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Abstract The Plasmodium falciparum erythrocyte-membrane-protein-1 (PF3D7_1150400/PF11_0521) contains both domain cassette DC13 and DBLβ3 domain binding to EPCR and ICAM-1 receptors, respectively. This type of PfEMP1 proteins with dual binding specificity mediate specific interactions with brain micro-vessels endothelium leading to the development of cerebral malaria (CM). Using plasma collected from children at time of hospital admission and after 30 days, we study an acquisition of IgG response to PF3D7_1150400/PF11_0521 DC13 and DBLβ3_D4 recombinant constructs, and five peptides located within these constructs, specifically in DBLα1.7_D2 and DBLβ3_D4 domains. We found significant IgG responses against the entire DC13, PF11_0521_DBLβ3_D4 domain, and peptides. The responses varied against different peptides and depended on the clinical status of children. The response was stronger at day 30, and mostly did not differ between CM and uncomplicated malaria (UM) groups. Specifically, the DBLβ3 B3-34 peptide that contains essential residues involved in the interaction between PF11_0521 DBLβ3_D4 domain and ICAM-1 receptor demonstrated significant increase in reactivity to IgG1 and IgG3 antibodies at convalescence. Further, IgG reactivity in CM group at time of admission against functionally active (ICAM-1-binding) PF11_0521 DBLβ3_D4 domain was associated with protection against severe anemia. These results support development of vaccine based on the PF3D7_1150400/PF11_0521 structures to prevent CM.

A recombinant Duffy binding-like (DBL)- gamma domain from a previously identified placental isolate, 732, was expressed by use of the baculovirus/insect cell system and was purified in milligram quantities. The recombinant protein binds specifically to chondroitin sulfate A (CSA) and inhibits CSA binding by placental infected erythrocytes (IEs). Polyclonal antibodies raised against the domain recognized the surfaces of live IEs from CSA-adherent clinical placental isolates. These antibodies also abrogated the in vitro binding of IEs to CSA. The 732 DBL-3 gamma domain was specifically recognized by plasma from pregnant women but not by plasma from control subjects. In addition, the protein was, comparatively, significantly more reactive with plasma from women with infected placentas, strongly suggesting that the 732 DBL-3 gamma domain carries preferentially IE-expressed immunogenic epitopes. High levels of plasma antibodies to the recombinant domain were associated with reduced placental parasite density. This is the first report of a recombinant DBL- gamma domain derived from a placental isolate that shows CSA-binding properties. Copyright © 2005 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Background: Early pregnancy Zika virus (ZIKV) infection is associated with major brain damage in fetuses, leading to microcephaly in 0.6-5.0% of cases, but the underlying mechanisms remain largely unknown., Methods: To understand the kinetics of ZIKV infection during fetal development in a nonhuman primate model, four cynomolgus macaque fetuses were exposed in utero through echo-guided intramuscular inoculation with 10 3 PFU of ZIKV at 70-80 days of gestation, 2 controls were mock inoculated. Clinical, immuno-virological and ultrasound imaging follow-ups of the mother/fetus pairs were performed until autopsy after cesarean section 1 or 2 months after exposure (n = 3 per group)., Results: ZIKV was transmitted from the fetus to the mother and then replicate in the peripheral blood of the mother from week 1 to 4 postexposure. Infected fetal brains tended to be smaller than those of controls, but not the femur lengths. High level of viral RNA ws found after the first month in brain tissues and placenta. Thereafter, there was partial control of the virus in the fetus, resulting in a decreased number of infected tissue sections and a decreased viral load. Immune cellular and humoral responses were effectively induced., Conclusions: ZIKV infection during the second trimester of gestation induces short-term brain injury, and although viral genomes persist in tissues, most of the virus is cleared before delivery., (© 2024. The Author(s).)

