- Email: [email protected]
Flow cytometric analysis of IgG reactive to parasitized red blood cell membrane antigens in Plasmodium falciparum-immune individuals
Acta Tropica 73 (1999) 175 – 181 www.elsevier.com/locate/actatropica
Flow cytometric analysis of IgG reactive to parasitized red blood cell membrane antigens in Plasmodium falciparum-immune individuals Idrissa Drame a, Babacar Diouf a, Andre´ Spiegel b, Olivier Garraud a, Ronald Perraut a,* a
Unite´ d’Immunologie, Institut Pasteur de Dakar, 36 A6. Pasteur, BP-220 Dakar, Senegal b Unite´ d’Epide´miologie, Institut Pasteur de Dakar, BP-220 Dakar, Senegal
Received 10 December 1998; received in revised form 4 March 1999; accepted 2 April 1999
Abstract Antigens exposed at the surface of Plasmodium falciparum parasitized red blood cells (pRBCs) represent potential targets for protective antibodies involved in opsonization and immune phagocytosis of pRBCs. We measured the recognition of parasitized red blood cell membrane associated antigens by IgG in the plasma of clinically immune individuals by flow cytometry and ELISA. The plasmas were selected on the basis of preexisting IgG antibodies to pRBC membrane associated recombinant proteins. In every plasma sample IgG could bind the surface of live pRBCs in flow cytometry. In addition, there was a significant correlation between the level of IgG recognition of live pRBCs and of pRBC membrane ghost proteins or major identified antigens by ELISA. Flow cytometry thus represents a technique suitable to test for the accessibility and potential functionality of IgG antibodies directed to antigens expressed by the surface of pRBCs. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Plasmodium falciparum; Erythrocyte membrane; Surface antigens; IgG; Flow cytometry
* Corresponding author. Tel.: + 221-839-92-45; fax: + 221-839-92-10. E-mail address: [email protected] (R. Perraut) 0001-706X/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 9 9 ) 0 0 0 2 6 - 1
176
I. Drame et al. / Acta Tropica 73 (1999) 175–181
1. Introduction There is some evidence that protection from Plasmodium falciparum infection is partly based on antibody (Ab) responses to diverse parasite antigens (Ag) involving several effector mechanisms such as immune phagocytosis of opsonized parasitized red blood cells (pRBCs) which targets Ags expressed at the surface of pRBCs (Celada et al., 1982; Groux et al., 1990; Gysin et al., 1993; Dubois and Pereira da Silva, 1995). Several Ags were shown to be constitutive of pRBC membranes such as PfEMP3 (Le Scanf et al., 1997) or to be associated with pRBC membranes, such as PfEB200 and R23 (Gysin et al., 1993; Perraut et al., 1995). Recombinant proteins derived from these Ags were capable of inducing strong Ab responses in immunized monkeys (Perraut et al., 1995, 1997). Further, they were consistently recognized by naturally acquired Abs in humans continuously exposed to P. falciparum (Perraut et al., 1999). The present work has focused on naturally acquired IgG Abs to pRBC membrane associated proteins, in a number of selected individuals living in endemic areas, who proved clinically immune. The binding of naturally acquired plasma IgG to the surface of live P. falciparum-infected RBCs was assessed by means of flow cytometry.
2. Materials and methods Blood donors were individuals followed up for many years (Trape et al., 1994) and who did not suffer from malaria attack for at least 6 months prior the present study. They were over 12 years of age in the village of Dielmo where P. falciparum transmission is intense and perennial, and over 33 years of age in Ndiop, where transmission is moderate and seasonal (Trape et al., 1994). The age thresholds were estimated to correlate with stable acquired immunity in these locations (Rogier et al., 1996). Sampling was done after informed consent, at the end of the rainy season. A selection of individual samples with or without IgG Abs reactive to the pRBC membrane associated Ags under consideration was done by means of ELISA. Microtiter plates were coated with 1 mg/ml of R23, PfEB200, PfEMP3/ clone 5 or 20 mg/ml of crude pRBC ‘ghost’ proteins, prepared according to the technique described by Fairbanks et al. (1971). Plasma IgG detection of crude Ag and of recombinant proteins was done exactly as described previously (Aribot et al., 1996; Nguer et al., 1997). The PfEB200 antigen derives from Pf332 and it contains 13 repeats with characteristic Glu–Glu dimers and is accessible onto the infected red blood cell surface in late shizonts (Mattei and Scherf, 1992); R23 derives from the central domain of the R45 antigen: it consists of a series of stretches of 11 repeats whose consensus sequence is HKSDS N/S/H (Bonnefoy et al., 1992). Clone 5 is a recombinant protein derived from the PfEMP3 C-terminal domain and was identified as a target of variant immune response (Le Scanf et al., 1999); expression level of PfEMP3 was shown to be modulated during antigenic variation, with some variants expressing high levels while others expressed reduced amounts of protein (Le Scanf et al., 1997).
