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Previous Article | Next Article 
Clinical and Diagnostic Laboratory Immunology, September 1998, p. 721-724, Vol. 5, No. 5
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Immune Response against the Exp-1 Protein of Plasmodium
falciparum Results in Antibodies That Cross-React with Human
T-Cell Lymphotropic Virus Type 1 Proteins
Kevin R.
Porter,1,*
Joao
Aguiar,2
Allen
Richards,3
B.
Sandjaya,4
H.
Ignatias,3
H.
Hadiputranto,3
Robert G.
Ridley,5
Bela
Takacs,5
F. Stephen
Wignall,3
Stephen L.
Hoffman,2 and
Curtis
G.
Hayes1
Viral and Rickettsial Diseases
Program1 and
Malaria
Program,2 Naval Medical Research Institute,
Bethesda, Maryland;
Naval Medical Research Unit 2,
Jakarta,3 and
Department of Health,
Jayapura, Irian Jaya,4 Indonesia; and
Department of Infectious Diseases, F. Hoffmann-La Roche Ltd.,
CH-4070 Basel, Switzerland5
Received 9 February 1998/Returned for modification 17 March
1998/Accepted 11 June 1998
 |
ABSTRACT |
To examine the role of the Plasmodium falciparum Exp-1
blood-stage protein in producing antibodies that cross-react with human T-cell lymphotropic virus type I (HTLV-I) proteins, we studied sera
from Indonesian volunteers who seroconverted to malaria after transmigrating to an area where malaria is hyperendemic. Samples from
Philippine volunteers, that were used in a prior study that examined
malaria antibodies that cross-react with HTLV-I proteins, were also
used. Eighty-three percent of the Indonesian transmigrants developed antibodies against the malaria Exp-1 protein by 6 months postmigration. Of these malaria seroconverters, 27% developed false-positive HTLV-I enzyme immunoassay (EIA) immunoreactivity, as
indicated by indeterminate HTLV-I Western blot banding patterns. Five
of the six Philippine samples tested were HTLV-I EIA false positive and
Western blot indeterminate. When a recombinant Exp-1 protein was used
in blocking experiments, the HTLV-I Western blot immunoreactivity of
sera from both groups was either completely eliminated or greatly
reduced. No effect on the Western blot immunoreactivity of truly
HTLV-I-positive sera was seen. To determine if immunization with
the recombinant Exp-1 protein could elicit the production of HTLV-I
antibodies, six mice were inoculated with the recombinant protein.
Following administration of three 50-µg doses of the protein, four of
the six mice developed antibodies that cross-reacted with HTLV-I
proteins on Western blot. These results indicate that the immune
response against the malaria Exp-1 protein may result in
HTLV-I-cross-reacting antibodies that can lead to
false-positive EIA and indeterminant Western blotting results.
| |
INTRODUCTION |
Plasmodium falciparum is
capable of inducing antibodies that cross-react with human T-cell
lymphotropic virus type I (HTLV-I) proteins to give false-positive
enzyme immunoassay (EIA) results and indeterminate Western blot
patterns (3, 4, 6). The specific malaria proteins
responsible for this immunologic response are unknown. Recent peptide
mapping studies identified a seven-amino-acid epitope, located at the
carboxy-terminal end of the gag-encoded p19 protein of
HTLV-I, that is recognized by P. falciparum antibodies (5). Through a computerized sequence homology search, this p19 epitope was found to be similar to a stretch of seven amino acids
on P. falciparum blood-stage antigen Exp-1. The present study was conducted to clarify the role the Exp-1 antigen in the development of HTLV-I-cross-reacting antibodies. The results provide direct evidence that it is the immune response against this antigen that may produce antibodies that cross-react with several HTLV-I proteins.
| |
MATERIALS AND METHODS |
Study population.
