Var2CSA DBL6-epsilon domain expressed in HEK293 induces limited cross-reactive and blocking antibodies to CSA binding parasites

<p>Abstract</p> <p>Background</p> <p>Pregnancy-associated malaria (PAM) is a serious consequence of <it>Plasmodium falciparum</it>-infected erythrocytes sequestration in the placenta through the adhesion to the placental receptor chondroitin sulfate A (CSA). Although women become resistant to PAM as they acquire transcending inhibitory immunity against CSA-binding parasites, hundreds of thousands of lives could be saved if a prophylactic vaccine targeting the surface proteins of placental parasites could be designed. Recent works point to the variant protein var2CSA as the key target for the development of a pregnancy-associated malaria vaccine. However, designing such a prophylactic vaccine has been hindered by the difficulty in identifying regions of var2CSA that could elicit broadly neutralizing and adhesion-blocking antibodies.</p> <p>Methods</p> <p>Var2CSA is a very large protein with an estimated molecular weight of 350 kDa, and can be divided into six cysteine rich Duffy binding-like domains (DBL). The human embryonic kidney 293 cell line (HEK293) was used to produce secreted soluble recombinant forms of var2CSA DBL domains. The <it>Escherichia coli </it>expression system was also assessed for the domains not expressed or expressed in low amount in the HEK293 system. To investigate whether var2CSA binding DBL domains can induce biologically active antibodies recognizing the native var2CSA and blocking the interaction, mice were immunized with the refolded DBL3-X or the HEK293 secreted DBL6-ε domains.</p> <p>Results</p> <p>Using the HEK293 expression system, DBL1-X, DBL4-ε and DBL6-ε were produced at relatively high levels in the culture supernatant, while DBL3-X and DBL5-ε were produced at much lower levels. DBL2-X and DBL3-X domains were obtained after refolding of the inclusion bodies produced in <it>E. coli</it>. Importantly, mice antisera raised against the recombinant DBL6-ε domain, specifically reacted against the surface of CSA-binding parasites and revealed adhesion blocking activity.</p> <p>Conclusion</p> <p>This is the first report showing inhibitory binding antibodies obtained through a var2CSA recombinant DBL domain immunization protocol. These results support the current strategies using var2CSA as immunogen in the aim of blocking placental sequestration of malaria parasites. This work is a step towards the development of a var2CSA based vaccine that will prevent pregnancy-associated malaria and improve pregnancy outcomes.</p

Coxiella burnetii, the Agent of Q Fever, Replicates within Trophoblasts and Induces a Unique Transcriptional Response

Q fever is a zoonosis caused by Coxiella burnetii, an obligate intracellular bacterium typically found in myeloid cells. The infection is a source of severe obstetrical complications in humans and cattle and can undergo chronic evolution in a minority of pregnant women. Because C. burnetii is found in the placentas of aborted fetuses, we investigated the possibility that it could infect trophoblasts. Here, we show that C. burnetii infected and replicated in BeWo trophoblasts within phagolysosomes. Using pangenomic microarrays, we found that C. burnetii induced a specific transcriptomic program. This program was associated with the modulation of inflammatory responses that were shared with inflammatory agonists, such as TNF, and more specific responses involving genes related to pregnancy development, including EGR-1 and NDGR1. In addition, C. burnetii stimulated gene networks organized around the IL-6 and IL-13 pathways, which both modulate STAT3. Taken together, these results revealed that trophoblasts represent a protective niche for C. burnetii. The activation program induced by C. burnetii in trophoblasts may allow bacterial replication but seems unable to interfere with the development of normal pregnancy. Such pathophysiologocal processes should require the activation of immune placental cells associated with trophoblasts

Platelets reorient Plasmodium falciparum-infected erythrocyte cytoadhesion to activated endothelial cells.

Severe malaria is characterized by the sequestration of Plasmodium falciparum-infected erythrocytes (IEs). Because platelets can affect tumor necrosis factor (TNF)-activated endothelial cells (ECs), we investigated their role in the sequestration of IEs, using IEs that were selected because they can adhere to endothelial CD36 (IE(CD36)), a P. falciparum receptor that is expressed on platelets. The results of coincubation studies indicated that platelets can induce IE(CD36) binding to CD36-deficient brain microvascular ECs. This induced cytoadhesion resisted physiological shear stress, was increased by EC stimulation with TNF, and was abolished by anti-CD36 monoclonal antibody. Immunofluorescence and scanning electron microscopy results showed that platelets serve as a bridge between IEs and the surface of ECs and may therefore provide receptors for adhesion to microvascular beds that otherwise lack adhesion receptors. This novel mechanism of cytoadhesion may reorient the sequestration of different parasite phenotypes and play an important role in the pathogenesis of severe malaria

A single member of the Plasmodium falciparum var multigene family determines cytoadhesion to the placental receptor chondroitin sulphate A