I. Drame et al. / Acta Tropica 73 (1999) 175–181
177
To test for the recognition of live pRBCs by individual plasma, a cytofluorimetric technique was used as described by Jouin et al. (1995). A ‘knob’-positive Uganda Palo Alto FUP strain of P. falciparum maintained in continuous culture was used a source of pRBCs. Schizonts were harvested after Plasmagel® concentration when parasitaemia reached approximately 10%. Membrane bound IgG were revealed by means of a first incubation of 30 min at 37°C with plasma diluted 1/20 followed by an additional 30 min incubation at 37°C with phycoerythrin (PE)-conjugated goat anti-human IgG (1/200; Cappel Organon Teknica, West Chester, PA). Live parasites were then labelled for 30 min at 37°C in the dark with Thiazol Orange (TO) (Retic-Count®; Becton-Dickinson, San Jose, CA). Fluorescence was read within 1 h. After gating on the TO + pRBCs (FL1] 101; Fig. 1A), PE-labelled membrane bound IgG were measured in the FL2 channel where two fluorescence levels were
Fig. 1. Histograms of flow cytometry data acquisition. Acquisition of 10 000 events was done after gating on FL1. TO-positive cells (dot plot A) were recognized by human IgG and measured in the FL2 channel (dot plot B). Positive fluorescence values were selected in the P2 region (B). (C) This shows pRBCs recognized by a reference negative control. (D) This shows pRBCs recognized by a positive control. (E, F) This show characteristic data obtained with samples from Dielmo (E) and Ndiop (F). The data are expressed as mean levels of positive fluorescence and percent of labelled cells.
178
I. Drame et al. / Acta Tropica 73 (1999) 175–181
discerned, corresponding to the negative background (B 101; P1) and to the positive values (] 101; P2) (Fig. 1B). A pool of 30 plasmas with elevated IgG levels to R23, PfEB200 and/or PfEMP3/clone 5 was referred to as the positive control and a pool of plasma obtained from P. falciparum-unexposed Europeans was referred to as the negative control (Senegalese unexposed individuals were checked negative as well). An indice (iP) for the quantification of pRBC recognition was calculated from the mean fluorescence intensities (MFI) multiplied by the percent of positive events in region ‘P2’. These iP values were then compared individually with actual OD values for plasma IgG to ‘ghost’ proteins, and to arbitrary values corresponding to IgG reactive to recombinant proteins: OD values B 0.1 were scored 0; OD values ranging from 0.1 to 0.5 were scored 1; and OD values over 0.5 were scored 2. Statistical correlation between non-normally distributed paired data was performed by means of the Spearman rank test.