The Indonesian samples used in this study
were pre- and postmigration serum samples that had previously been
obtained from 18 Indonesian volunteers who had migrated from Java,
where malaria is not endemic, to the Arso region of Irian Jaya, where
malaria is hyperendemic. The samples were collected as part of an
earlier study examining malaria transmission rates in Indonesia. All
postmigration samples were positive for P. falciparum
antibodies by immunofluorescence assay. Premigration and 6-month
postmigration serum samples from all volunteers were available. Three-
and 12-month postmigration samples from only two and six volunteers,
respectively, were available.
Previously collected serum samples from six volunteers living in the
Philippines were also used. These samples were obtained as part of a
prior study that examined the cross-reactivity between malaria
antibodies and HTLV-I proteins (3). Two samples that were
malaria and HTLV-I antibody negative and two Western blot HTLV-I-positive serum samples were used as controls. All samples were
stored at 
70°C until used and were obtained after informed consent
had been obtained and used in accordance with approved human use
protocols. The participation of the volunteers was in accordance with
U.S. Navy regulations governing the use of human subjects in medical
research.
Detection of HTLV-I and Exp-1 antibodies.
Samples were
tested for anti-HTLV-I antibodies by EIA (Abbott Laboratories, Abbott
Park, Ill.). A Western blot assay (HTLV-I Blot 2.4; Genelabs
Diagnostics, Singapore, Singapore) was used to confirm EIA-positive
samples. To be classified as Western blot positive, sera had to be
reactive against a gag-encoded protein (p19 or p24) and two
env-encoded proteins (gp46 or rgp46 and GD21). Samples not
meeting these criteria but showing reactivity were considered
indeterminate, and samples with no reactivity were considered negative.
All serum samples were screened by EIA for reactivity against a
recombinant Exp-1 protein and a recombinant DR4a/b antigen as a
control. The Exp-1 recombinant protein (Hoffmann-La Roche, Basel,
Switzerland) was produced in vitro from recombinant plasmid pUC8-5.1
and purified as previously described (1). The Exp-1-encoding gene used to construct the recombinant plasmid was derived from P. falciparum K1 from Thailand. The Exp-1 protein
encoded by this gene is also known as the 5.1 antigen (8).
The control DR4a/b protein consisted of the N-terminal half of the HLA
DR4a1 protein ligated to the N-terminal half of the HLA DR4b1 protein.
Both the Exp-1 and DR4a/b proteins contained a C-terminal hexahistidine tail for purification.
The recombinant proteins were used to coat 96-well plates at a
concentration of 2 µg/ml and reacted with serum samples diluted 1:6,250 with phosphate-buffered saline (PBS). This serum dilution was
chosen after tests with serial fivefold dilutions of negative and
positive control samples showed that a 1:6,250 dilution produced the
lowest signal-to-noise ratio (data not shown). A sample was considered
positive if the Exp-1 optical density (OD) value was at least fivefold
greater than the DR4a/b OD and the mean OD obtained with the negative
control sera. This stringent criterion was chosen to ensure the
elimination of false-positive results due to nonspecific immunoreactivity.
Western blot blocking assays.
Experiments were conducted to
see if the recombinant Exp-1 protein could block the HTLV-I
Western blot immunoreactivity of the study sera. A serum sample from a
Philippine volunteer, that produced a strong but indeterminate HTLV-I
Western blot banding pattern, was first tested to determine the amount
of Exp-1 protein needed to block HTLV-I immunoreactivity. The serum was
diluted 1:500 in PBS alone and in PBS containing 102,
103, and 104 µg of recombinant Exp-1 protein
per ml. The total volume of the diluted serum-protein solution was 500 µl. The diluted samples were then incubated at 4°C overnight with
shaking at 100 rpm. A Western blot HTLV-I-positive serum sample was
used as a control and treated identically. Following the overnight
incubation, the samples were assayed by HTLV-I Western blotting as
described previously (6). To accomplish this, 1.5 ml of kit
blocking solution, containing Exp-1 protein at the concentrations used
in the overnight incubation step, were added to each sample and
subsequently assayed in accordance with the manufacturer's
instructions.