We investigated a COVID-19 outbreak at a fire station in Marseille, France. Confirmed cases were defined as individuals with positive SARS-CoV-2 reverse transcription (RT)-PCR and/or neutralising antibodies. All 85 firefighters at work during the outbreak period were included after questioning and sampled for RT-PCR and viral neutralisation assay. Twenty-three firefighters were confirmed positive, 19 of them were symptomatic, and four asymptomatic cases were confirmed by virus neutralisation. A total of 22 firefighters had specific neutralising antibodies against SARS-CoV-2. Neutralising antibodies were found in four asymptomatic and 18 symptomatic cases. Eleven symptomatic cases had high titres (≥ 1:80). The earliest detection of neutralising antibodies was 7 days after symptom onset, and 80% had neutralising antibodies 15 days after onset. One viral culture was positive 13 days after onset. The attack rate was 27%. We identified two introductions of the virus in this outbreak, through a presymptomatic and a paucisymptomatic case. Asymptomatic cases were not the source of a third generation of cases, although they worked without wearing a mask, indicating that asymptomatic cases did not play a significant role in this outbreak. Management and strategy based on early research of clinical signs associated with self-quarantine was effective.

Background: Delay in receiving treatment for uncomplicated malaria (UM) is often reported to increase the risk of developing severe malaria (SM), but access to treatment remains low in most high-burden areas. Understanding the contribution of treatment delay on progression to severe disease is critical to determine how quickly patients need to receive treatment and to quantify the impact of widely implemented treatment interventions, such as 'test-and-treat' policies administered by community health workers (CHWs). We conducted a pooled individual-participant meta-analysis to estimate the association between treatment delay and presenting with SM., Methods and Findings: A search using Ovid MEDLINE and Embase was initially conducted to identify studies on severe Plasmodium falciparum malaria that included information on treatment delay, such as fever duration (inception to 22nd September 2017). Studies identified included 5 case-control and 8 other observational clinical studies of SM and UM cases. Risk of bias was assessed using the Newcastle-Ottawa scale, and all studies were ranked as 'Good', scoring ≥7/10. Individual-patient data (IPD) were pooled from 13 studies of 3,989 (94.1% aged <15 years) SM patients and 5,780 (79.6% aged <15 years) UM cases in Benin, Malaysia, Mozambique, Tanzania, The Gambia, Uganda, Yemen, and Zambia. Definitions of SM were standardised across studies to compare treatment delay in patients with UM and different SM phenotypes using age-adjusted mixed-effects regression. The odds of any SM phenotype were significantly higher in children with longer delays between initial symptoms and arrival at the health facility (odds ratio [OR] = 1.33, 95% CI: 1.07-1.64 for a delay of >24 hours versus ≤24 hours; p = 0.009). Reported illness duration was a strong predictor of presenting with severe malarial anaemia (SMA) in children, with an OR of 2.79 (95% CI:1.92-4.06; p < 0.001) for a delay of 2-3 days and 5.46 (95% CI: 3.49-8.53; p < 0.001) for a delay of >7 days, compared with receiving treatment within 24 hours from symptom onset. We estimate that 42.8% of childhood SMA cases and 48.5% of adult SMA cases in the study areas would have been averted if all individuals were able to access treatment within the first day of symptom onset, if the association is fully causal. In studies specifically recording onset of nonsevere symptoms, long treatment delay was moderately associated with other SM phenotypes (OR [95% CI] >3 to ≤4 days versus ≤24 hours: cerebral malaria [CM] = 2.42 [1.24-4.72], p = 0.01; respiratory distress syndrome [RDS] = 4.09 [1.70-9.82], p = 0.002). In addition to unmeasured confounding, which is commonly present in observational studies, a key limitation is that many severe cases and deaths occur outside healthcare facilities in endemic countries, where the effect of delayed or no treatment is difficult to quantify., Conclusions: Our results quantify the relationship between rapid access to treatment and reduced risk of severe disease, which was particularly strong for SMA. There was some evidence to suggest that progression to other severe phenotypes may also be prevented by prompt treatment, though the association was not as strong, which may be explained by potential selection bias, sample size issues, or a difference in underlying pathology. These findings may help assess the impact of interventions that improve access to treatment., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: PH works for Medicines for Malaria Venture (MMV), which has a Research Collaboration Agreement in place with Imperial College. LCO declares grant funding from the World Health Organization, the Bill and Melinda Gates Foundation, and Medicines for Malaria Venture.

