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Imbalanced distribution of IgM and IgG antibodies against Plasmodium falciparum antigens and merozoite surface protein-1 (MSP1) in pregnancy
Immunology Letters 61 (1998) 197 – 199
Rapid Note
Imbalanced distribution of IgM and IgG antibodies against Plasmodium falciparum antigens and merozoite surface protein-1 (MSP1) in pregnancy Wilfrid S. Nambei a, Moctar Goumbala b, Andre´ Spiegel c, Alioune Die`ye a, Ronald Perraut a, Olivier Garraud a,* a
Laboratoire d’Immunologie, Institut Pasteur, BP 220, Dakar, Senegal Department of Obstetrics, Hoˆpital Principal de Dakar, Dakar, Senegal c Laboratoire d’Epide´miologie du Paludisme, Institut Pasteur, BP 220, Dakar, Senegal b
Received 19 November 1997; accepted 28 November 1997
Abstract In malaria endemic areas, pregnancy is assumed to be associated with a specific reduction in immunity to Plasmodium. falciparum malaria. To understand some of the mechanisms which underlie such a poor immunity, we have attempted to examine the frequency and distribution of IgM and IgG antibodies to a crude antigenic extract of parasitized erythrocytes and to the merozoite surface protein-1 (MSP1), in a population of mothers compared to control non-pregnant women, all living in Dakar and suburbs. Specifically, this work describes: (i) the responses of mothers and control women; (ii) the balance between IgM and IgG responses; and (iii) responses to malarial antigen and to MSP1. An unexpected balance between P. falciparum-specific IgM and IgG is shown, associated with a substantial increase in anti-MSP1 IgM, and a decrease in anti-MSP1 IgG in parturients. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Malaria; Plasmodium falciparum; Pregnancy; Newborn; Cord blood; Antibodies; MSP1; Se´ne´gal
In malaria endemic areas, pregnancy is assumed to be associated with a specific reduction in immunity to Plasmodium falciparum malaria and to an increased risk of clinical episodes which affects both pregnancy and fetal development [1]. It appears of critical importance to understand some of the mechanisms which underlie such a poor immunity. In the present report, we have attempted to examine the frequency and distribution of IgM and IgG antibodies (Ab) to P. falciparum blood stage antigens (Ag) in a population of mothers comAbbre6iations: Ab, antibody; Ag, antigen; OD, optical density; PBMC, peripheral blood mononuclear cell; Pf.sAg, somatic P. falciparum Ag; rAg, recombinant Ag. * Corresponding author. Fax.: + 221 8239210; e-mail: [email protected] 0165-2478/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0165-2478(97)00168-5
pared to control non-pregnant women, all living in Dakar and suburbs, an area with low seasonal transmission. The study population consisted of parturients having given informed consent and delivering at the Hoˆpital Principal de Dakar and of volunteer age-matched nonpregnant women with no debilitating diseases consulting at the Institut Pasteur de Dakar for standard labwork. The control population has been chosen on the basis of similar socioeconomic status and proximity of habitation. Peripheral blood recovery was done on heparinized tubes before or at the beginning of the malaria transmission period. Of note, no circulating parasite was detected in both populations by means of the QBC® test. The Ags used were: (i) a P. falciparum
198
W.S. Nambei et al. / Immunology Letters 61 (1998) 197–199
Fig. 1. Relationship between IgM antibody titers specific to Pf.sAg and MSP1 in parturients and controls. Plasma samples from parturients (right panel) and controls (left panel), were assayed for IgM Abs to both Pf.sAg (x-axis) and MSP1 (y-axis). Data (expressed as OD ratios) for both Ags are plotted against each other (each dot representing an individual plasma). Dashed lines indicate thresholds for positivity (1.5).