Based on the results obtained with this single sample, a serum dilution
of 1:100 and a protein concentration of 102 µg/ml were
used to assay the remaining samples. A lower serum dilution was used
with these samples because the intensity of the Western blot
indeterminate immunoreactivity was considerably less than that of the
Philippine sample (Fig. 1, lane 11) used to titrate the antigen. Two
samples, one Indonesian and one Philippine, that were HTLV-I EIA
negative and Exp-1 EIA positive were also tested. To control for
nonspecific blocking, samples were also blocked with DR4a/b recombinant
protein at 50 µg/ml.
Statistical analysis.
To look for a correlation between
HTLV-I and Exp-1 antigen EIA positivity, the sample OD values obtained
with these proteins and the DR4a/b protein were grouped according to
the times when they were collected and subjected to correlation
analysis by the software StatView, version 4.5 for Macintosh (Abacus
Concepts, Berkeley, Calif.). The P values for each
comparison were calculated by using Fisher's r to z test.
Mouse immunization with recombinant Exp-1.
Four- to
6-week-old BALB/cByJ mice were immunized with the recombinant Exp-1
protein to elicit HTLV-I-cross-reactive antibodies. Each of six mice
was given three 50-µg subcutaneous injections at 2-week intervals.
The first injection was prepared in complete Freund's adjuvant, and
the subsequent injections were prepared in incomplete Freund's
adjuvant. Serum samples were collected 2 weeks after the final
immunization and tested for immunoreactivity against HTLV-I proteins by
Western blotting. To adapt this assay for use with mouse serum, the
anti-human immunoglobulin G-horseradish peroxidase conjugate was
replaced with an anti-mouse immunoglobulin G-horseradish peroxidase
conjugate. Serum from a mouse immunized with a recombinant dengue virus
antigen in complete Freund's adjuvant was used as a negative control.
This animal was shown to have high titers of dengue virus antibodies,
as demonstrated by enzyme-linked immunosorbent assay and plaque
reduction neutralization assay (data not shown).
| |
RESULTS |
Detection of HTLV-I and Exp-1 antibodies.
All samples from the
Indonesian volunteers were HTLV-I negative by EIA premigration. At 6 months postmigration, 3 of the 18 volunteers had seroconverted to
HTLV-I, as shown by EIA (Table 1).
One volunteer seroconverted 12 months postmigration. Confirmatory Western blot analysis of the EIA-positive samples showed
indeterminate banding patterns, as shown in Fig.
1. Five of the six Philippine samples
tested were HTLV-I positive by EIA, and all five showed indeterminate
Western blot banding patterns (Table 1). One of the Philippine samples
HTLV-I positive by EIA showed relatively weak Western blot
immunoreactivity that was barely visible at the serum dilutions used in
the blocking assay (Fig. 1, lane 7B).
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FIG. 1.
HTLV-I Western blot blocking results obtained with
Indonesian transmigrant and Philippine samples. Lanes: 1, 2, 4, and 5, samples from the four Indonesian transmigrant HTLV-I EIA seroconverters
in Table 1; 3, sample from a volunteer who was HTLV-I negative but
Exp-1 positive by EIA; 6 to 11, Philippine samples; 12, HTLV-I-positive
control; 13, kit positive (P) and negative (N) controls. (A) Samples
preabsorbed and blocked with recombinant Exp-1 protein. (B) Samples
preabsorbed and blocked with HLA DR control recombinant protein. Note
that GD21 is a recombinant env-encoded
glycoprotein that migrates at a faster rate than the other
HTLV-I proteins.
|
|
Serum samples from all Indonesian volunteers were negative for
immunoreactivity to the Exp-1 protein prior to migration. Fifteen of
the 18 Indonesian volunteers seroconverted to the P. falciparum Exp-1 antigen by 6 months postmigration, including the
four who were also shown to have seroconverted to HTLV-I by EIA. No
immunoreactivity was seen against the DR4a/b recombinant protein with
any of the samples (data not shown).
