P. malariae blood stage parasites in up to 31% of the children in one village. Infections with P. malariae in rates in excess of 50% have been seen in other villages within the Saradidi project area (A. D. Brandling-Bennett and C. Oster, personal communication). Because the CS proteins are species-specific, application of antibody detection assays using repeating epitopes from P. malariae or P. ovale should provide useful information about exposure to the other Plasmodium species. A combination of data on antibody acquisition rates, percentage of infection, and infectivity rates in mosquitoes will allow a more complete assessment of transmission patterns of malaria parasites. This study has shown the usefulness of an ELISA using a synthetic peptide to define the presence of antibody in children at risk of infection with malaria parasites.

ACKNOWLEDGMENTS

The authors thank M. Mugambi, Director, Kenya Medical Research Institute, for permission to publish these results. These studies were supported in part by USAID PASA BST-8453-0453-P-HC-2086.

The use of trade names is for identification only and does not constitute endorsement by the Public Health Service or the U.S. Department of Health and Human Services.

  • 1.

    Nardin E. H., Nussenzweig V., Nussenzweig R. S., Collins W. E., Harinasuta K. T., Tapchaisri P., Chomcharn Y., 1982. Circumsporozoite proteins of human malaria parasites Plasmodium falciparum and P. vivax. J. Exp. Med. 156: 2030.

    • Search Google Scholar
    • Export Citation
  • 2.

    Zavala F., Cochrane A. H., Nardin E. H., Nussenzweig R. S., Nussenzweig V., 1983. Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes. J. Exp. Med., 157: 19471957.

    • Search Google Scholar
    • Export Citation
  • 3.

    Ballou W. R., Rothbard J., Wirtz R. A., Gordon D. M., Williams J. S., Gore R. W., Schneider I., Hollingdale M. R., Beaudoin R. L., Maloy W. L., Miller L. H., Hockmeyer W. T., 1985. Immunogenicity of synthetic peptides from circumsporozoite protein of Plasmodium falciparum. Science, 228: 996999.

    • Search Google Scholar
    • Export Citation
  • 4.

    Dame J. B., Williams J. L., McCutchan T. F., Weba J. L., Wirtz R. A., Hockmeyer W. T., Maloy W. L., Haynes J. D., Schneider I., Roberts D., Sanders G. S., Reddy E. P., Diggs C. L., Miller L. H., 1984. Structure of the gene encoding the irnmunodorninant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum. Science, 225: 593599.

    • Search Google Scholar
    • Export Citation
  • 5.

    Enea V., Ellis J., Zavala F., Arnot D. E., Asavanich A., Masuda A., Quakyi I., Nussenzweig R. S., 1984. DNA cloning of Plasmodium falciparum circumsporozoite gene: Amino acid sequence of repetitive epitope. Science, 225: 628630.

    • Search Google Scholar
    • Export Citation
  • 6.

    Zavala F., Tam P., Hollingdale M. R., Cochrane A. H., Quakyi I., Nussenzweig R. S., Nussenzweig V., 1985. Rationale for development of a synthetic vaccine against Plasmodium falciparum malaria. Science, 228:14361440.

    • Search Google Scholar
    • Export Citation
  • 7.

    Campbell G. H., Brandling-Bennett A. D., Roberts J. M., Collins F. H., Barber A. M., Turner A., 1987. Detection of antibodies in human sera to the repeating epitope of the circumsporozoite protein of Plasmodium falciparum using the synthetic peptide, (NANP)3, in an enzyme-linked immunosorbent assay (ELISA). Am. J. Trop. Med. Hyg., 37: 1923.

    • Search Google Scholar
    • Export Citation
  • 8.

    Hoffman S. L., Wistar R., Ballou W. R., Hollingdale M. R., Wirtz R. A., Schneider I., Marwoto H. A., Hockmeyer W. T., 1986. Immunity to malaria and naturally acquired antibodies to the circumsporozoite protein of Plasmodium falciparum. N. Engl. J. Med., 315: 601606.

    • Search Google Scholar
    • Export Citation
  • 9.

    Druilhe P., Pradier O., Marc J-P., Miltgen F., Mazier D., Parent G., 1986. Levels of antibodies to Plasmodium falciparum sporozoite surface antigens reflect malaria transmission rates and are persistent in the absence of reinfection. Infect. Immun., 53: 393397.

    • Search Google Scholar
    • Export Citation
  • 10.

    Nardin E. H., Nussenzweig R., McGregor I., Bryan J., 1979. Antibodies to sporozoites: Their frequent occurrence in individuals living in an area of hyperendemic malaria. Science, 206: 597-599.

    • Search Google Scholar
    • Export Citation
  • 11.

    Tapchaisri P., Chomcharn Y., Poonthong C., Asavanich A., Limsuwan S., Maleevan O., Tharavanij S., Harinasuta T., 1983. Antisporozoite antibodies induced by natural infection. Am. J. Trop. Med. Hyg., 32: 12031208.

    • Search Google Scholar
    • Export Citation
  • 12.

    Nardin E. H., Nussenzweig R. S., Bryan J. H., McGregor I. A., 1981. Congenital transfer of antibodies against malarial sporozoites detected in Gambian infants. Am. J. Trop. Med. Hyg., 30: 11591163.

    • Search Google Scholar
    • Export Citation
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