Placental Malaria is Associated with Higher LILRB2 Expression in Monocyte Subsets and Lower Anti-Malarial IgG Antibodies During Infancy.

Background: Placental malaria (PM) is associated with a higher susceptibility of infants to Plasmodium falciparum (Pf) malaria. A hypothesis of immune tolerance has been suggested but no clear explanation has been provided so far. Our goal was to investigate the involvement of inhibitory receptors LILRB1 and LILRB2, known to drive immune evasion upon ligation with pathogen and/or host ligands, in PM-induced immune tolerance. Method: Infants of women with or without PM were enrolled in Allada, southern Benin, and followed-up for 24 months. Antibodies with specificity for five blood stage parasite antigens were quantified by ELISA, and the frequency of immune cell subsets was quantified by flow cytometry. LILRB1 or LILRB2 expression was assessed on cells collected at 18 and 24 months of age. Findings: Infants born to women with PM had a higher risk of developing symptomatic malaria than those born to women without PM (IRR=1.53, p=0.040), and such infants displayed a lower frequency of non-classical monocytes (OR=0.74, p=0.01) that overexpressed LILRB2 (OR=1.36, p=0.002). Moreover, infants born to women with PM had lower levels of cytophilic IgG and higher levels of IL-10 during active infection. Interpretation: Modulation of IgG and IL-10 levels could impair monocyte functions (opsonisation/phagocytosis) in infants born to women with PM, possibly contributing to their higher susceptibility to malaria. The long-lasting effect of PM on infants' monocytes was notable, raising questions about the capacity of ligands such as Rifins or HLA-I molecules to bind to LILRB1 and LILRB2 and to modulate immune responses, and about the reprogramming of neonatal monocytes/macrophages.

Peripheral spectrum neurological disorder after arbovirus infection is associated with HLA-F variants among Northeastern Brazilians.

INTRODUCTION: Non-classical class I human leukocyte antigens (HLA) molecules are known to modulate the function of cytotoxic cells (NK and T CD8+) during viral infection by interacting with inhibitory/activating receptors. However, little is known about the HLA-E/-F genetic variability on arbovirus infections. METHODS: We evaluated by massive parallel sequencing the full HLA-E/-F genetic diversity among patients infected during the arbovirus (ZIKV, DENV, and CHIKV) outbreak leading to a broad range of neurological complications in the Brazilian State of Pernambuco. In parallel, healthy blood donors from the same area were also studied. Plink and R software were used for genetic association study. To limit the false-positive results and enhance the reliability of the results, we adopted P-values <0.01 as significant levels. RESULTS: Compared to controls, the HLA-F alleles: -1610 C (rs17875375), +1383 G (rs17178385), and +3537 A (rs17875384), all in complete linkage disequilibrium with each other (r2 = 1), were overrepresented in patients presenting peripheral spectrum disorders (PSD). The HLA-F*Distal-D haplotype that harbored the -1610 C allele exhibited a trend increase in PSD group. No associations were found for HLA-E. CONCLUSIONS: Our findings showed that the HLA-F genetic background seems to be more important than HLA-E on the susceptibility to PSD complications.

Cytokines and Soluble HLA-G Levels in the Acute and Recovery Phases of Arbovirus-Infected Brazilian Patients Exhibiting Neurological Complications.