Four of the six Philippine samples tested were positive for Exp-1 by
EIA. One additional sample showed Exp-1 immunoreactivity, but the level
of reactivity was not strong enough to be considered positive. This
sample, however, was positive for HTLV-I by EIA. The two Western blot
HTLV-I-positive control samples did not react with either the Exp-1
protein or the DR4a/b protein.
When the raw OD values for the samples HTLV-I positive by EIA, grouped
according to the times when they were collected, were compared, a
statistically significant correlation between the OD values for the
Exp-1 and HTLV-I proteins was seen (Table
2). The correlation coefficient for this
comparison was 0.755 (P < 0.02). There was no
correlation between the OD values obtained with the HTLV-I and DR4a/b
proteins (data not shown).
Western blot blocking assays.
Figure 1 shows the blocking
results obtained with five Indonesian (four 6-month, one 12-month)
postmigration samples and six Philippine samples by using the
Exp-1 protein and the negative control DR4a/b protein. Two of the
samples used in the blocking assay, one Indonesian (lane 3) and one
Philippine (lane 6), although HTLV-I negative by EIA, did
show indeterminate Western blot patterns with the HTLV-I Blot 2.4 kit.
The Exp-1 protein blocked or greatly reduced the indeterminate
immunoreactivity of all of the Philippine and Indonesian sera tested,
whereas the DR4a/b protein had no effect. The immunoreactivity of the
truly HTLV-I-positive sample was not affected.
Mouse immunization with recombinant Exp-1.
Mice were given
three immunizations with the recombinant Exp-1 protein to see if
HTLV-I-cross-reactive antibodies could be produced. Two weeks after the
final immunization, all animals developed Exp-1 antibody titers of
approximately 1:20,000, as measured by EIA (data not shown). When
assayed by HTLV-I Western blotting, immunoreactivity against the
recombinant GD21 env-encoded protein was seen with four of
the six samples (Fig. 2). One of these
samples also showed weak immunoreactivity against the p24 antigen. The
mouse serum sample that contained high titers of dengue virus
antibodies showed no Western blot immunoreactivity (data not shown).
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FIG. 2.
HTLV-I Western blots of sera from mice immunized with
recombinant Exp-1 proteins (stripes 1 to 4). Sera from two
Exp-1-immunized mice showed no Western blot immunoreactivity, as did
sera from a dengue virus-immune mouse (data not shown).
|
|
| |
DISCUSSION |
Prior studies showed that anti-P. falciparum
antibodies can cross-react with HTLV-I proteins by EIA and Western
blotting. These cross-reactive antibodies recognize a seven-amino-acid
epitope on the HTLV-I p19 protein. The sequence of this epitope is
similar to a stretch of seven amino acids located on the P. falciparum Exp-1 protein, suggesting that this protein has a role
in generating HTLV-I-cross-reacting antibodies. The function of this
protein, as it relates to the P. falciparum life cycle,
is not completely understood. However, it is known that this protein is
exported from the malaria parasite to the parasitophorous vacuole
membrane and to membranous compartments of the erythrocyte
(2). This protein is also expressed on the
infected-hepatocyte surface (7).
Eighty-three percent of the Indonesian transmigrants we studied clearly
demonstrated seroconversion to the P. falciparum Exp-1 blood-stage antigen postmigration to an area where malaria is endemic.
Of those who seroconverted, 27% simultaneously seroconverted to
HTLV-I, as shown by EIA. These seroconversions were false positives, as
indicated by indeterminate Western blot banding patterns obtained upon
confirmatory testing. The simultaneous development of a
false-positive HTLV-I EIA result and a statistically significant
correlation between HTLV-I and Exp-1 OD values provides
suggestive evidence that this malaria protein is responsible for
the production of the HTLV-I antibodies.