Three autochthonous cases of Zika virus occurred in southern France in August 2019. Diagnosis relied on serology and transcription-mediated amplification. Attempts for virus isolation and ZIKV genome RT-PCR detection remained negative. Since the index case was not identified, we addressed the issue of genotyping and geographical origin by performing hemi-nested PCR and sequencing in the Pr gene. Analysis of 16 genotype-specific Single Nucleotides Polymorphisms identified the Asian genotype and suggested a Southeast Asia origin.

Background: Zika virus (ZIKV) and Dengue virus (DENV) are often co-endemic. The high protein-sequence homology of flaviviruses renders IgG induced by and directed against them highly cross-reactive against their antigen(s), as observed on a large set of sera, leading to poorly reliable sero-diagnosis., Methods: We selected Domain III of the ZIKV Envelope (ZEDIII) sequence, which is virus specific. This recombinant domain was expressed and purified for the specific detection of ZEDIII-induced IgG by ELISA from ZIKV-RT-PCR-positive, ZIKV-IgM-positive, flavivirus-positive but ZIKV-negative, or flavivirus-negative sera. We also assessed the reactivity of ZEDIII-specific human antibodies against EDIII of DENV serotype 4 (D4EDIII) as a specific control. Sera from ZEDIII-immunized mice were also tested., Results: Cross-reactivity of IgG from 5,600 sera against total inactivated DENV or ZIKV was high (71.0% [69.1; 72.2]), whereas the specificity and sensitivity calculated using a representative cohort (242 sera) reached 90% [84.0; 95.8] and 92% [84.5; 99.5], respectively, using a ZEDIII-based ELISA. Moreover, purified human IgG against D2EDIII or D4EDIII did not bind to ZEDIII and we observed no D4EDIII reactivity with ZIKV-induced mouse polyclonal IgGs., Conclusions: We developed a ZEDIII-based ELISA that can discriminate between past or current DENV and ZIKV infections, allowing the detection of a serological scar from other flaviviruses. This could be used to confirm exposure of pregnant women or to follow the spread of an endemic disease., Competing Interests: The authors have declared that no competing interests exist.

Background: The three members of the ring-infected erythrocyte surface antigen (RESA) proteins family share high sequence homologies, which impair the detection and assignment to one or another protein of some pathogenic processes inherent to Plasmodium falciparum malaria. The present study was intended to determine if the antibody and inflammatory responses of children living in a malaria-endemic area varied depending on the RESA-1, RESA-2 or RESA-3 proteins and the severity of the disease, two groups of severe and uncomplicated malaria cases being considered., Methods: Two synthetic peptides representing predicted B cell epitopes were designed per RESA protein, all located outside of the 3' and 5' repetition blocks, in order to allow an antibody detection specific of each member of the family. Recombinant rRESA-1B and rRESA-3B proteins were also engineered. Two groups of Beninese children admitted to hospital in 2009 for either uncomplicated or severe malaria were compared for their plasma levels of IgG specifically recognizing each recombinant RESA protein or synthetic peptide, and for their plasma inflammatory cytokine levels (IFN-γ, TNF-α and IL-10), taking into account host and parasite genetic factors., Results: The absence of IgG cross-reactivity between rRESA proteins and their protein carrier as well as between each RESA peptide and a non-epitopic RESA control peptide validated the use of the engineered recombinant proteins and peptides for the measurement of plasma IgG. Taking into account age, fever duration and parasitaemia, a multiple logistic regression performed on children clustered according to their antibody responses' profiles concluded to an increased risk of severe malaria for P2 (representative of RESA-1) responders (P = 0.007). Increased IL-10 plasma levels were found in children harbouring multiclonal P. falciparum infections on the basis of the T1526G resa2 gene polymorphism (P = 0.004)., Conclusions: This study provided novel tools to dissect the seroreactivity against the three members of the RESA protein family and to describe its relation to protection against malaria. It suggested the measurement of plasma antibodies raised against specific peptides to serve as predictive immunologic markers for disease severity. Lastly, it reinforced previous observations linking the T1526G resa2 gene mutation to severe malaria.

