Immunology Letters, 25 (1990), 237-242

Elsevier

IMLET 01459

Reduced cellular immune reactivity in healthy individuals during

the malaria transmission season
T h o r G. T h e a n d e r ~,2, Lars Hviid 1, 2, Yousif A. Abu-Zeid 5, N a s r E l d i n H. A b d u l h a d i 5, Bakri O.
Saeed 5, Palle H. J a k o b s e n 3, Claus M. Reimert 1, Soren Jepsen 4, Riad A. L. B a y o u m i 5 a n d James
B. Jensen 6
1Department of Infectious Diseases, State University Hospital, Copenhagen, Denmark, 2Institute of Medical Microbiology,
University of Copenhagen, Copenhagen, Denmark, 3Malaria Research Laboratory and 4WHO National Malaria Diagnostic
Center, State Serum Institute, Copenhagen, Denmark, 5Department of Biochemistry, University of Khartoum, Khartoum, Sudan
and 6Department of Microbiology, Brigham Young University, Provo, UT, US.A.

1. Summary years. Where malaria occurs in its unstable extreme

the entire population is vulnerable regardless of age,
Antigen-induced cellular immune responses are and inoculation tends to be followed by clinical dis-
suppressed during acute malaria. The present study ease. In areas where the population is exposed to fre-
engages the possibility that malaria-induced altera- quent heavy inoculations, the individuals gradually
tions in cellular immune reactivity extend beyond the acquire protection against clinical attacks over sever-
clinical disease. Thus, lymphoproliferative responses al years [2- 4], and the disease is thus mainly affect-
of healthy individuals were diminished during the ing children. These observations indicate that malar-
malaria transmission period in individuals living in ia parasites obstruct immune development by
an area of highly seasonal, unstable malaria trans- processes in which both antigenic diversity of the
mission. This finding may have important implica- parasites, and immunosuppression caused by the
tions for the design of studies of stimulatory proper- parasites are involved. The objective of our studies
ties of antigens using lymphocytes of endemic in the Sudan is to investigate these matters by follow-
origin. ing the interaction between the parasite and the hu-
man immune system by relating the parasite popula-
2. Introduction tions, the intensity of malaria transmission, and the
malaria morbidity to immune parameters over a
The protection against malaria acquired by in- number of years.
dividuals living in malaria endemic areas is largely The purpose of the present study was to evaluate
governed by the transmission pattern. MacDonald the impact of malaria transmission on the immune
[1] pointed to two epidemiological extremes between reactivity of healthy adults living in an area of unsta-
which the disease exists, one where instability is the ble and highly seasonal malaria-transmission. Im-
dominant feature and malaria is characterized by mune parameters of 46 healthy donors were com-
either substantial seasonal fluctuations in incidence pared before and during the malaria transmission
or by cyclical epidemics, and the other where trans- season. It was found that the in vitro proliferative re-
mission and morbidity is stable over the seasons and sponse of peripheral blood mononuclear cells
(PBMC) after stimulation with malaria antigen
(SPag) and purified protein derivative of tuberculin
Key words." Plasmodium falciparum; Malaria; Lymphocyte; Im- (PPD) were significantly depressed during the
munosuppression; Epidemiology malaria transmission season, wheras the responses
Correspondence to: Thor G. Theander, Lymphocyte Laboratory, to phytohemagglutanin (PHA) were unaffected.
Dept. of Infect. Dis. M7641, State University Hospital, Tagensvej
20, DK-2200 Copenhagen N., Denmark. Fax: +45-31-39 87 66.