3. Results and discussion We have evaluated the capacity of naturally acquired IgG responses that contained defined Abs to Ags expressed on the surface of P. falciparum-parasitized RBCs (Gysin et al., 1993; Jouin et al., 1995), to bind live pRBCs by means of a fluorescence flow cytometric technique. We have compared this data with individual levels of plasma Abs to three recombinant proteins known to be associated with P. falciparum blood stage Ags and shown to either protect monkeys in eliciting strong opsonizing Ab responses (Perraut et al., 1995) or to be associated with a certain degree of protection (Le Scanf et al., 1997; Perraut et al., 1997). Plasma samples from individuals living in Dielmo (n=18) and in Ndiop (n= 6) were selected with different levels of specific reactivity to R23 and/or PfEB200 and/or PfEMP3/clone 5, three villagers were selected as negative individuals. As can be seen in Fig. 1, individual plasma was capable of binding the surface of pRBCs (Fig. 1E and F), compared with the positive (Fig. 1D) and the negative control plasma (Fig. 1C). Of note, every plasma tested contained functionally active IgG capable of specifically binding the surface of pRBCs despite some individual variation; furthermore, most of the fluorescent Ag – Ab complexes were labelled with high intensity (FL2] 102). We have then calculated the iP indices, showing that the mean indices in Dielmo and in Ndiop individuals were 99 and 106, respectively; this difference is not significant (for comparisons, indices for the positive and negative controls were 156 and 10, respectively; P =0.64). This confirms that clinically immune individuals have acquired strong IgG responses directed to determinants expressed by pRBC membranes. The heterogeneity within individual indices could be related to the possibility that a fraction of Abs has bound circulating pRBCs (Trape and Rogier, 1996). A limitation of the technique was found to be the antigenic variation of parasites in cultures since long-term cultured parasites constantly modify the expression of surface Ags (Iqbal et al., 1997). Such a variation in the availability of parasites originating from in vitro culture and suitable for flow cytometry was not observed in previous work which used ex vivo obtained pRBCs in the Saimiri
I. Drame et al. / Acta Tropica 73 (1999) 175–181
179
Fig. 2. Comparison of pRBC membrane specific IgG levels by flow cytometry and ELISA in individuals living in Dielmo and Ndiop. (A) Individual samples were classified according to the magnitude of live pRBC recognition: the histograms indicate individual percent of specific IgG responses compared to the positive control (100%), by means of flow cytometry performed on live pRBCs (dark histograms) and detection of Abs to crude P. falciparum ‘ghost’ proteins by specific ELISA (white histograms). (B) OD values of IgG to recombinant proteins in individual plasma are shown as follows: open boxes, no detectable Ab (OD B 0.1); shadowed boxes, positive Ab (0.1 5OD B0.5); filled boxes, high Ab levels (OD ]0.5).
monkey (Jouin et al., 1995; Perraut et al., 1995, 1997). These technical difficulties could be overcome by improving the selection of parasite strains suitable for pRBC membrane analyses on a wider scale. To further examine the fine specificity of this set of data obtained using flow cytometry, we have compared individual iP indices with conventional P. falciparumspecific IgG detection by mean of ELISAs. Fig. 2.A shows the comparison of percent responses versus controls (100%) of individual plasma tested for pRBCmembrane Ag binding (flow cytometry) and protein recognition (ELISA where captured Ags consisted of a crude ‘ghost’ membrane pRBC preparation). Despite individual fluctuations, IgG responses measured by means of these two different techniques are consistent, and the positive correlation was statistically significant (P= 0.012). In addition, Fig. 2B shows individual scores for plasma IgG to R23, PfEB200 and PfEMP3/clone5. The comparison between iP indices and individual scores for IgG Abs to either recombinant protein (measured by ELISA) proved to be non significantly associated. However, when individual iP indices were compared with the mathematic summation of individual scores for all three recombinant proteins, there was a significant positive relationship with pRBC membrane associated Ab recognition (P =0.042), irrespective of P. falciparum transmission conditions in the field. This study also reveals the relatively constant prevalence of Abs to membrane associated pRBC Ags in the plasma of individuals living in endemic areas; it further shows that such Abs, which include IgG Abs to defined proteins considered as
180
I. Drame et al. / Acta Tropica 73 (1999) 175–181
vaccine candidates (Dubois and Pereira da Silva, 1995; Perraut et al., 1997), may contribute to the effective binding of live pRBCs, demonstrating their potential functional properties.
Acknowledgements The authors are indebted to Drs O. Puijalon, D. Mattei, P. Dubois, S. Bonnefoy, J. Gysin and H. Jouin (Institut Pasteur, Paris) for fruitful discussions and for providing essential reagents. They acknowledge Dr C. Le Scanf (Institut Pasteur de la Guyane Franc¸aise) and M. Guillotte for technical help. They also thank Dr G. Yap (NIAID/NIH, Bethesda) for fruitful comments and a critical review of the manuscript. This work was supported in part by grants from Institut Pasteur Fondation and from the French Ministe`re de la Coope´ration et du De´veloppement.