Ag lysate from a schizont rich P. falciparum-parasitized red blood cell culture [2]; (ii) the C-terminal domain of the P. falciparum merozoite surface protein-1 (MSP1) (the first EGF-like motif), obtained as an E. coli-recombinant Ag (rAg) cleaved from the vector protein [3]. These Ags are subsequently referred to as Pf.sAg and MSP1, respectively. Individual plasma samples were diluted 1/200 in PBS, 1% bovine serum albumin and 0.1% Tween-20. Unless otherwise stated, reagents were purchased from Sigma (Saint-Louis, MO). ELISA-tests have been performed as described elsewhere, using MaxiSorp® plates to detect anti-Pf.sAg Abs (Nunc, Roskilde, Denmark) [3] and Immulon-4® plates to detect anti-MSP1 Abs (Dynatech, Springfield, VA) [4]. Secondary polyclonal goat anti-human IgG (1/6000) and IgM Ab (1/4000) were purchased from Cappel (Organon-Technika, Turnhout, Belgium). Optical densities (OD) were read at 450 nm on a Titertech-Multiskan® ELISA reader, and the results were expressed as OD ratios deduced from OD values of a negative reference serum obtained from pooled individual sera from Europeans never exposed to malaria (Institut Pasteur, Paris, France), in order to minimize the plate to plate and the day to day variations [4]. Comparisons of Ab titers between individuals were done using paired t-tests and the frequencies of positive responses were analysed using x 2- or Fisher’s exact tests. The distribution and levels of plasma IgM and IgG specific to Pf.sAg and/or to MSP1 were compared in up to 54 parturients and 42 controls. The % of individuals with specific IgM was more elevated in parturients than in controls: 21 vs. 5% (P = 0.023) for Pf.sAg, and 28 vs.
5% (P= 0.004) for MSP1, suggesting that parturients developed specific IgM more frequently than controls (Fig. 1). Further, there was no significant association between anti-Pf.sAg and anti-MSP1 IgM scores in parturients (P= 0.9), compared to controls (P = 0.002), suggesting that parturients developed IgM Abs in a manner different than controls (Fig. 1). Specific IgM might thus reflect Abs to new alleles or to a selection of P. falciparum-malaria parasites, as recently suggested [5]. Alternatively, parturients enrolled in this study may have developed cross-reacting IgM autoantibodies, as often occurs both during pregnancy and malaria infection [6,7]. In this line, it is interesting to note that MSP1 contains two EGF-like motifs, hologous to intermediate filament proteins [3]. No evidence of cross-reacting IgM against non-infected erythrocytes has been found experimentally, however (data not shown). Further, it has been suggested that protection against asexual blood stages in P. falciparum malaria is largely IgG Ab-mediated [8,9]. The percentage of individuals with IgG specific to Pf.sAg was, in contrast to IgM, lower in parturients than in controls: 21 vs. 33% (P= 0.19). Though still not significant, the percentage of individuals with IgG specific to MSP1 was much smaller in parturients than in controls: 4 vs. 17% (P= 0.073). It is however, interesting to note that there was a significant association between the distribution of IgG specific to Pf.sAg and to MSP1 in controls (P=0.002), whereas such an association was no longer found in parturients (P= 0.2), though the number of parturients with Pf.sAg-specific IgG was significantly higher in the anti-MSP1 positives (P= 0.049), suggesting a selective
W.S. Nambei et al. / Immunology Letters 61 (1998) 197–199
199
Fig. 2. Relationship between IgG antibody titers specific to Pf.sAg and MSP1 in parturients and controls. Plasma samples from parturients (right panel) and controls (left panel), were assayed for IgG Abs to both Pf.sAg (x-axis) and MSP1 (y-axis). Data (expressed as OD ratios) for both Ags are plotted against each other (each dot representing an individual plasma). Dashed lines indicate thresholds for positivity (1.5).
loss in anti-MSP1 IgG Abs. As can be seen in addition (Fig. 2), there are clearly different profiles for antiPf.sAg and anti-MSP1 specific IgG in parturients and controls. The cellular and molecular immunological events responsible for the downregulation of P. falciparum-Ag specific Ab formation by memory and/or naive B lymphocytes in parturients are not yet elucidated. There has been a suggestion that pregnancy was associated with a skewing of type-1 towards type-2 responses, i.e. with a weakening of cell-mediated immunity, an increased susceptibility to intracellular pathogens, and a strengthening of humoral immunity [10]. However, those Abs are often autoantibodies, of the IgM isotype. Malaria is often considered to favor type-1 responses at the onset of infection and the further development of type-2 responses [11]. Such type-2 responses are characterized by the production of Abs of every class and subclass, apart from the IgM and IgG2 [12], and are associated with the development of protection, at least in relevant experimental models [13]. Data presented herein do not seem to confirm the skewing of immunological responses towards biased IgG production in response to MSP1, an Ag which generally elicits prominent IgG1/IgG3 Ab production [4,14]. It is therefore possible that type-2 responses in materno-fetal relationships, if confirmed, are mostly targetted at ensuring a successful pregnancy. The likely discrepancy between the formation during pregnancy of IgM versus IgG specific Abs, and their respective role in terms of protection and/or in utero exposure of the newborns, would thus merit further investigation.
