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Clinical and Diagnostic Laboratory Immunology, November 2001, p. 1289-1291, Vol. 8, No. 6
Centre for Medical Parasitology at Department
of Infectious Diseases, Copenhagen University Hospital
(Rigshospitalet), and Institute for Medical Microbiology and
Immunology, University of Copenhagen, Copenhagen,
Denmark1; London School of Hygiene and
Tropical Medicine, London, United
Kingdom2; and National Institute of
Medical Research, Amani Medical Research Centre, Amani,
Tanzania3
Received 14 January 2001/Returned for modification 5 April
2001/Accepted 21 August 2001
The use of insecticide-treated bed nets (ITN) has been documented
to reduce malaria morbidity and mortality in areas with endemic
malaria, but concerns have been raised that ITN usage could affect the
acquisition of malaria immunity. Several lines of evidence have
indicated that antibodies against variant surface antigens (VSA) are
important in the development of naturally acquired immunity to
Plasmodium falciparum malaria and may thus be good indicators of immune status. We have compared the levels of VSA antibodies in plasma from children who have used ITN for 4 years to
levels in plasma from children from a nearby village not using ITN. A
total of 97 plasma samples were analyzed using 13 different P. falciparum isolates. We found that the children using ITN had significantly lower VSA antibody levels and recognized a smaller proportion of the VSA expressed by the tested parasite isolates than
children not using ITN.
Malaria constitutes one of the
largest global health problems. Through the Roll Back Malaria Program,
the World Health Organization has set ambitious targets of halving the
malaria burden before 2010. One of the important strategies used to
reach this target is to promote the use of insecticide-treated bed nets
(ITN). ITN have been documented to reduce malaria morbidity and
mortality (8). ITN reduce contact between the mosquito
vector and the human host. When used widely in a community, ITN reduce
the population of sporozoite-positive mosquitoes (4),
which is reflected in a reduction in the plasma antibody levels to
circum sporozoite protein among the bed net users (11).
Even though the short- and medium-term beneficial effect of ITN are
well documented, concerns have been raised about the long-term effect,
since the reduction in infectious inoculations may affect both the
development and the maintenance of malaria immunity (16).
Malaria immunity is developed after repeated exposures to the parasite,
and clinical malaria immunity is acquired at a much earlier age in
areas of intense malaria transmission than in areas of more moderate
transmission intensity (13).
The functional background for malaria immunity is not fully understood,
but several studies have indicated that it relies on the acquisition of
a repertoire of agglutinating antibodies, which recognize a broad
spectrum of variant surface antigens (VSA) expressed on infected
erythrocytes (2, 7, 9, 10). The best characterized of
these is the molecule responsible for sequestration of parasitized
erythrocytes, Plasmodium falciparum erythrocyte membrane
protein 1 (PfEMP1) (1, 15). PfEMP1 is encoded by var genes (18). Each parasite genome contains
about 40 var genes; only one var gene product is
expressed at a time, and through each generation some parasites will
switch PfEMP1 expression phenotype (14).