0165-2478 / 90 / $ 3.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division) 237
3. Materials and Methods trophoresis. Proliferative responses to SPag has
earlier been shown to reflect a specific activation of
3.1. Study area in vivo primed T cells [8 - 11]. SPag was used at dilu-
tions of the stock solution previously shown to in-
This study was done in Daraweesh village in the duce optimal proliferation in vitro.
Eastern Province, Sudan. The region is characterized
by highly seasonal, unstable malaria transmission 3.4. Blood sampfing and PBMC preparation
[5, 6]. Daraweesh is inhabited by 42 families, of
which the gene for sickle cell anemia (HbS) is present Blood was collected by venipuncture into
in 14 families. The study population for this report heparinized vacutainers (Becton-Dickinson Ltd.,
was the 257 individuals of the 28 families in which Rutherford, N J, U.S.A.). Following centrifugation,
the sickle cell gene was absent. plasma was collected and stored frozen at - 2 0 °C.
The malaria endemicity and age-related distribu- The pellet was resuspended to twice the original vol-
tion of parasite burdens were estimated by measure- ume in RPMI-1640 supplemented with 58.4/xg/ml
ments of parasite point prevalence by collection and L-glutamine, 20 U/penicillin and 20 txg/ml strep-
examination of blood films, once in the dry season, tomycin (all from Gibco, Paisley, U.K.) PBMC were
once in the beginning and once towards the end of then isolated by density centrifugation on Lym-
the malaria transmission (wet season). Malaria phoprep (Nyegaard, Oslo, Norway), washed 3 × in
chemoprophylaxis is not used in Daraweesh village. medium supplemented with 10% heat-inactivated
fetal calf serum (FCS), and frozen by controlled gra-
3.2. Donors dient freezing to - 1 9 6 ° C in RPMI 1640 sup-
plemented with 10% dimethyl-sulfoxide and 20%
Forty-six adults (31 women and 15 men) volun- FCS. At the day of use, PBMC were quickly thawed
teered to be bled once during the dry season (June) to 37 °C and washed 3 x in the medium used for the
1988) and once during the wet period assays. Cell viability upon thawing was > 90%.
( O c t o b e r - D e c e m b e r 1988). The age range of the
donors was 1 5 - 6 0 years. Prior to each blood sam- 3.5. Lymphoproliferation assay
piing, all donors were examined clinically, including
measurement of body temperature, and anamnestic For each donor the PBMC collected during the
information was obtained (previous disease epi- dry and the wet season were thawed and assayed in
sodes, malaria therapy, complaints of chronic or in- parallel to avoid day-to-day variation.
termittent fevers, headaches, joint pains, vomiting, Each well of 96-well round-bottomed microtiter
diarrhea, cough). None of the donors enrolled in this plates (Nunc, Roskilde, Denmark) received 1 x 105
study had clinical symptoms of malaria at the time PBMC in 150 #1 RPMI-t640 supplemented with
of either sampling. Parasites were found in 5 donors 15°70 heat-inactivated pooled human serum (NHS),
at the time of the wet-season bleed; no donors were 58.4 #g/m L-glutamine, 20 I.U./ml penicillin and
parasitemic at the time of the dry season sampling. 20/~g/ml streptomycin. Twenty #1 of antigen were
added to test cultures, whereas control cultures
3.3. Antigens received 20 ~1 medium. The cultures were incubated
at 37 °C in a humidified atmosphere containing 5%
PPD. Purified protein derivative of tuberculin CO 2 for 7 days, and pulsed by 20/~l/well
(PPD) (State Serum Institute, Copenhagen, Den- [3H]thymidine (New England Nuclear, Boston,
mark) was used at a final concentration of 12/zg/ml. MA, U.S.A.) (1.85 MBq/ml) for the last 24 h of incu-
bation. Cultures were harvested onto glassfiber
SPag. Soluble purified antigens (SPag) were isolated filters and the incorporation of [3H]thymidine into
from supernatants of P. falciparum in vitro cultures DNA was determined by liquid scintillation spec-
by affinity chromatography as described elsewhere trometry. All tests were done in triplicate.
[7]. The resulting preparation contains 7 distinct sin- Responses were considered significant if the incre-
gle antigens detectable by crossed immunoelec- ment (kCPMstim - kCPMunstim), A kCPM > 1, and