Acknowledgements The authors are grateful to nurses and midwives at the Department of Obstetrics (Hoˆpital Principal de Dakar); to Drs F. Spiegel, C. Coudert, G. Raphenon, H. Sartelet, I. Milko-Sartelet, C. Roussilhon (Dakar) and A.A. Holder (London) for their help in obtaining blood samples, clinical information and critical reagents; to Dr C.M. Nguer, and to M.M.A. Diouf, B. Diouf, E.M. Fall and A. Thiam for their technical help; to Drs J.Y. Le Hesran (Dakar), S. Longacre (Paris), and M. Marovich (Bethesda) for helpful suggestions. References [1] I.A. McGregor, Am. J. Trop. Med. Hyg. 33 (1984) 517–525. [2] R. Perraut, P. Berthe, B. Diouf, et al., Dakar Me´d. 42 (1997) 30–35. [3] C.M. Nguer, T.O. Diallo, A. Diouf, et al., Infect. Immun. 65 (1997) 4873 – 4876. [4] P.A. Burghaus, A.A. Holder, Mol. Biochem. Parasitolol. 64 (1994) 165 – 169. [5] M. Fried, P.E. Duffy, Science 272 (1996) 1502 – 1504. [6] J.A. Da Silva, T.D. Spector, Clin. Rheumatol. 11 (1992) 189–194. [7] D. Mattei, A. Scherf, Res. Immunol. 142 (1991) 698 – 702. [8] P. Druilhe, H. Bouharoun-Tayoun, Res. Immunol. 142 (1991) 637 – 643. [9] O. Garraud, Bull. Inst. Pasteur 91 (1993) 143 – 159. [10] T.G. Wegmann, H. Lin, L. Guilbert, T.M. Mossmann, Immunol. Today 14 (1993) 353 – 355. [11] G.E. Grau, C. Behr, Res. Immunol. 145 (1994) 441 – 455. [12] O. Garraud, T.B. Nutman, Bull. Inst. Pasteur 94 (1996) 285–309. [13] R.S. Phillips, K.E. Mathers, A.W. Taylor-Robinson, Res. Immunol. 145 (1994) 406 – 411. [14] A.F. Egan, J. Morris, G. Barnish, et al., J. Infect. Dis. 173 (1996) 765 – 769.
Rapid Note
Imbalanced distribution of IgM and IgG antibodies against Plasmodium falciparum antigens and merozoite surface protein-1 (MSP1) in pregnancy Wilfrid S. Nambei a, Moctar Goumbala b, Andre´ Spiegel c, Alioune Die`ye a, Ronald Perraut a, Olivier Garraud a,* a
Laboratoire d’Immunologie, Institut Pasteur, BP 220, Dakar, Senegal Department of Obstetrics, Hoˆpital Principal de Dakar, Dakar, Senegal c Laboratoire d’Epide´miologie du Paludisme, Institut Pasteur, BP 220, Dakar, Senegal b
Received 19 November 1997; accepted 28 November 1997
Abstract In malaria endemic areas, pregnancy is assumed to be associated with a specific reduction in immunity to Plasmodium. falciparum malaria. To understand some of the mechanisms which underlie such a poor immunity, we have attempted to examine the frequency and distribution of IgM and IgG antibodies to a crude antigenic extract of parasitized erythrocytes and to the merozoite surface protein-1 (MSP1), in a population of mothers compared to control non-pregnant women, all living in Dakar and suburbs. Specifically, this work describes: (i) the responses of mothers and control women; (ii) the balance between IgM and IgG responses; and (iii) responses to malarial antigen and to MSP1. An unexpected balance between P. falciparum-specific IgM and IgG is shown, associated with a substantial increase in anti-MSP1 IgM, and a decrease in anti-MSP1 IgG in parturients. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Malaria; Plasmodium falciparum; Pregnancy; Newborn; Cord blood; Antibodies; MSP1; Se´ne´gal
In malaria endemic areas, pregnancy is assumed to be associated with a specific reduction in immunity to Plasmodium falciparum malaria and to an increased risk of clinical episodes which affects both pregnancy and fetal development [1]. It appears of critical importance to understand some of the mechanisms which underlie such a poor immunity. In the present report, we have attempted to examine the frequency and distribution of IgM and IgG antibodies (Ab) to P. falciparum blood stage antigens (Ag) in a population of mothers comAbbre6iations: Ab, antibody; Ag, antigen; OD, optical density; PBMC, peripheral blood mononuclear cell; Pf.sAg, somatic P. falciparum Ag; rAg, recombinant Ag. * Corresponding author. Fax.: + 221 8239210; e-mail: [email protected] 0165-2478/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0165-2478(97)00168-5
pared to control non-pregnant women, all living in Dakar and suburbs, an area with low seasonal transmission. The study population consisted of parturients having given informed consent and delivering at the Hoˆpital Principal de Dakar and of volunteer age-matched nonpregnant women with no debilitating diseases consulting at the Institut Pasteur de Dakar for standard labwork. The control population has been chosen on the basis of similar socioeconomic status and proximity of habitation. Peripheral blood recovery was done on heparinized tubes before or at the beginning of the malaria transmission period. Of note, no circulating parasite was detected in both populations by means of the QBC® test. The Ags used were: (i) a P. falciparum