References Aribot, G., Rogier, C., Sarthou, J.-L., et al., 1996. Pattern of immunoglobulin isotype response to Plasmodium falciparum blood-stage antigens in individuals living in a holoendemic area of Senegal (Dielmo, West Africa). Am. J. Trop. Med. Hyg. 54, 449 – 457. Bonnefoy, S., Guillotte, M., Langsley, G., Mercereau-Puijalon, O., 1992. Plasmodium falciparum: Characterization of gene R45 encoding a trophozoite antigen containing a central block of six amino acid repeats. Exp. Parasitol. 74, 441– 451. Celada, A., Cruchaud, A., Perrin, L., 1982. Opsonic activity of human immune serum on in vitro phagocytosis of Plasmodium falciparum infected red blood cells by monocytes. Clin. Exp. Immunol. 47, 630–635. Dubois, P., Pereira da Silva, L., 1995. Towards a vaccine against asexual blood stage infection by Plasmodium falciparum. Res. Immunol. 146, 263 – 275. Fairbanks, G., Steck, T.L., Wallach, D.F., 1971. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 17, 2606 – 2617. Groux, H., Perraut, R., Garraud, O., Poingt, J.P., Gysin, J., 1990. Functional characterisation of the antibody-mediated protection against blood stages of Plasmodium falciparum in the monkey Saimiri sciureus. Eur. J. Immunol. 20, 2317–2323. Gysin, J., Gavoille, S., Mattei, D., et al., 1993. In vitro phagocytosis inhibition assay for the screening of potential candidate antigens for sub-unit vaccines against the asexual blood stage of Plasmodium falciparum. J. Immunol. Methods 159, 209 – 219. Iqbal, J., Siripoon, N., Snounou, V.G., Perlmann, P., Berzins, K., 1997. Plasmodium falciparum: selection of parasite subpopulations with decreased sensitivity for antibody-mediated growth inhibition in vitro. Parasitology 114, 317–334. Jouin, H., Goguet de la Salmoniere, Y.O., Behr, C., et al., 1995. Flow cytometry detection of surface antigen on fresh, unfixed red blood cells infected by Plasmodium falciparum. J. Immunol. Methods 179, 1–12. Le Scanf, C., Fandeur, T., Morales-Betoulle, M.E., Mercereau-Puijalon, O., 1997. Plasmodium falciparum: altered expression of erythrocyte membrane-associated antigens during antigenic variation. Exp. Parasitol. 85, 135–148. Le Scanf, C., Fandeur, T., Bonnefoy, S., Guillotte, M., Mercereau-Puijalon, O., 1999. Novel target antigens of the strain-specific immune response to Plasmodium falciparum identified by differential screening of an expression library. Infect. Immun. 67, 64 – 73.
I. Drame et al. / Acta Tropica 73 (1999) 175–181
181
Mattei, D., Scherf, A., 1992. The Pf332 gene of Plasmodium falciparum codes for a giant protein that is translocated from the parasite to the membrane of infected erythrocytes. Gene 110, 71 – 79. Nguer, C.M., Diallo, T.O., Diouf, A., Tall, A., Die`ye, A., Perraut, R., Garraud, O., 1997. Plasmodium falciparum- and merozoite surface protein 1-specific antibody isotype balance in immune Senegalese adults. Infect. Immun. 65, 4873–4876. Perraut, R., Mercereau-Puijalon, O., Mattei, D., Bourreau, E., Garraud, O., Bonnemains, B., Pereira da Silva, L., Michel, J.C., 1995. Induction of opsonizing antibodies after injection of recombinant Plasmodium falciparum vaccine candidate antigens in preimmune Saimiri sciureus monkeys. Infect. Immun. 63, 554–562. Perraut, R., Mercereau-Puijalon, O., Mattei, D., et al., 1997. Immunogenicity and protective efficacy of recombinant Plasmodium falciparum vaccine candidate antigens in naive squirrel monkey Saimiri sciureus. Am. J. Trop. Med. Hyg. 56, 343 – 350. Perraut et al., 1999 (submitted). Rogier, C., Commenge, D., Trape, J.-F., 1996. Evidence for an age-dependent pyrogenic threshold of Plasmodium falciparum parasitemia in highly endemic populations. Am. J. Trop. Med. Hyg. 54, 613–619. Trape, J.-F., Rogier, C., 1996. Combating malaria morbidity and mortality by reducing transmission. Parasitol. Today 12, 236–240. Trape, J.F., Rogier, C., Konate, L., et al., 1994. The Dielmo project: a longitudinal study of natural malaria infection and the mechanisms of protective immunity in a community living in a holoendemic area of Senegal. Am. J. Trop. Med. Hyg. 51 (2), 123 – 137.