This study was designed to investigate whether the plasma levels and
the repertoire of VSA antibodies were affected by the use of ITN by
comparing the plasma antibody levels in children living in two nearby
villages in northern Tanzania. The two villages are situated on the
coastal plain about 30 km from Tanga in an area characterized by
perennial and very intense malaria transmission. ITN were distributed
to the inhabitants of Mafere village in February 1996 as part of a
comparative trial of ITN versus house spraying (4). Since
then there has been annual reimpregnation of the nets with
alphacypermethrin and monitoring of the malaria morbidity among the
children in the villages (5, 6). In April 2000 bed nets
had not yet been distributed in Kibaoni village, and only a few
children were sleeping under nets in this village. Before the
introduction of ITN in Mafere the entomological inoculation rates (EIR)
in the two villages were similar. The EIR in a group of villages
including Mafere was 540 for the year before provision of ITN and 29 for the year after (4). In 1999 and 2000 the EIR in a
group of villages including Kibaoni was 397 (20). In April
2000 plasma samples were obtained from 48 children aged 2 to 6 years
and 48 children aged 3 to 7 years from Mafere and Kibaoni,
respectively. The children were randomly selected from a computerized
census list. Only asymptomatic children who were not receiving
antimalarial treatment were included. In addition, a 9-year-old child
from Mafere was sampled and included in the study. All children from
Mafere used bed nets on a regular basis, whereas the children from
Kibaoni did not. We measured the levels of antibodies to 13 different
parasite isolates by flow cytometry (17). The isolates
were originally obtained from malaria patients contracting malaria in
Ghana (n = 11; isolates: L1127, L1051, L1156,
L1114, L1094, L1070, L1093, L1106, L1047, L1052, and Q0016) or Sudan
(n = 2; isolates: G4 and G12) and were cultured
according to standard procedures with slight modifications (3,
19). On the day of assay erythrocytes infected with mature
blood-stage parasites were purified by exposure to a strong magnetic
field, resulting in material having >75% parasitaemia
(17). Aliquots of 2 × 105 purified
late-stage-infected erythrocytes labeled by ethidium bromide were
sequentially exposed to 5 µl of plasma, 0.4 µl of goat anti-human
immunoglobulin G (Dako, Glostrup, Denmark), and 4 µl of fluorescein
isothiocyanate-conjugated rabbit anti-goat immunoglobulin G (Dako).
Samples were washed twice in phosphate-buffered saline between each
incubation step. Twofold dilution of a hyperimmune plasma pool from
adult Ghanaians and individual plasma samples from 10 Danish adults
without exposure to malaria were included for each parasite isolate.
For each parasite-plasma combination, data from 5,000 cells were
collected on a FACScan (Becton Dickinson, Franklin Lakes, N.J.), and
the mean late-stage-infected erythrocyte fluorescence index (MFI) was
recorded. Nonspecific labeling was evaluated by analysis of uninfected
erythrocytes. It has previously been shown that the main reactivity
measured in this assay is directed against large (molecular mass, 200 to 300 kDa) polymorphic surface-expressed molecules (17)
and depends on expression of PfEMP1 (12).
For each child the overall VSA antibody reactivity was calculated as
the accumulated MFI of each of the 13 parasite isolates tested. Figure
1 shows the overall VSA antibody
reactivity in plasma from children sleeping under ITN and unprotected
children. In both groups the VSA antibody levels increased with age
(rs = 0.40 and P <0.005 and
rs = 0.41 and P < 0.004,
for bed net users and unprotected children, respectively). However
independently of this age correlation, the level of reactivity also
depended on bed net usage, with reactivity being lower in the children using ITN (P < 0.0001 [analysis of covariance]). The
plasma VSA antibody reactivities to the individual parasite isolates
were also compared between the two groups. For all isolates the plasma VSA antibody levels were higher among unprotected children than among
the bed net users (P < 0.002 for each isolate). Figure
2A shows the plasma VSA antibody
reactivity for a representative isolate and the corresponding values
obtained using a twofold titrated hyperimmune plasma pool and Danish
control plasma. For all isolates the difference between the regression
lines of the two groups was between 1 and 2 dilutions of the
hyperimmune plasma pool (Fig. 2A and data not shown), corresponding to
two- to four-fold-lower antibody levels among bed net users than among
the unprotected children. Taken together the data in Fig. 1 and 2A show
that both the overall amount of antibodies to VSA and the antibody
level to VSA expressed by individual parasite strains was affected by the use of ITN. To assess whether bed net usage also affected the
number of parasite isolates recognized, we counted the number of
parasite isolates for which each donor had plasma VSA reactivity above
the cutoff level (mean reactivity of Danish controls + 2standard deviations [SD]). Figure 2B shows that most of the unprotected children recognized VSA on all 13 parasite isolates tested, and among
these children there was no association between age and the number of
isolates recognized. Among the bed net users the percentage of
parasites recognized-was generally lower but increased with donor age
(rs= 0.36; P < 0.012). When
comparing children 40 to 70 months old, there was a statistically
significant difference between the percentage of parasites recognized
between unprotected children and bed net users (P < 0.001 [Mann-Whitney rank sum test]).