238
the stimulation index (kCPMstim/kCPMunstim), ma/cm 2. Plates were washed and pressed 3 x and
SI > 1/A kCPM) + 2. The algorithm was devised to stained by Coomassie Brilliant Blue.
discriminate against cultures showing only little in-
crease in [3H]thymidine incorporation likely to be 3.8. Statistical analysis
due to stocastic variation, despite an SI > 2.5, a com-
mon significance criterion. The Wilcoxon signed ranks test for paired differ-
ences (T +) was used for comparisons of lym-
3.6. Pf155/RESA IFA phoproliferative responses. Ln(CPM) values were
used in the testing, and responses regarded non-
Plasma titres of anti-Pf155/RESA antibodies significant according to the criteria described above
were determined by the method described by were arbitrarily assigned the CPM-value 100. Two-
Perlmann et al. [12]. Briefly, 8-well Toxoplasma tailed P-values < 0.05 were considered significant.
slides (Bellco Glass Co., Vineland, N J, U.S.A.) were The Spearman rank-order correlation coefficient
coated with carbonate-bicarbonate buffer (pH 9.6), (rs) was used for evaluation of parameter associa-
followed by addition of 20 ~1 Tris-buffered Hanks' tion. The fact that pairwise association between
saline (TBH) containing a 0.5% suspension of many parameters were tested for should be consid-
young ring-stage P.falciparum-infected erythrocytes ered when evaluating the significance of the ob-
(parasitemia 5-10°70). After 30 min, the slides were tained P-values. Thus the significance of associa-
dipwashed in TBH and air-dried. Wells were then tions should be confirmed in future studies before
treated sequentially with 20 tzl test plasma, bio- firm conclusions can be drawn.
tinylated anti-human IgG antibodies, and fluores-
cein isothiocyanate conjugated avidin. Slides were 4. Results
washed between incubations in TBH supplemented
with 0.05°7o Tween 20. 4.1. Parasite point prevalence rates

3.7. Crossed immuno-electrophoresis No parasites were found in samples collected dur-

ing the dry season. Table 1 shows results of the
Detection of antibodies to SPag-components by parasite-screenings in the beginning and towards the
crossed immuno-electrophoresis (CIE) has been end of the transmission season. The point preva-
described in detail previously [13]. In brief, 20/~1 lences of parasitemic individuals were 18% and
SPag was run in the first dimension gel at 10070, respectively. The point prevalence rates varied
10-15 V/cm until a parallel albumin marker had somewhat between the age groups, but no pattern
migrated 2.6 cm. The second dimension electrophor- was apparent, even when examining data from in-
esis was then run perpendicular to the first at dividuals with more than one parasite per field only.
2 V/cm for 18 h into a gel containing 16/~1 test plas-

TABLE 1

Results of the two malaria screenings (October/December) in Daraweesh village during the malaria transmission season.

Age interval Individuals Parasitemic > I parasite/field Gametocytemic

(years) (number) (%) (o70) (%)

0- 3 29/20 10/10 3/10 0/5

4- 7 37/27 16/ 7 14/ 4 0/4
8-12 36/ 32 22/12 14/ 9 6/6
13 - 20 30/23 17/13 13/13 4/9
2 1 - 30 21/15 24/14 10/ 7 10/0
31 - 80 29/28 17/ 7 10/ 4 0/0

All 182/145 18/10 11/ 8 3/4

239
 SPog PPD
60 -A 100 B,

45 75.