We studied all consensus sequences within the four least 'variable blocks' (VB) present in the DBL6ε domain of VAR2CSA, the protein involved in the adhesion of infected red blood cells by Plasmodium falciparum that causes the Pregnancy-Associated Malaria (PAM). Characterising consensus sequences with respect to recognition of antibodies and percentage of responders among pregnant women living in areas where P. falciparum is endemic allows the identification of the most antigenic sequences within each VB. When combining these consensus sequences among four serotypes from VB1 or VB5, the most often recognized ones are expected to induce pan-reactive antibodies recognizing VAR2CSA from all plasmodial strains. These sequences are of main interest in the design of an immunogenic molecule. Using a similar approach than for DBL6ε, we studied the five other DBL and the CIDRpam from VAR2CSA, and again identified VB segments with highly conserved consensus sequences. In addition, we identified consensus sequences in other var genes expressed by non-PAM parasites. This finding paves the way for vaccine design against other pathologies caused by P. falciparum.

A specific and sensitive serodiagnostic test for Mycobacterium ulcerans infection would greatly assist the diagnosis of Buruli ulcer and would also facilitate seroepidemiological surveys. By comparative genomics, we identified 45 potential M. ulcerans specific proteins, of which we were able to express and purify 33 in E. coli. Sera from 30 confirmed Buruli ulcer patients, 24 healthy controls from the same endemic region and 30 healthy controls from a non-endemic region in Benin were screened for antibody responses to these specific proteins by ELISA. Serum IgG responses of Buruli ulcer patients were highly variable, however, seven proteins (MUP045, MUP057, MUL&#95;0513, Hsp65, and the polyketide synthase domains ER, AT propionate, and KR A) showed a significant difference between patient and non-endemic control antibody responses. However, when sera from the healthy control subjects living in the same Buruli ulcer endemic area as the patients were examined, none of the proteins were able to discriminate between these two groups. Nevertheless, six of the seven proteins showed an ability to distinguish people living in an endemic area from those in a non-endemic area with an average sensitivity of 69% and specificity of 88%, suggesting exposure to M. ulcerans. Further validation of these six proteins is now underway to assess their suitability for use in Buruli ulcer seroepidemiological studies. Such studies are urgently needed to assist efforts to uncover environmental reservoirs and understand transmission pathways of the M. ulcerans.

Background: Placental malaria is a disease linked to the sequestration of Plasmodium falciparum infected red blood cells (IRBC) in the placenta, leading to reduced materno-fetal exchanges and to local inflammation. One of the virulence factors of P. falciparum involved in cytoadherence to chondroitin sulfate A, its placental receptor, is the adhesive protein VAR2CSA. Its localisation on the surface of IRBC makes it accessible to the immune system. VAR2CSA contains six DBL domains. The DBL6epsilon domain is the most variable. High variability constitutes a means for the parasite to evade the host immune response. The DBL6epsilon domain could constitute a very attractive basis for a vaccine candidate but its reported variability necessitates, for antigenic characterisations, identifying and classifying commonalities across isolates., Methodology/principal Findings: Local alignment analysis of the DBL6epsilon domain had revealed that it is not as variable as previously described. Variability is concentrated in seven regions present on the surface of the DBL6epsilon domain. The main goal of our work is to classify and group variable sequences that will simplify further research to determine dominant epitopes. Firstly, variable sequences were grouped following their average percent pairwise identity (APPI). Groups comprising many variable sequences sharing low variability were found. Secondly, ELISA experiments following the IgG recognition of a recombinant DBL6epsilon domain, and of peptides mimicking its seven variable blocks, allowed to determine an APPI cut-off and to isolate groups represented by a single consensus sequence., Conclusions/significance: A new sequence approach is used to compare variable regions in sequences that have extensive segmental gene relationship. Using this approach, the VAR2CSA DBL6 domain is composed of 7 variable blocks with limited polymorphism. Each variable block is composed of a limited number of consensus types. Based on peptide based ELISA, variable blocks with 85% or greater sequence identity are expected to be recognized equally well by antibody and can be considered the same consensus type. Therefore, the analysis of the antibody response against the classified small number of sequences should be helpful to determine epitopes.