.
Flow cytometric analysis of IgG reactive to parasitized red blood cell membrane antigens in Plasmodium falciparum-immune individuals Idrissa Drame a, Babacar Diouf a, Andre´ Spiegel b, Olivier Garraud a, Ronald Perraut a,* a
Unite´ d’Immunologie, Institut Pasteur de Dakar, 36 A6. Pasteur, BP-220 Dakar, Senegal b Unite´ d’Epide´miologie, Institut Pasteur de Dakar, BP-220 Dakar, Senegal
Received 10 December 1998; received in revised form 4 March 1999; accepted 2 April 1999
Abstract Antigens exposed at the surface of Plasmodium falciparum parasitized red blood cells (pRBCs) represent potential targets for protective antibodies involved in opsonization and immune phagocytosis of pRBCs. We measured the recognition of parasitized red blood cell membrane associated antigens by IgG in the plasma of clinically immune individuals by flow cytometry and ELISA. The plasmas were selected on the basis of preexisting IgG antibodies to pRBC membrane associated recombinant proteins. In every plasma sample IgG could bind the surface of live pRBCs in flow cytometry. In addition, there was a significant correlation between the level of IgG recognition of live pRBCs and of pRBC membrane ghost proteins or major identified antigens by ELISA. Flow cytometry thus represents a technique suitable to test for the accessibility and potential functionality of IgG antibodies directed to antigens expressed by the surface of pRBCs. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Plasmodium falciparum; Erythrocyte membrane; Surface antigens; IgG; Flow cytometry
* Corresponding author. Tel.: + 221-839-92-45; fax: + 221-839-92-10. E-mail address: [email protected] (R. Perraut) 0001-706X/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 9 9 ) 0 0 0 2 6 - 1
176
I. Drame et al. / Acta Tropica 73 (1999) 175–181
1. Introduction There is some evidence that protection from Plasmodium falciparum infection is partly based on antibody (Ab) responses to diverse parasite antigens (Ag) involving several effector mechanisms such as immune phagocytosis of opsonized parasitized red blood cells (pRBCs) which targets Ags expressed at the surface of pRBCs (Celada et al., 1982; Groux et al., 1990; Gysin et al., 1993; Dubois and Pereira da Silva, 1995). Several Ags were shown to be constitutive of pRBC membranes such as PfEMP3 (Le Scanf et al., 1997) or to be associated with pRBC membranes, such as PfEB200 and R23 (Gysin et al., 1993; Perraut et al., 1995). Recombinant proteins derived from these Ags were capable of inducing strong Ab responses in immunized monkeys (Perraut et al., 1995, 1997). Further, they were consistently recognized by naturally acquired Abs in humans continuously exposed to P. falciparum (Perraut et al., 1999). The present work has focused on naturally acquired IgG Abs to pRBC membrane associated proteins, in a number of selected individuals living in endemic areas, who proved clinically immune. The binding of naturally acquired plasma IgG to the surface of live P. falciparum-infected RBCs was assessed by means of flow cytometry.
2. Materials and methods Blood donors were individuals followed up for many years (Trape et al., 1994) and who did not suffer from malaria attack for at least 6 months prior the present study. They were over 12 years of age in the village of Dielmo where P. falciparum transmission is intense and perennial, and over 33 years of age in Ndiop, where transmission is moderate and seasonal (Trape et al., 1994). The age thresholds were estimated to correlate with stable acquired immunity in these locations (Rogier et al., 1996). Sampling was done after informed consent, at the end of the rainy season. A selection of individual samples with or without IgG Abs reactive to the pRBC membrane associated Ags under consideration was done by means of ELISA. Microtiter plates were coated with 1 mg/ml of R23, PfEB200, PfEMP3/ clone 5 or 20 mg/ml of crude pRBC ‘ghost’ proteins, prepared according to the technique described by Fairbanks et al. (1971). Plasma IgG detection of crude Ag and of recombinant proteins was done exactly as described previously (Aribot et al., 1996; Nguer et al., 1997). The PfEB200 antigen derives from Pf332 and it contains 13 repeats with characteristic Glu–Glu dimers and is accessible onto the infected red blood cell surface in late shizonts (Mattei and Scherf, 1992); R23 derives from the central domain of the R45 antigen: it consists of a series of stretches of 11 repeats whose consensus sequence is HKSDS N/S/H (Bonnefoy et al., 1992). Clone 5 is a recombinant protein derived from the PfEMP3 C-terminal domain and was identified as a target of variant immune response (Le Scanf et al., 1999); expression level of PfEMP3 was shown to be modulated during antigenic variation, with some variants expressing high levels while others expressed reduced amounts of protein (Le Scanf et al., 1997).