The ability of Exp-1 protein to block indeterminate Western blot HTLV-I
immunoreactivity of the Indonesian sera and not the immunoreactivity of
Western blot HTLV-I-positive sera provides conclusive evidence that
this protein elicits antibodies that cross-react with HTLV-I
antibodies. This cross-reactive immune response is not seen in all
Exp-1 antibody-positive individuals. Although the reason for this is
not clear, this observation does suggest that other factors are
involved, such as HLA type and number of malaria re-exposures. However,
the data generated in our study are not sufficient to make any
conclusions regarding the influence of these factors on the generation
of HTLV-I cross-reactive antibodies. Thus, risk factors related to the
development of these antibodies cannot be established. The successful
blocking of indeterminate Western blot HTLV-I immunoreactivity in sera
from Philippine volunteers does indicate that this phenomenon is not
confined to Indonesia.
It is unlikely that the only Exp-1 epitope responsible for these
antibodies is the seven-amino-acid sequence similar to the previously identified HTLV-I p19 malaria antibody-cross-reactive epitope. This view is supported by the fact that although the other
Western blot HTLV-I-reactive proteins have amino acid sequences different from that of the p19 protein, immunoreactivity to these proteins was also blocked or reduced by the recombinant Exp-1 antigen.
The recombinant Exp-1 antigen had no effect on the p19 Western blot
immunoreactivity of truly HTLV-I-positive sera. This result was
not unexpected. We speculate that during HTLV-I infection, antibodies are generated against p19 epitopes different from the malaria antibody-cross-reactive p19 epitopes. Therefore, adsorption with the Exp-1 malaria protein would have little to no effect on the
Western blot p19 immunoreactivity of truly HTLV-I-positive sera.
Two of the samples (one Philippine and one Indonesian), used in the
blocking assay as EIA HTLV-I-negative; Exp-1-positive controls, did
show indeterminate immunoreactivity to HTLV-I by Western blotting. This
Western blot immunoreactivity was also completely eliminated by
blocking with the recombinant Exp-1 protein (Fig. 1, lanes 3 and 6).
This indicates that the cross-reactive immune response of these
volunteers was not strong enough to produce a positive reaction in the
HTLV-I EIA.
The ability of the Exp-1 protein to elicit antibodies that
cross-react with HTLV-I proteins was demonstrated by immunizing mice with the recombinant protein. Immunoreactivity against the HTLV-I
GD21 recombinant env-encoded protein developed in 67% of the mice, and one mouse also showed immunoreactivity against p24. The
immunoreactivity elicited in mice against predominantly a single
HTLV-I protein (GD21) is contrary to the broad HTLV-I
immunoreactivity seen with the human sera. Further, immunoreactivity
against GD21 was not seen with any of the human samples used in this
study, although prior studies with human samples did show definite but infrequent immunoreactivity against this glycoprotein
(6). The differences in the number and type of HTLV-I
proteins recognized by murine compared to human sera could, perhaps, be
explained by the obvious differences between murine and human immune
systems. The longer duration of P. falciparum antigen
exposure in humans compared to the mice we immunized could also explain
why the human sera reacted to more HTLV-I proteins. Volunteers who
transmigrated to an area where malaria is endemic were presumably
exposed repeatedly to P. falciparum over a 6-month
period, unlike the mice, which were immunized over a shorter period of
time. Because data supporting any of these hypotheses are lacking, we
cannot say definitely why murine anti-Exp-1 antibodies react
predominately with the HTLV-I GD21 protein.
We have shown that the unique phenomenon of P. falciparum generating an immune response against HTLV-I proteins
is due to an antibody response directed against epitopes located on the Exp-1 blood-stage protein. Although this immune response may have a
profound impact on screening assays for HTLV-I, the impact of these
antibodies on the biology of the virus remains to be determined.
| |
ACKNOWLEDGMENT |
This work was supported by the U.S. Naval Medical Research and
Development Command for Work Units 61152N MR00001 001 2108 and 63105A
3M263105 DH29 AA1.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: U.S. NAMRU 2, Box 3, Unit 8132, APO AP, 96520-8132. Phone: 62 21 421-4457. Fax: 62 21 424-4507. E-mail: porterkr{at}namru2.com.
| |
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Clinical and Diagnostic Laboratory Immunology, September 1998, p. 721-724, Vol. 5, No. 5
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