13_ 30 13_ 50
o (D

15 25

0
DRY WET DRY WET

PHA

120 Fig. 1. Lymphoproliferativeresponses of paired PBMC-samples

C obtained in the dry and wet season. Panel A, SPag; Panel B, PPD;
Panel C, PHA. The responses of each donor are connected by
lines, and the dry and wet season median responses of all donors
are shown as columns.
90

the wet season (Fig. 1A, B). The responses during the
wet season were significantly lower than the
13_ 60 responses during the dry season (P(T+)<0.001
o
(SPag) and P ( T + ) < 0 . 0 2 (PPD). The median re-
sponse to SPag and P P D during the dry and the wet
season decreased from 9.9 kCPM to 3.0 kCPM, and
30 from 20.1 kCPM to 11.5 kCPM, respectively. To in-
vestigate the possibility that low responses in donors
with asymptomatic parasitemia could explain the
observed differences, the 5 donors with parasitemia
0 at the time of the wet season sampling were excluded
DRY WET
from the statistical analysis. Despite this, the differ-
ences between the responses during the dry and wet
period remained significant (P(T +) < 0.003 (SPag),
4.2. Lymphoproliferation P(T +) < 0.01 (PPD)). The responses to P H A were
not significantly different between the dry and the
The lymphoproliferative responses of the 46 adult wet period (Fig. IC).
donors tested before (dry season) and during (wet The proliferative responses of samples obtained
season) the malaria transmission are shown in Fig. 1. during the wet and the dry season were positively
The majority of donors had lower responses to SPag correlated (PHA, P(r S = 0.63)<1x10-5); PPD,
and P P D in the wet season than in the dry season, P(r s = 0 . 5 5 ) < 2 x 10-4); SPag, P(r = 0.49)<0.02).
although some donors had higher responses during The dry season responses to P H A were negatively

240
correlated to age (P(rs = - 0 . 3 4 ) < 0 . 0 3 ) . Apart dividuals suffering from acute malaria. In those
from this no conspicuous correlations were found studies lymphocytes from individuals with acute P.
between proliferative responses and sex or age of the falciparum malaria were shown to be unable to re-
donors. No correlations were found between any spond or to respond only weakly to malaria antigens
anamnestic information and proliferative responses. in proliferative assays, but that the ability to respond
was restored after treatment [16-22]. The present
4.3. Serological parameters study indicates that malaria-induced alterations of
immune reactivity can extend beyond the acute
Pf155/RESA titres were generally low or absent. stages of the disease. Several possibilities could ex-
Antibodies were detected in 11/46 dry season sam- plain the observed reduction in responsiveness of
ples, and in 16/46 wet season samples. Positive titres wet-season PBMC to PPD and SPag. One possibility
ranged from 1:20-1:2560. is a lowered frequency of reactive lymphocytes in the
In the dry season samples age and Pf155/RESA peripheral circulation, maybe due to homing to lym-
titres correlated, (P(rs = 0.45)<0.002), whereas a phoid tissues [23], another that the lymphocytes may
parallel correlation was not found in the wet season be less responsive, perhaps due to immunosuppres-
samples. Dry season Pf155/RESA titres were also sive mechanisms. In support of the second hypothe-
correlated to the lymphoproliferative responses to sis, Riley et al. [24] showed that removal of CD8 ÷
PPD and SPag during the wet season (both cells increased SPag-induced proliferative responses
P(r s = 0.32) < 0.03). in Gambian donors with low SPag responses. Fur-
Antibodies against one or more of the compo- thermore other studies have shown that im-
nents of SPag were detected by CIE in 32/43 dry sea- munosuppressive factors can be purified from P fal-
son samples and in 27/44 wet season samples. No ciparum cultures [25], and are present in the serum
correlation was found between the presence of anti- of patients infected with malaria [26]. If im-
bodies to SPag and the proliferative response to any munosuppressive mechanisms are operating, it is
of the antigens. tempting to suggest that these contribute to the slug-
gish immune development in the individuals of our
5. Discussion study area, who, despite frequent exposure to the
parasites, seem unable to develop an immunity that
In this longitudinal study, we compared the controls the multiplication of the parasites in the
proliferative responses of PBMC obtained before peripheral blood.
(dry season) and during the malaria transmission We did not find any correlations between any of
period (wet season) from healthy adults living in an the anamnestic information obtained and antigen-
area of highly seasonal and unstable malaria trans- induced proliferative responses. It has previously
mission. Our results demonstrate that the lym- been shown that anamnestic information is an in-
phoproliferative responses to both the malaria anti- sufficient instrument when evaluating malaria mor-
gen SPag and to PPD were significantly lowered in bidity [27], and the absence of expected correlations
the samples collected in the wet period, whereas in this study may be due to this.
PHA-induced responses were unaffected. It has The correlation between the presence of
previously been suggested that asymptomatic Pf155/RESA antibodies in the dry season and the
parasitemia is associated with reduced lym- proliferative responses to PPD and SPag in the wet
phoproliferative responsiveness to malaria antigen season suggests that individuals with antibodies
[14, 15]. However, the differences found in this study against Pf155/RESA at the beginning of the trans-
can not be explained by that phenomenon alone, mission season are less prone to immunosuppression
since the differences between the results obtained be- during the transmission season. However, the sig-
fore and during transmission remained when data nificance of this result should be confirmed in future
from donors with asymptomatic parasitemia were studies.
excluded from analysis. Proliferation assays are subject to considerable
Most earlier studies on malaria-induced altera- day to day variation [28], and the possibility of de-
tions of immunoreactivity have focussed on in- tecting differences in cellular immune reactivities in