Background: Antibodies, particularly cytophilic IgG subclasses, with specificity for asexual blood stage antigens of Plasmodium falciparum, are thought to play an important role in acquired immunity to malaria. Evaluating such responses in longitudinal sero-epidemiological field studies, allied to increasing knowledge of the immunological mechanisms associated with anti-malarial protection, will help in the development of malaria vaccines., Methods and Findings: We conducted a 1-year follow-up study of 305 Senegalese children and identified those resistant or susceptible to malaria. In retrospective analyses we then compared post-follow-up IgG responses to six asexual-stage candidate malaria vaccine antigens in groups of individuals with clearly defined clinical and parasitological histories of infection with P. falciparum. In age-adjusted analyses, children resistant to malaria as well as to high-density parasitemia, had significantly higher IgG1 responses to GLURP and IgG3 responses to MSP2 than their susceptible counterparts. Among those resistant to malaria, high anti-MSP1 IgG1 levels were associated with protection against high-density parasitemia. To assess functional attributes, we used an in vitro parasite growth inhibition assay with purified IgG. Samples from individuals with high levels of IgG directed to MSP1, MSP2 and AMA1 gave the strongest parasite growth inhibition, but a marked age-related decline was observed in these effects., Conclusion: Our data are consistent with the idea that protection against P. falciparum malaria in children depends on acquisition of a constellation of appropriate, functionally active IgG subclass responses directed to multiple asexual stage antigens. Our results suggest at least two distinct mechanisms via which antibodies may exert protective effects. Although declining with age, the growth inhibitory effects of purified IgG measurable in vitro reflected levels of anti-AMA1, -MSP1 and -MSP2, but not of anti-GLURP IgG. The latter could act on parasite growth via indirect parasiticidal pathways.

In the Saimiri sciureus monkey, erythrocytes infected with the varO antigenic variant of the Plasmodium falciparum Palo Alto 89F5 clone bind uninfected red blood cells (rosetting), form autoagglutinates, and have a high multiplication rate, three phenotypic characteristics that are associated with severe malaria in human patients. We report here that varO parasites express a var gene having the characteristics of group A var genes, and we show that the varO Duffy binding-like 1alpha(1) (DBL1alpha(1)) domain is implicated in the rosetting of both S. sciureus and human erythrocytes. The soluble varO N-terminal sequence (NTS)-DBL1alpha(1) recombinant domain, produced in a baculovirus-insect cell system, induced high titers of antibodies that reacted with varO-infected red blood cells and disrupted varO rosettes. varO parasites were culture adapted in vitro using human erythrocytes. They formed rosettes and autoagglutinates, and they had the same surface serotype and expressed the same varO gene as the monkey-propagated parasites. To develop an in vitro model with highly homogeneous varO parasites, rosette purification was combined with positive selection by panning with a varO NTS-DBL1alpha(1)-specific mouse monoclonal antibody. The single-variant, clonal parasites were used to analyze seroprevalence for varO at the village level in a setting where malaria is holoendemic (Dielmo, Senegal). We found 93.6% (95% confidence interval, 89.7 to 96.4%) seroprevalence for varO surface-reacting antibodies and 86.7% (95% confidence interval, 82.8 to 91.6%) seroprevalence for the recombinant NTS-DBL1alpha(1) domain, and virtually all permanent residents had seroconverted by the age of 5 years. These data imply that the varO model is a relevant in vivo and in vitro model for rosetting and autoagglutination that can be used for rational development of vaccine candidates and therapeutic strategies aimed at preventing malaria pathology.

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-alpha stimulation. This suggests that placental malaria could increase the risk of HIV-1 transmission in utero.

H-NS is a protein of the bacterial nucleoid involved in DNA compaction and transcription regulation. In vivo, H-NS selectively silences specific genes of the bacterial chromosome. However, many studies have concluded that H-NS binds sequence-independently to DNA, leaving the molecular basis for its selectivity unexplained. We show that the negative regulatory element (NRE) of the supercoiling-sensitive Escherichia coliproU gene contains two identical high-affinity binding sites for H-NS. Cooperative binding of H-NS is abrogated by changes in DNA superhelical density and temperature. We further demonstrate that the high-affinity sites nucleate cooperative binding and establish a nucleoprotein structure required for silencing. Mutations in these sites result in loss of repression by H-NS. In this model, silencing at proU, and by inference at other genes directly regulated by H-NS, is tightly controlled by the cooperativity between bound H-NS molecules.