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.6.1289-1291.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Insecticide-Treated Bed Nets Reduce Plasma Antibody Levels and
Limit the Repertoire of Antibodies to Plasmodium falciparum
Variant Surface Antigens
ABSTRACT
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FIG. 1.
VSA plasma antibody levels in relation to age among
children sleeping under ITN ( 
) (n = 48) and
unprotected children (
) (n = 49). The antibody
levels were measured by flow cytometry, and the figure shows the
cumulative MFI for 13 parasite isolates. The solid lines show linear
regression, and dotted lines represent the 95% confidence interval.
View larger version (14K):
[in a new window]
FIG. 2.
VSA plasma antibody levels and repertoire in children
sleeping under ITN ( ) and unprotected children (). (A) Plasma
antibody levels to VSA carried by the parasite isolate L1047. The
y axis at left shows the MFI measured by flow cytometry. The
y axis at right shows the reactivity in dilutions of a
hyperimmune plasma pool (HIP). The reactivity of Danish control
plasma + 2 SD (DC) is indicated by the dotted line. The
x axis shows the plasma donor age. (B) Percentage of
parasite isolates recognized in relation to age, assessed by counting
how many of the 13 parasite isolates the donor had plasma VSA antibody
reactivity to above the cutoff level (mean reactivity of Danish
controls + 2 SD).
Our findings indicate that the use of ITN even in areas of very high malaria endemicity reduces the level of VSA antibodies and narrows the repertoire of recognized VSA. The clinical consequences of this are not known, but it could delay acquisition of malaria immunity and thereby influence the disease pattern. The reduction of the plasma VSA antibody repertoire could make bed net users more vulnerable to parasites that express rare VSA variants (2). Widespread use of ITN decreases the malaria disease burden in communities in the short run (8). Our data indicate that the immune responses to an important target of protective malaria immunity are influenced by bed net usage and suggest that ITN in the longer run could influence malaria disease patterns. However, the ongoing clinical monitoring in the mentioned villages shows that there continues to be health benefits after using ITN for 2 to 5 years, including a reduction of anemia prevalence (5, 6; Maxwell et al., unpublished data).
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ACKNOWLEDGMENTS |
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This work was supported by the European Commission (QLK2-CT-1999-01293), the Danish Medical Research Council (52-00-1086), and the Danish Development Agency (Danida, Enreca). This work was done as part of the Joint Malaria Programme, a collaboration between Kilimanjaro Christian Medical College, London School of Hygiene and Tropical Medicine, National Institute of Medical Research (Tanzania), Centre for Medical Parasitology at the University of Copenhagen and Rigshospitalet.
We thank Jimmy Weng, Anne Corfitz, Gitte Grauert, and Kirsten Pihl for technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Medical Microbiology and Immunology, University of Copenhagen, Panum Institute Building 24.2, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. Phone: 45 35 32 76 77. Fax: 45 35 32 78 51. E-mail: theander{at}biobase.dk.
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REFERENCES |
|---|
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Abstract
Text
References
|
|---|
| 1. | Baruch, D. L., B. L. Pasloske, H. B. Singh, X. Bi, X. C. Ma, M. Feldman, T. F. Taraschi, and R. J. Howard. 1995. Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes. Cell 82:77-87[CrossRef][Medline]. |
| 2. |
Bull, P. C.,
B. S. Lowe,
M. Kortok, and K. Marsh.
1999.
Antibody recognition of Plasmodium falciparum erythrocyte surface antigens in Kenya: evidence for rare and prevalent variants.
Infect. Immun.