241
l o n g i t u d i n a l studies are i n c r e a s e d if i n t e r a s s a y v a r i a - Y. A., Abdulhadi, N. H., Jepsen, S., Bayoumi, R. A. L.,
t i o n is m i n i m i z e d [29]. C o n s e q u e n t l y P B M C were Bendtzen, K., Jensen (1990) Acta Pathol. Microbiol. Scand.,
c r y o p r e s e r v e d in t h e field, a n d d r y a n d wet s e a s o n in press.
[12] Perlmann, H., Berzins, K., Wahlgren, M., Carlsson, J.,
s a m p l e s o f e a c h d o n o r were t h a w e d a n d tested in
Bj6rkman, A., Patarroyo, M. E. and Perlmann, P. (1984) J.
parallel. Exp. Med. 159, 1686.
In c o n c l u s i o n we f o u n d t h a t l y m p h o c y t e r e a c t i v i t y [13] Jepsen, S. and Axelsen, N. H. (1980) ActaPathol. Microbiol.
to a n t i g e n s f l u c t u a t e a c c o r d i n g to m a l a r i a t r a n s m i s - Immunol. Scand. (C) 88, 263.
sion. T h i s e m p h a s i z e s t h a t it is i m p o r t a n t to c h a r a c - [14] Theander, T. G., Bygbjerg, I. C., Jacobsen, L., Jepsen, S.,
Larsen, P. B. and Kharazmi, A. (1986) Trans. R. Soc. Trop.
terize the epidemiological setting when evaluating Med. Hyg. 80, 1000.
t h e a b i l i t y o f a n t i g e n s to s t i m u l a t e l y m p h o c y t e s . [15] Petersen, E., Hogh, B., Perlmann, H., Kabilan, L., Troye-
Blomberg, M., Marbiah, N. T., Hanson, A. P., Bj6rkman,
Acknowledgements A. and Perlmann, P. (1989) Am. J. Trop. Med. Hyg. 41,394.
[16] Wyler, D. J. and Brown, J. (1977) Clin. Exp. Immunol. 29,
401.
J. D a l s t e n is t h a n k e d for excellent t e c h n i c a l as- [17] Troye-Blomberg, M., Perlmann, H., Patarroyo, M. E. and
sistance. T h i s s t u d y r e c e i v e d f i n a n c i a l s u p p o r t f r o m Perlmann, P. (1983) Clin. Exp. Immunol. 53, 345.
the UNDP/World Bank/WHO Special Programme [18] Troye-Blomberg, M., Romero, P., Patarroyo, M. E., Bj6rk-
f o r R e s e a r c h a n d T r a i n i n g in T r o p i c a l D i s e a s e s g r a n t man, A. and Perlmann, P. (1984) Clin. Exp. Immunol. 58,
380.
no. 870398, t h e D a n i s h M e d i c a l R e s e a r c h C o u n c i l [19] Theander, T. G., Bygbjerg, I. C., Andersen, B. J., Jepsen, S.,
g r a n t no. 12-7311, t h e D a n i s h R e s e a r c h C o u n c i l for Kharazmi, A., Odum, N. (1986) Scand. J. Immunol. 24, 73.
D e v e l o p m e n t a l R e s e a r c h g r a n t no. 104. D a n . 8 / 3 5 7 [20] Ho, M., Webster, H. K., Looareesuwan, S., Supanaranond,