At several E. coli promoters, initiation of transcription is repressed by a tight nucleoprotein complex formed by the assembly of the H-NS protein. In order to characterize the relationship between the structure of H-NS oligomers in solution and on relevant DNA fragments, we have compared wild-type H-NS and several transdominant H-NS mutants using gel shift assays, DNase I footprinting, analytical ultracentrifugation, and reactivity toward a cross-linking reagent. In solution, oligomerization occurs through two protein interfaces, one necessary to construct a dimeric core (and involving residues 1-64) and the other required for subsequent assembly of these dimers. We show that, as well as region 64-95, residues present in the NH(2)-terminal coiled coil domain also participate in this second interface. Our results support the view that the same interacting interfaces are also involved on the DNA. We propose that the dimeric core recognizes specific motifs, with the second interface being critical for their correct head to tail assembly. The COOH-terminal domain of the protein contains the DNA binding motif essential for the discrimination of this specific functional assembly over competitive nonspecific H-NS polymers.

Escherichia coli K-12, the most widely used laboratory bacterium, does not secrete proteins into the extracellular medium under standard growth conditions, despite possessing chromosomal genes encoding a putative type II secretion machinery (secreton). We show that in wild-type E.coli K-12, divergent transcription of the two operons in the main chromosomal gsp locus, encoding the majority of the secreton components, is silenced by the nucleoid-structuring protein H-NS. In mutants lacking H-NS, the secreton genes cloned on a moderate-copy-number plasmid are expressed and promote efficient secretion of the endogenous, co-regulated endochitinase ChiA. This is the first time that secretion of an endogenous extracellular protein has been demonstrated in E.coli K-12.

During the last decade, the hns gene and its product, the H-NS protein, have been extensively studied in Escherichia coli. H-NS-like proteins seem to be widespread in gram-negative bacteria. However, unlike in E. coli and in Salmonella enterica serovar Typhimurium, little is known about their role in the physiology of those organisms. In this report, we describe the isolation of vicH, an hns-like gene in Vibrio cholerae, the etiological agent of cholera. This gene was isolated from a V. cholerae genomic library by complementation of different phenotypes associated with an hns mutation in E. coli. It encodes a 135-amino-acid protein showing approximately 50% identity with both H-NS and StpA in E. coli. Despite a low amino acid conservation in the N-terminal part, VicH is able to cross-react with anti-H-NS antibodies and to form oligomers in vitro. The vicH gene is expressed as a single gene from two promoters in tandem and is induced by cold shock. A V. cholerae wild-type strain expressing a vicHDelta92 gene lacking its 3' end shows pleiotropic alterations with regard to mucoidy and salicin metabolism. Moreover, this strain is unable to swarm on semisolid medium. Similarly, overexpression of the vicH wild-type gene results in an alteration of swarming behavior. This suggests that VicH could be involved in the virulence process in V. cholerae, in particular by affecting flagellum biosynthesis.

The chromosome of Escherichia coli K-12 contains a putative gene, yheB (chiA), at centisome 74.7, whose product shows sequence similarity with chitinases of bacterial and viral origin. We cloned the chiA (yheB) gene and demonstrated that it codes for a 94.5 kDa periplasmic protein with endochitinase/lysozyme activity. Under standard laboratory growth conditions, chiA expression is very low, as shown by the Lac- phenotype of a chiA transcriptional fusion to a promoterless lacZ reporter. To identify factors that control chitinase gene expression, we generated random Tn10 insertions in the chromosome of the fusion-containing strain, selecting for a Lac+ phenotype. The majority of the mutations that caused a Lac+ phenotype mapped to the hns gene, encoding the nucleoid-structuring protein H-NS. Transcription of chiA in vivo is driven by a single sigma70 promoter and is derepressed in an hns mutant. Using a competitive gel retardation assay, we demonstrated that H-NS binds directly and with high affinity to the chiA promoter region. In addition to hns, other E. coli mutations causing defects in global regulatory proteins, such as fis, crp or stpA in combination with hns, increased chiA expression to different extents, as did decreasing the growth temperature from 37 degrees C to 30 degrees C. A possible physiological function of ChiA (YheB) endochitinase in E. coli K-12 is discussed.