67:733-739 |
| 3. | Cranmer, S. L., C. Magowan, J. Liang, R. L. Coppel, and B. M. Cooke. 1997. An alternative to serum for cultivation of Plasmodium falciparum in vitro. Trans. R. Soc. Trop. Med. Hyg. 91:363-365[CrossRef][Medline]. |
| 4. | Curtis, C. F., C. A. Maxwell, R. J. Finch, and K. J. Njunwa. 1998. A comparison of use of a pyrethroid either for house spraying or for bednet treatment against malaria vectors. Trop. Med. Int. Health 3:619-631[Medline]. |
| 5. | Curtis, C. F., and A. E. P. Mnzava. 2000. Comparison of house spraying and insecticide treated nets for malaria control. Bull. W. H. O. 78:1389-1400[Medline]. |
| 6. | Curtis, C. F. 1998. The public health importance of malaria worldwide. Nytt. Norwegian Int. Health Assoc 4:3-5. |
| 7. | Giha, H. A., T. Staalsoe, D. Dodoo, C. Roper, G. M. Satti, D. E. Arnot, L. Hviid, and T. G. Theander. 2000. Antibodies to variable Plasmodium falciparum-infected erythrocyte surface antigens are associated with protection from novel malaria infections. Immunol. Lett. 71:117-126[CrossRef][Medline]. |
| 8. | Lengeler, C. 2000. Insecticide-treated bednets and curtains for preventing malaria. Cochrane Database Syst. Rev. 2:CD000363[Medline]. |
| 9. | Marsh, K., L. Otoo, R. J. Hayes, D. C. Carson, and B. M. Greenwood. 1989. Antibodies to blood stage antigens of Plasmodium falciparum in rural Gambians and their relation to protection against infection. Trans. R. Soc. Trop. Med. Hyg. 83:293-303[CrossRef][Medline]. |
| 10. |
Marsh, K., and R. J. Howard.
1986.
Antigens induced on erythrocytes by P. falciparum: expression of diverse and conserved determinants.
Science
231:150-153 |
| 11. | Metzger, W. G., C. A. Maxwell, and C. F. Curtis. 1998. Anti-sporozoite immunity and impregnated bednets in Tanzanian villages. Ann. Trop. Med. Parasitol. 92:727-729[CrossRef][Medline]. |
| 12. | Piper, K. P., D. J. Roberts, and K. P. Day. 1999. Plasmodium falciparum: analysis of the antibody specificity to the surface of the trophozoite-infected erythrocyte. Exp. Parasitol. 91:161-169[CrossRef][Medline]. |
| 13. | Riley, E. M., L. Hviid, and T. G. Theander. 1994. Malaria, p. 119-143. In F. Kierszenbaum (ed.), Parasitic infections and the immune system. Academic Press, New York, N.Y. |
| 14. | Roberts, D. J., A. G. Craig, A. R. Berendt, R. Pinches, G. Nash, K. Marsh, and C. I. Newbold. 1992. Rapid switching to multiple antigenic and adhesive phenotypes in malaria. Nature 357:689-692[CrossRef][Medline]. |
| 15. | Smith, J. D., C. E. Chitnis, A. G. Craig, D. J. Roberts, D. E. Hudson-Taylor, D. S. Peterson, R. Pinches, C. I. Newbold, and L. H. Miller. 1995. Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes. Cell 82:101-110[CrossRef][Medline]. |
| 16. | Snow, R. W., and K. Marsh. 1995. Will reducing Plasmodium falciparum transmission alter malaria mortality among African children? Parasitol. Today 11:188-190[CrossRef]. |
| 17. | Staalsoe, T., H. A. Giha, D. Dodoo, T. G. Theander, and L. Hviid. 1999. Detection of antibodies to variant antigens on Plasmodium falciparum infected erythrocytes by flow cytometry. Cytometry 35:329-336[CrossRef][Medline]. |
| 18. | Su, X., V. M. Heatwole, S. P. Wertheimer, F. Guinet, J. A. Herrfeldt, D. S. Peterson, J. A. Ravetch, and T. E. Wellems. 1995. The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes. Cell 82:89-100[CrossRef][Medline]. |
| 19. |
Trager, W., and J. B. Jensen.
1976.
Human malaria parasites in continuous culture.
Science
193:673-675 |
| 20. | Tsang, L., C. F. Curtis, and C. A. Maxwell. 2001. The effect of alphacypermethrin impregnated nets on sporozoite rates in Tanzania. Trans. R. Soc. Trop. Med. Hyg. 95:249[CrossRef]. |
Clinical and Diagnostic Laboratory Immunology, November 2001, p. 1289-1291, Vol. 8, No. 6
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.6.1289-1291.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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