a n d N I A I D ( N I H ) g r a n t no. AI-16312. W., Phillips, R. E., Chanthavanich, P., Warrell, D. A. (1986)
J. Infect. Dis. 153, 763.
[21] Ho, M., Webster, H. K., Green, B., Looareesuwan, S., Kong-
References chareon, S., White, N. (1988) J. Immunol. 141, 2755.
[22] Riley, E. M., Andersson, G., Otoo, L. N., Jepsen, S. and
[1] MacDonald, G. (1957) The Epidemiology and Control of Greenwood, B. M. (1988) Clin. Exp. Immunol. 73, 17.
Malaria. Oxford University Press, London. [23] Playfair, J. H. L. and De Souza, J. B. (1982) Immunology
[2] Christophers, S. R. (1924) Ind. J. Med. Res. 12, 273. 46, 125.
[3] Miller, M. J. (1958) Trans. R. Soc. Trop. Med. Hyg. 52, 152. [24] Riley, E. M., Jobe, O. and Whittle, H. C. (1989) Infect. Im-
[4] Bruce-Chwatt, L. J. (1963) W. Aft. Med. J. 12, 141 and 199. mun. 57, 1281.
[5] Bayoumi, R. A., Bashir, A. H. and Abdulhadi, N. H. (1986) [25] Riley, E. M., Jobe, O., Blackman, M., Whittle, H. C. and
Am. J. Trop. Med. Hyg. 35, 45. Greenwood, B. M. (1989) Infect. Immun. 57, 3181.
[6] Jensen, J. B., Boland, M. T., Allan, J.S., Carlin, J. M., [26] Theander, T. G., Svenson, M., Bygbjerg, i. C., Kharazmi,
Vande Waa, J. A., Divo, A. and Akood, M. A. S. (1983) In- A., Jepsen, S., Andersen, B. J. and Larsen, P. B. (1987) Acta
fect. Immun. 41, 1302. Pathol. Microbiol. Immunol. Scand. (C) 95, 257.
[7] Jepsen, S., Hindersson, P. and Axelsen, A. (1985) Trans. R. [27] Snow, R. W., Menon, A. and Greenwood, B. M. (1989) Ann.
Soc. Trop. Med. Hyg. 80, 534. Trop. Med. Parasitol. 83, 321.
[8] Bygbjerg, I. C., Jepsen, S., Theander, T. G. and Odum, N. [28] Merker, R., Check, 1. and Hunter, R. L. (1979) Clin. Exp.
(1985) Clin. Exp. Immunol. 59, 421. Immunol. 38, 116.
[9] Theander, T. G., Bygbjerg, I. C., Jepsen, S., Svenson, M., [29] Oldham, R. K., Dean, J. H., Cannon, G. B., Ortaldo, J. R.,
Kharazmi, A., Larsen, P. B. and Bendtzen, K. (1986) Infect. Dunston, G., Applebaum, E, McCoy, J. L., Djeu, J. and
Immun. 53, 221. Herberman, R. B. (1976) Int. J. Cancer 18, 145.
[10] Riley, E. M., Jepsen, S., Andersson, G., Otoo, L. N. and
Greenwood, B. M. (1988) Clin. Exp. Immunol. 71, 377.
[11] Hviid, L., Theander, T.G., Jakobsen, P. H., Abu-Zeid, (Accepted for publication 15 May 1990)

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