I. Drame et al. / Acta Tropica 73 (1999) 175–181
177
To test for the recognition of live pRBCs by individual plasma, a cytofluorimetric technique was used as described by Jouin et al. (1995). A ‘knob’-positive Uganda Palo Alto FUP strain of P. falciparum maintained in continuous culture was used a source of pRBCs. Schizonts were harvested after Plasmagel® concentration when parasitaemia reached approximately 10%. Membrane bound IgG were revealed by means of a first incubation of 30 min at 37°C with plasma diluted 1/20 followed by an additional 30 min incubation at 37°C with phycoerythrin (PE)-conjugated goat anti-human IgG (1/200; Cappel Organon Teknica, West Chester, PA). Live parasites were then labelled for 30 min at 37°C in the dark with Thiazol Orange (TO) (Retic-Count®; Becton-Dickinson, San Jose, CA). Fluorescence was read within 1 h. After gating on the TO + pRBCs (FL1] 101; Fig. 1A), PE-labelled membrane bound IgG were measured in the FL2 channel where two fluorescence levels were
Fig. 1. Histograms of flow cytometry data acquisition. Acquisition of 10 000 events was done after gating on FL1. TO-positive cells (dot plot A) were recognized by human IgG and measured in the FL2 channel (dot plot B). Positive fluorescence values were selected in the P2 region (B). (C) This shows pRBCs recognized by a reference negative control. (D) This shows pRBCs recognized by a positive control. (E, F) This show characteristic data obtained with samples from Dielmo (E) and Ndiop (F). The data are expressed as mean levels of positive fluorescence and percent of labelled cells.
178
I. Drame et al. / Acta Tropica 73 (1999) 175–181
discerned, corresponding to the negative background (B 101; P1) and to the positive values (] 101; P2) (Fig. 1B). A pool of 30 plasmas with elevated IgG levels to R23, PfEB200 and/or PfEMP3/clone 5 was referred to as the positive control and a pool of plasma obtained from P. falciparum-unexposed Europeans was referred to as the negative control (Senegalese unexposed individuals were checked negative as well). An indice (iP) for the quantification of pRBC recognition was calculated from the mean fluorescence intensities (MFI) multiplied by the percent of positive events in region ‘P2’. These iP values were then compared individually with actual OD values for plasma IgG to ‘ghost’ proteins, and to arbitrary values corresponding to IgG reactive to recombinant proteins: OD values B 0.1 were scored 0; OD values ranging from 0.1 to 0.5 were scored 1; and OD values over 0.5 were scored 2. Statistical correlation between non-normally distributed paired data was performed by means of the Spearman rank test.