In high-transmission regions, protective clinical immunity to Plasmodium falciparum develops during the early years of life, limiting serious complications of malaria in young children. Pregnant women are an exception and are especially susceptible to severe P. falciparum infections resulting from the massive adhesion of parasitized erythrocytes to chondroitin sulphate A (CSA) present on placental syncytiotrophoblasts. Epidemiological studies strongly support the feasibility of an intervention strategy to protect pregnant women from disease. However, different parasite molecules have been associated with adhesion to CSA. In this work, we show that disruption of the var2csa gene of P. falciparum results in the inability of parasites to recover the CSA-binding phenotype. This gene is a member of the var multigene family and was previously shown to be composed of domains that mediate binding to CSA. Our results show the central role of var2CSA in CSA adhesion and support var2CSA as a leading vaccine candidate aimed at protecting pregnant women and their fetuses

Persistence of <i>Coxiella burnetii</i>, the Agent of Q Fever, in Murine Adipose Tissue

<div><p><i>Coxiella burnetii</i>, the agent of Q fever, is known to persist in humans and rodents but its cellular reservoir in hosts remains undetermined. We hypothesized that adipose tissue serves as a <i>C. burnetii</i> reservoir during bacterial latency. BALB/c and C57BL/6 mice were infected with <i>C. burnetii</i> by the intraperitoneal route or the intracheal route. Adipose tissue was tested for the presence of <i>C. burnetii</i> several months after infection. <i>C. burnetii</i> was detected in abdominal, inguinal and dorsal adipose tissue 4 months post-infection, when no bacteria were detected in blood, liver, lungs and spleen, regardless of the inoculation route and independently of mouse strain. The transfer of abdominal adipose tissue from convalescent BALB/c mice to naïve immunodeficient mice resulted in the infection of the recipient animals. It is likely that <i>C. burnetii</i> infects adipocytes <i>in vivo</i> because bacteria were found in adipocytes within adipose tissue and replicated within <i>in vitro</i>-differentiated adipocytes. In addition, <i>C. burnetii</i> induced a specific transcriptional program in <i>in-vivo</i> and <i>in vitro</i>-differentiated adipocytes, which was enriched in categories associated with inflammatory response, hormone response and cytoskeleton. These changes may account for bacterial replication in <i>in-vitro</i> and chronic infection <i>in-vivo</i>. Adipose tissue may be the reservoir in which <i>C. burnetii</i> persists for prolonged periods after apparent clinical cure. The mouse model of <i>C. burnetii</i> infection may be used to understand the relapses of Q fever and provide new perspectives to the follow-up of patients.</p></div

Mean DNA copies of <i>C. burnetii</i> in the organs of nude mice transplanted with BALB/c-infected abdominal adipose tissue.

<p>*DNA was extracted from 10 mg of tissue.</p><p>AT: adipose tissue.</p

Cell culture and immunostaining of <b><i>C. burnetii</i></b> within adipose tissue.

<p><b>A</b>, <i>C. burnetii</i> was detected in dilacerated inguinal AT at day 60 p.i. incubated with L929 cells after 40 days of culture. Bacteria were detected using specific antibodies conjugated with Alexa 488 (in green). Original magnification: X100. <b>B</b>, The presence of <i>C. burnetii</i> was determined in sections of paraffin-embedded abdominal AT at day 30 p.i. using a rabbit anti-<i>C. burnetii</i> polyclonal antibodies. Bacteria were revealed using biotin-conjugated antibodies and peroxidase-labeled streptavidin. Labeled bacteria appear in red in the inflammatory infiltrate (circle) and in adipocytes (arrows). Original magnification: X400. <b>C</b>, Immunodetection of <i>C. burnetii</i> in sections of paraffin-embedded abdominal AT at day 30 p.i. Bacteria were revealed using rabbit anti-<i>C. burnetii</i> polyclonal antibodies and <u>Alexa 555-conjugted anti-rabbit IgG (red).</u> Labeled bacteria in red (arrows) were observed in AT (green, <u>artificially attributed color</u>) using confocal microscopy. <b>D</b>, Sections of paraffin-embedded abdominal AT from C57BL/6 transgenic mice with GFP-labeled immune cells inoculated with <i>C. burnetii</i> via the IP route at day 10 p.i. were incubated with rabbit anti-<i>C. burnetii</i> polyclonal antibodies. Bacteria were revealed using Alexa 555-conjugated F(ab') anti-rabbit IgG. Bacteria appear in red in the macrophages and in adipocytes. Original magnification: X100.</p

Microarray analysis of adipocytes stimulated by <b><i>C. burnetii</i></b>.

<p>Adipocytes differentiated from the fibroblast cell line 3T3-L1 were incubated with 50 bacteria per cell for 8 hours. <b>A</b>, Heatmap representation of modulated genes selected by supervised analysis in stimulated and unstimulated adipocytes. Genes (in rows) and samples (in columns) were organized by hierarchical clustering (Pearson correlation distance, average linkage). Gene expression levels are color coded from blue (down-modulated) to red (up-modulated). <b>B-C</b>, Functional annotation of modulated genes and qRT-PCR results. The modulated genes in the most enriched GO terms are shown (B). qRT-PCR confirmation of some genes of the most enriched GO terms identified by microarray analysis (C). qRT-PCR ratios are presented as bleu bars and microarray ratios are presented as red bars. Both methods gave similar results (Spearman correlation coefficient  = 0.85, <i>P</i> value  = 0.006).</p