198
W.S. Nambei et al. / Immunology Letters 61 (1998) 197–199
Fig. 1. Relationship between IgM antibody titers specific to Pf.sAg and MSP1 in parturients and controls. Plasma samples from parturients (right panel) and controls (left panel), were assayed for IgM Abs to both Pf.sAg (x-axis) and MSP1 (y-axis). Data (expressed as OD ratios) for both Ags are plotted against each other (each dot representing an individual plasma). Dashed lines indicate thresholds for positivity (1.5).
Ag lysate from a schizont rich P. falciparum-parasitized red blood cell culture [2]; (ii) the C-terminal domain of the P. falciparum merozoite surface protein-1 (MSP1) (the first EGF-like motif), obtained as an E. coli-recombinant Ag (rAg) cleaved from the vector protein [3]. These Ags are subsequently referred to as Pf.sAg and MSP1, respectively. Individual plasma samples were diluted 1/200 in PBS, 1% bovine serum albumin and 0.1% Tween-20. Unless otherwise stated, reagents were purchased from Sigma (Saint-Louis, MO). ELISA-tests have been performed as described elsewhere, using MaxiSorp® plates to detect anti-Pf.sAg Abs (Nunc, Roskilde, Denmark) [3] and Immulon-4® plates to detect anti-MSP1 Abs (Dynatech, Springfield, VA) [4]. Secondary polyclonal goat anti-human IgG (1/6000) and IgM Ab (1/4000) were purchased from Cappel (Organon-Technika, Turnhout, Belgium). Optical densities (OD) were read at 450 nm on a Titertech-Multiskan® ELISA reader, and the results were expressed as OD ratios deduced from OD values of a negative reference serum obtained from pooled individual sera from Europeans never exposed to malaria (Institut Pasteur, Paris, France), in order to minimize the plate to plate and the day to day variations [4]. Comparisons of Ab titers between individuals were done using paired t-tests and the frequencies of positive responses were analysed using x 2- or Fisher’s exact tests. The distribution and levels of plasma IgM and IgG specific to Pf.sAg and/or to MSP1 were compared in up to 54 parturients and 42 controls. The % of individuals with specific IgM was more elevated in parturients than in controls: 21 vs. 5% (P = 0.023) for Pf.sAg, and 28 vs.
5% (P= 0.004) for MSP1, suggesting that parturients developed specific IgM more frequently than controls (Fig. 1). Further, there was no significant association between anti-Pf.sAg and anti-MSP1 IgM scores in parturients (P= 0.9), compared to controls (P = 0.002), suggesting that parturients developed IgM Abs in a manner different than controls (Fig. 1). Specific IgM might thus reflect Abs to new alleles or to a selection of P. falciparum-malaria parasites, as recently suggested [5]. Alternatively, parturients enrolled in this study may have developed cross-reacting IgM autoantibodies, as often occurs both during pregnancy and malaria infection [6,7]. In this line, it is interesting to note that MSP1 contains two EGF-like motifs, hologous to intermediate filament proteins [3]. No evidence of cross-reacting IgM against non-infected erythrocytes has been found experimentally, however (data not shown). Further, it has been suggested that protection against asexual blood stages in P. falciparum malaria is largely IgG Ab-mediated [8,9]. The percentage of individuals with IgG specific to Pf.sAg was, in contrast to IgM, lower in parturients than in controls: 21 vs. 33% (P= 0.19). Though still not significant, the percentage of individuals with IgG specific to MSP1 was much smaller in parturients than in controls: 4 vs. 17% (P= 0.073). It is however, interesting to note that there was a significant association between the distribution of IgG specific to Pf.sAg and to MSP1 in controls (P=0.002), whereas such an association was no longer found in parturients (P= 0.2), though the number of parturients with Pf.sAg-specific IgG was significantly higher in the anti-MSP1 positives (P= 0.049), suggesting a selective
W.S. Nambei et al. / Immunology Letters 61 (1998) 197–199
199
Fig. 2. Relationship between IgG antibody titers specific to Pf.sAg and MSP1 in parturients and controls. Plasma samples from parturients (right panel) and controls (left panel), were assayed for IgG Abs to both Pf.sAg (x-axis) and MSP1 (y-axis). Data (expressed as OD ratios) for both Ags are plotted against each other (each dot representing an individual plasma). Dashed lines indicate thresholds for positivity (1.5).