3. Results and discussion We have evaluated the capacity of naturally acquired IgG responses that contained defined Abs to Ags expressed on the surface of P. falciparum-parasitized RBCs (Gysin et al., 1993; Jouin et al., 1995), to bind live pRBCs by means of a fluorescence flow cytometric technique. We have compared this data with individual levels of plasma Abs to three recombinant proteins known to be associated with P. falciparum blood stage Ags and shown to either protect monkeys in eliciting strong opsonizing Ab responses (Perraut et al., 1995) or to be associated with a certain degree of protection (Le Scanf et al., 1997; Perraut et al., 1997). Plasma samples from individuals living in Dielmo (n=18) and in Ndiop (n= 6) were selected with different levels of specific reactivity to R23 and/or PfEB200 and/or PfEMP3/clone 5, three villagers were selected as negative individuals. As can be seen in Fig. 1, individual plasma was capable of binding the surface of pRBCs (Fig. 1E and F), compared with the positive (Fig. 1D) and the negative control plasma (Fig. 1C). Of note, every plasma tested contained functionally active IgG capable of specifically binding the surface of pRBCs despite some individual variation; furthermore, most of the fluorescent Ag – Ab complexes were labelled with high intensity (FL2] 102). We have then calculated the iP indices, showing that the mean indices in Dielmo and in Ndiop individuals were 99 and 106, respectively; this difference is not significant (for comparisons, indices for the positive and negative controls were 156 and 10, respectively; P =0.64). This confirms that clinically immune individuals have acquired strong IgG responses directed to determinants expressed by pRBC membranes. The heterogeneity within individual indices could be related to the possibility that a fraction of Abs has bound circulating pRBCs (Trape and Rogier, 1996). A limitation of the technique was found to be the antigenic variation of parasites in cultures since long-term cultured parasites constantly modify the expression of surface Ags (Iqbal et al., 1997). Such a variation in the availability of parasites originating from in vitro culture and suitable for flow cytometry was not observed in previous work which used ex vivo obtained pRBCs in the Saimiri
I. Drame et al. / Acta Tropica 73 (1999) 175–181
179
Fig. 2. Comparison of pRBC membrane specific IgG levels by flow cytometry and ELISA in individuals living in Dielmo and Ndiop. (A) Individual samples were classified according to the magnitude of live pRBC recognition: the histograms indicate individual percent of specific IgG responses compared to the positive control (100%), by means of flow cytometry performed on live pRBCs (dark histograms) and detection of Abs to crude P. falciparum ‘ghost’ proteins by specific ELISA (white histograms). (B) OD values of IgG to recombinant proteins in individual plasma are shown as follows: open boxes, no detectable Ab (OD B 0.1); shadowed boxes, positive Ab (0.1 5OD B0.5); filled boxes, high Ab levels (OD ]0.5).
monkey (Jouin et al., 1995; Perraut et al., 1995, 1997). These technical difficulties could be overcome by improving the selection of parasite strains suitable for pRBC membrane analyses on a wider scale. To further examine the fine specificity of this set of data obtained using flow cytometry, we have compared individual iP indices with conventional P. falciparumspecific IgG detection by mean of ELISAs. Fig. 2.A shows the comparison of percent responses versus controls (100%) of individual plasma tested for pRBCmembrane Ag binding (flow cytometry) and protein recognition (ELISA where captured Ags consisted of a crude ‘ghost’ membrane pRBC preparation). Despite individual fluctuations, IgG responses measured by means of these two different techniques are consistent, and the positive correlation was statistically significant (P= 0.012). In addition, Fig. 2B shows individual scores for plasma IgG to R23, PfEB200 and PfEMP3/clone5. The comparison between iP indices and individual scores for IgG Abs to either recombinant protein (measured by ELISA) proved to be non significantly associated. However, when individual iP indices were compared with the mathematic summation of individual scores for all three recombinant proteins, there was a significant positive relationship with pRBC membrane associated Ab recognition (P =0.042), irrespective of P. falciparum transmission conditions in the field. This study also reveals the relatively constant prevalence of Abs to membrane associated pRBC Ags in the plasma of individuals living in endemic areas; it further shows that such Abs, which include IgG Abs to defined proteins considered as
180
I. Drame et al. / Acta Tropica 73 (1999) 175–181
vaccine candidates (Dubois and Pereira da Silva, 1995; Perraut et al., 1997), may contribute to the effective binding of live pRBCs, demonstrating their potential functional properties.
Acknowledgements The authors are indebted to Drs O. Puijalon, D. Mattei, P. Dubois, S. Bonnefoy, J. Gysin and H. Jouin (Institut Pasteur, Paris) for fruitful discussions and for providing essential reagents. They acknowledge Dr C. Le Scanf (Institut Pasteur de la Guyane Franc¸aise) and M. Guillotte for technical help. They also thank Dr G. Yap (NIAID/NIH, Bethesda) for fruitful comments and a critical review of the manuscript. This work was supported in part by grants from Institut Pasteur Fondation and from the French Ministe`re de la Coope´ration et du De´veloppement.
