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AIDS:
doi: 10.1097/QAD.0b013e3282f560ee
Research Letters

Specific stimulation of HIV-1 replication in human placental trophoblasts by an antigen of Plasmodium falciparum

Ayouba, Ahidjoa; Badaut, Cyrilb; Kfutwah, Anfumbomc; Cannou, Claudea; Juillerat, Alexandreb; Gangnard, Stéphaneb; Behr, Charlotted; Mercereau-Puijalon, Odiled; Bentley, Graham Ab; Barré-Sinoussi, Françoisea; Menu, Elisabetha

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aUnité de Régulation des Infections Rétrovirales, Institut Pasteur, 75724 Paris Cedex 15, France

bUnité d'Immunologie Structurale (CNRS URA 2185), Institut Pasteur, 75724 Paris Cedex 15, France

cLaboratoire de Virologie, Centre Pasteur du Cameroun, BP1274 Yaoundé, Cameroon

dUnité d'Immunologie Moléculaire des Parasites (CNRS URA 2581), Institut Pasteur, 75724 Paris Cedex 15, France.

Received 28 October, 2007

Revised 5 December, 2007

Accepted 11 December, 2007

Correspondence to Ahidjo Ayouba, Unité de Régulation des Infections Rétrovirales, Institut Pasteur, 75724 Paris, Cedex 15, France. Tel: +33 467 416305; fax: +33 467 416146; e-mail: ahidjo.ayouba@mpl.ird.fr

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Abstract

Epidemiological data point to an increased risk of HIV-1 mother-to-child transmission in pregnant women with malaria, by unknown mechanisms. We show here that surface binding of a recombinant Plasmodium falciparum adhesin to chondroitin sulphate A proteoglycans increases HIV-1 replication in the human placental cell line BeWo, probably by a P. falciparum adhesin-induced long-terminal repeat-driven TNF-α stimulation. This suggests that placental malaria could increase the risk of HIV-1 transmission in utero.

Field and laboratory studies have highlighted an interplay between HIV-1 status and malarial parasitaemia, with increased plasma viral loads being present during malaria infection [1]. Malaria complications during pregnancy arise from an accumulation of Plasmodium falciparum in the placenta (placental malaria) [2]. During an HIV-1 mother-to-child transmission (MTCT) prevention study we noticed a strong correlation between rainfall (associated with high P. falciparum transmission) and HIV-1 MTCT [3]. HIV-positive pregnant women with P. falciparum placental infections can show accentuated viral MTCT in utero/peripartum [4]. Parasite accumulation in the placenta results from cytoadherence of infected erythrocytes to chondroitin sulfate A (CSA) proteoglycans on the placental lining via a subset of P. falciparum PfEMP1 adhesins [5]. We hypothesized that this P. falciparum placental binding would stimulate local HIV-1 replication and contribute to MTCT in utero. To test this hypothesis, we explored the consequence of P. falciparum adhesion to the placenta-derived cell line BeWo [6] and HIV-1 replication in vitro.

Human placental trophoblasts and BeWo cells resist infection by cell-free HIV-1 [7] but are permissive to infection after contact with HIV-1-infected blood cells, leading to in-vivo transplacental passage of viruses [8]. To bypass this restriction and to mimic cell-to-cell infection, we used HIV-1 pseudotyped with the vesicular stomatis virus G protein (VSV-G) bearing the luciferase reporter gene to infect BeWo cells [7]. These cells were incubated with the recombinant DBL3γ-732 adhesin, which is derived from the P. falciparum placental adhesin varPAM-732 complementary DNA (GenBank entry AY679117) and binds to CSA and placental chondroitin sulfate proteoglycans [9].

Using immunofluorescence assays with an anti-CSA monoclonal antibody, we first confirmed that BeWo cells, like primary placental trophoblasts, expressed surface CSA (not shown). BeWo cells were infected with VSV-G pseudotyped HIV-1 and incubated with increasing concentrations of recombinant DBL3γ-732. A concentration-dependent effect on viral replication was observed for the two viral doses used (Fig. 1a). For both low and high viral doses, there was an inhibition of replication at low DBL3γ-732 concentration but above a 2.5 μM concentration, a significant increase of replication occurred. This increase was DBL3γ-732 specific because the addition of DBL1α-varO, an unrelated adhesin domain from the rosette-forming PfEMP1-varO variant [10], which does not bind CSA to infected BeWo cells, did not augment viral replication (not shown).