loss in anti-MSP1 IgG Abs. As can be seen in addition (Fig. 2), there are clearly different profiles for antiPf.sAg and anti-MSP1 specific IgG in parturients and controls. The cellular and molecular immunological events responsible for the downregulation of P. falciparum-Ag specific Ab formation by memory and/or naive B lymphocytes in parturients are not yet elucidated. There has been a suggestion that pregnancy was associated with a skewing of type-1 towards type-2 responses, i.e. with a weakening of cell-mediated immunity, an increased susceptibility to intracellular pathogens, and a strengthening of humoral immunity [10]. However, those Abs are often autoantibodies, of the IgM isotype. Malaria is often considered to favor type-1 responses at the onset of infection and the further development of type-2 responses [11]. Such type-2 responses are characterized by the production of Abs of every class and subclass, apart from the IgM and IgG2 [12], and are associated with the development of protection, at least in relevant experimental models [13]. Data presented herein do not seem to confirm the skewing of immunological responses towards biased IgG production in response to MSP1, an Ag which generally elicits prominent IgG1/IgG3 Ab production [4,14]. It is therefore possible that type-2 responses in materno-fetal relationships, if confirmed, are mostly targetted at ensuring a successful pregnancy. The likely discrepancy between the formation during pregnancy of IgM versus IgG specific Abs, and their respective role in terms of protection and/or in utero exposure of the newborns, would thus merit further investigation.
Acknowledgements The authors are grateful to nurses and midwives at the Department of Obstetrics (Hoˆpital Principal de Dakar); to Drs F. Spiegel, C. Coudert, G. Raphenon, H. Sartelet, I. Milko-Sartelet, C. Roussilhon (Dakar) and A.A. Holder (London) for their help in obtaining blood samples, clinical information and critical reagents; to Dr C.M. Nguer, and to M.M.A. Diouf, B. Diouf, E.M. Fall and A. Thiam for their technical help; to Drs J.Y. Le Hesran (Dakar), S. Longacre (Paris), and M. Marovich (Bethesda) for helpful suggestions. References [1] I.A. McGregor, Am. J. Trop. Med. Hyg. 33 (1984) 517–525. [2] R. Perraut, P. Berthe, B. Diouf, et al., Dakar Me´d. 42 (1997) 30–35. [3] C.M. Nguer, T.O. Diallo, A. Diouf, et al., Infect. Immun. 65 (1997) 4873 – 4876. [4] P.A. Burghaus, A.A. Holder, Mol. Biochem. Parasitolol. 64 (1994) 165 – 169. [5] M. Fried, P.E. Duffy, Science 272 (1996) 1502 – 1504. [6] J.A. Da Silva, T.D. Spector, Clin. Rheumatol. 11 (1992) 189–194. [7] D. Mattei, A. Scherf, Res. Immunol. 142 (1991) 698 – 702. [8] P. Druilhe, H. Bouharoun-Tayoun, Res. Immunol. 142 (1991) 637 – 643. [9] O. Garraud, Bull. Inst. Pasteur 91 (1993) 143 – 159. [10] T.G. Wegmann, H. Lin, L. Guilbert, T.M. Mossmann, Immunol. Today 14 (1993) 353 – 355. [11] G.E. Grau, C. Behr, Res. Immunol. 145 (1994) 441 – 455. [12] O. Garraud, T.B. Nutman, Bull. Inst. Pasteur 94 (1996) 285–309. [13] R.S. Phillips, K.E. Mathers, A.W. Taylor-Robinson, Res. Immunol. 145 (1994) 406 – 411. [14] A.F. Egan, J. Morris, G. Barnish, et al., J. Infect. Dis. 173 (1996) 765 – 769.