References Aribot, G., Rogier, C., Sarthou, J.-L., et al., 1996. Pattern of immunoglobulin isotype response to Plasmodium falciparum blood-stage antigens in individuals living in a holoendemic area of Senegal (Dielmo, West Africa). Am. J. Trop. Med. Hyg. 54, 449 – 457. Bonnefoy, S., Guillotte, M., Langsley, G., Mercereau-Puijalon, O., 1992. Plasmodium falciparum: Characterization of gene R45 encoding a trophozoite antigen containing a central block of six amino acid repeats. Exp. Parasitol. 74, 441– 451. Celada, A., Cruchaud, A., Perrin, L., 1982. Opsonic activity of human immune serum on in vitro phagocytosis of Plasmodium falciparum infected red blood cells by monocytes. Clin. Exp. Immunol. 47, 630–635. Dubois, P., Pereira da Silva, L., 1995. Towards a vaccine against asexual blood stage infection by Plasmodium falciparum. Res. Immunol. 146, 263 – 275. Fairbanks, G., Steck, T.L., Wallach, D.F., 1971. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 17, 2606 – 2617. Groux, H., Perraut, R., Garraud, O., Poingt, J.P., Gysin, J., 1990. Functional characterisation of the antibody-mediated protection against blood stages of Plasmodium falciparum in the monkey Saimiri sciureus. Eur. J. Immunol. 20, 2317–2323. Gysin, J., Gavoille, S., Mattei, D., et al., 1993. In vitro phagocytosis inhibition assay for the screening of potential candidate antigens for sub-unit vaccines against the asexual blood stage of Plasmodium falciparum. J. Immunol. Methods 159, 209 – 219. Iqbal, J., Siripoon, N., Snounou, V.G., Perlmann, P., Berzins, K., 1997. Plasmodium falciparum: selection of parasite subpopulations with decreased sensitivity for antibody-mediated growth inhibition in vitro. Parasitology 114, 317–334. Jouin, H., Goguet de la Salmoniere, Y.O., Behr, C., et al., 1995. Flow cytometry detection of surface antigen on fresh, unfixed red blood cells infected by Plasmodium falciparum. J. Immunol. Methods 179, 1–12. Le Scanf, C., Fandeur, T., Morales-Betoulle, M.E., Mercereau-Puijalon, O., 1997. Plasmodium falciparum: altered expression of erythrocyte membrane-associated antigens during antigenic variation. Exp. Parasitol. 85, 135–148. Le Scanf, C., Fandeur, T., Bonnefoy, S., Guillotte, M., Mercereau-Puijalon, O., 1999. Novel target antigens of the strain-specific immune response to Plasmodium falciparum identified by differential screening of an expression library. Infect. Immun. 67, 64 – 73.
I. Drame et al. / Acta Tropica 73 (1999) 175–181
181
Mattei, D., Scherf, A., 1992. The Pf332 gene of Plasmodium falciparum codes for a giant protein that is translocated from the parasite to the membrane of infected erythrocytes. Gene 110, 71 – 79. Nguer, C.M., Diallo, T.O., Diouf, A., Tall, A., Die`ye, A., Perraut, R., Garraud, O., 1997. Plasmodium falciparum- and merozoite surface protein 1-specific antibody isotype balance in immune Senegalese adults. Infect. Immun. 65, 4873–4876. Perraut, R., Mercereau-Puijalon, O., Mattei, D., Bourreau, E., Garraud, O., Bonnemains, B., Pereira da Silva, L., Michel, J.C., 1995. Induction of opsonizing antibodies after injection of recombinant Plasmodium falciparum vaccine candidate antigens in preimmune Saimiri sciureus monkeys. Infect. Immun. 63, 554–562. Perraut, R., Mercereau-Puijalon, O., Mattei, D., et al., 1997. Immunogenicity and protective efficacy of recombinant Plasmodium falciparum vaccine candidate antigens in naive squirrel monkey Saimiri sciureus. Am. J. Trop. Med. Hyg. 56, 343 – 350. Perraut et al., 1999 (submitted). Rogier, C., Commenge, D., Trape, J.-F., 1996. Evidence for an age-dependent pyrogenic threshold of Plasmodium falciparum parasitemia in highly endemic populations. Am. J. Trop. Med. Hyg. 54, 613–619. Trape, J.-F., Rogier, C., 1996. Combating malaria morbidity and mortality by reducing transmission. Parasitol. Today 12, 236–240. Trape, J.F., Rogier, C., Konate, L., et al., 1994. The Dielmo project: a longitudinal study of natural malaria infection and the mechanisms of protective immunity in a community living in a holoendemic area of Senegal. Am. J. Trop. Med. Hyg. 51 (2), 123 – 137.
.