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Equation (Uncited)
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Fig. 1
Fig. 1
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Stimulation of HIV-1 replication in BeWo cells was completely abolished when DBL3γ-732 (5 μM) was preincubated with 4 μM Fab of monoclonal antibody A5.13 (P = 0.03; Fig. 1b). This anti-DBL3γ-732-specific monoclonal antibody inhibits CSA binding by P. falciparum-infected erythrocytes (manuscript in preparation). The Fab was used because BeWo cells express IgG Fc receptors, which are known to modulate HIV-1 replication. When used alone, Fab A5.13 had no effect on HIV-1 replication or on BeWo cell viability (not shown). This indicates that blocking of DBL3γ-732 binding to CSA by Fab A5.13 prevented the modulation of viral replication. Preincubating DBL3γ-732 with the Fab of monoclonal antibody E4-2, specific for an irrelevant P. falciparum antigen (F. Nato, unpublished), did not abolish stimulation of HIV-1 replication (not shown). Furthermore, Fab E4-2 alone had no effect on HIV-1-infected BeWo cells (not shown). DBL3γ-732 had no effect on HIV-1 replication when we used CD4-positive T lymphocytes, which do not express surface-exposed CSA, in place of BeWo cells (not shown).

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Equation (Uncited)
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As a result of its known role in HIV-1 replication in T cells and HIV-1-infected placenta cells [11], as well as in placental malaria infection [2] we quantified TNF-α production in the BeWo cell culture supernatants. There was a dose-dependent relationship between added DBL3γ-732 and the quantity of TNF-α released into the supernatants irrespective of whether the BeWo cells were infected (VSV-G) or not (Delta Env control). TNF-α in these culture supernatants rose from undetectable in the absence of DBL3γ-732 up to 250 pg/ml at the highest dose. This indicates that TNF-α induction is DBL3γ-732 dependent. Furthermore, this was DBL3γ-732 specific, because the supernatants from parallel cultures incubated with DBL1α-varO contained no detectable TNF-α (not shown). The addition of recombinant TNF-α (0.5, 5 and 50 ng/ml) to pseudotyped HIV-1-infected BeWo cells resulted in a dose-dependent increase of HIV-1 replication (not shown). We next quantified IL-8 in the supernatant of infected BeWo cells cultivated in the presence of escalating doses of DBL3γ-732 (Fig. 1a); no detectable IL-8 was found (not shown).

We have shown here a marked increase in viral replication in BeWo cells at high DBL3γ-732 doses and a moderate inhibition with low DBL3γ-732 doses. It is noteworthy that this biphasic response is mirrored in field studies, in which high placental malaria parasitaemia is associated with an increased risk of in-utero HIV-1 MTCT, whereas low placental malaria parasitaemia is associated with a lower risk of transmission [12]. The demonstration that recombinant DBL3γ-732-induced TNF-α production in BeWo cells is, to our knowledge, the first evidence for the stimulation of a placental cell type for TNF-α production by a specific P. falciparum adhesin. Such a stimulation may contribute to the elevated TNF-α levels in the placenta of P. falciparum-infected women [2], thus generating a specific cytokine milieu that influences cellular immune response(s) within the placenta [13]. Furthermore, TNF-α increases the expression of additional receptors involved in Plasmodium-infected erythrocyte binding such as interstitial cell adhesion molecule type 1 also involved in the adhesion of peripheral blood monocytes to the trophoblast cells [14]. The DBL3γ-732-triggered TNF-α production in BeWo cells could stimulate HIV-1 replication, possibly via long-terminal repeat-mediated activation as reported in ACH-2 cells [15]. Recombinant exogenous TNF-α also stimulated HIV-1 replication in BeWo cells. Although this study strongly implicates TNF-α, we cannot at this stage exclude other factors.

We have studied here one CSA-binding adhesin of P. falciparum. We anticipate that other CSA-binding adhesins (e.g. var2CSA) might also modulate HIV-1 replication. Our data provide a direct link between placental cytoadherence and HIV-1 replication in a placental cell line. This suggests that specific parasite interactions within the placenta contribute to MTCT of HIV-1 in areas in which the two diseases co-localize. Improved implementation of antimalaria therapy in addition to antiretroviral therapies throughout pregnancy may help prevent MTCT of HIV-1.

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Acknowledgements

The authors thank Sévérine Loizon, Ines Vigan-Womas and Micheline Guillotte for communicating unpublished information, Ayman Khattab and Mo-Quen Klinkert for the DBL3γ-732 gene, and Farida Nato and Hélène Souchon for the gift of the mouse monoclonal antibodies A5.13 and E4-2.

Ahidjo Ayouba and Cyril Badaut contributed equally to this work.

Sponsorship: This work was supported by grants from the European Commission (QLK2-CT-2001-01302 and QLK2-CT-2002-01197), Institut Pasteur and CNRS. AA was supported by a fellowship of SIDACTION (France). CB was supported by a Sanofi-Pasteur fellowship.

Conflicts of interest: None.

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References

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