Severe neurological complications following arbovirus infections have been a major concern in seasonal outbreaks, as reported in the Northeast region of Brazil, where the same mosquito transmitted Zika (ZIKV), Dengue (DENV), and Chikungunya (CHIKV) viruses. In this study, we evaluated the levels of 36 soluble markers, including cytokines, chemokines, growth factors, and soluble HLA-G (Luminex and ELISA) in: i) serum and cerebrospinal fluid (CSF), during the acute phase and two years after the infection (recovery phase, only serum), ii) the relationship among all soluble molecules in serum and CSF, and iii) serum of infected patients without neurological complications, during the acute infection. Ten markers (sHLA-G, IL-10, IL-22, IL-8, MIP-1α, MIP-1ß, MCP-1, HGF, VEGF, and IL-1RA) exhibited differential levels between the acute and recovery phases, with pronounced increases in MIP-1α (P<0.0001), MCP-1 (P<0.0001), HGF (P= 0.0001), and VEGF (P<0.0001) in the acute phase. Fourteen molecules (IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-13, IL-15, IL-17A, IFN-α, TNF, and G-CSF) exhibited distinct levels between arbovirus patients presenting or not neurological complications. IL-8, EGF, IL-6, and MCP-1 levels were increased in CSF, while RANTES and Eotaxin levels were higher in serum. Soluble serum (IL-22, RANTES, Eotaxin) and CSF (IL-8, EGF, IL-3) mediators may discriminate putative risks for neurological complications following arbovirus infections. Neurological complications were associated with the presence of a predominant inflammatory profile, whereas in non-complicated patients an anti-inflammatory profile may predominate. Mediators associated with neuroregeneration (EGF and IL-3) may be induced in response to neurological damage. Broad spectrum immune checkpoint molecules (sHLA-G) interact with cytokines, chemokines, and growth factors. The identification of soluble markers may be useful to monitor neurological complications and may aid in the development of novel therapies against neuroinflammation.

Human leukocyte antigen (HLA)-F and -G gene polymorphisms and haplotypes are associated with malaria susceptibility in the Beninese Toffin children.

BACKGROUND: Little attention has been devoted to the role of the immunoregulatory HLA-E/-F/-G genes in malaria. We evaluated the entire HLA-E/-F/-G variability in Beninese children highly exposed to Plasmodium falciparum (P.f.) malaria. METHODS: 154 unrelated children were followed-up for six months and evaluated for the presence and number of malaria episodes. HLA-E/-F/-G genes were genotyped using massively parallel sequencing. Anti P.f. antibodies were evaluated using ELISA. RESULTS: Children carrying the G allele at HLA-F (-1499,rs183540921) showed increased P.f. asymptomatic/symptomatic ratio, suggesting that these children experienced more asymptomatic P.f. episodes than symptomatic one. Children carrying HLA-G-UTR-03 haplotype exhibited increased risk for symptomatic P.f. episodes and showed lower IgG2 response against P.f. GLURP-R2 when compared to the non-carriers. No associations were observed for the HLA-E gene. CONCLUSION: HLA-F associations may be related to the differential expression profiles of the encoded immunomodulatory molecules, and the regulatory sites at the HLA-G 3'UTR may be associated to posttranscriptional regulation of HLA-G and to host humoral response against P.f.

Increased HLA-G Expression in Tissue-Infiltrating Cells in Inflammatory Bowel Diseases.

BACKGROUND: Since HLA-G is an immune checkpoint molecule and since Crohn's disease (CD) and ulcerative colitis (UC) exhibit deregulated immune-mediated mechanisms, we aimed to evaluate intestinal HLA-G expression and soluble HLA-G (sHLA-G) levels in CD/UC patients stratified according to the CD phenotype/localization and UC extension. METHODS: HLA-G tissue expression was assessed by immunohistochemistry in biopsies collected from 151 patients (90 CD, 61 UC) and in surgical resection specimens (28 CD, 12 UC). Surgical material from 24 healthy controls was also assessed. Plasma sHLA-G levels (97 CD, 81 UC, and 120 controls) were evaluated using ELISA. RESULTS: HLA-G expression was similarly observed in the intestinal epithelial cells of control and CD/UC specimens. However, in biopsies, the plasma cells/lymphocytes infiltrating the lamina propria in CD/UC presented (1) increased HLA-G expression compared to controls (P < 0.0001), (2) greater cell staining in UC cells than in CD cells irrespective of disease extent (P = 0.0011), and (3) an increased number of infiltrating cells in the inflammatory CD phenotype compared to that in the stenosing and fistulizing phenotypes (P = 0.0407). In surgical specimens, CD/UC patients exhibited higher infiltrating cell HLA-G expression in lesion areas than in margins. sHLA-G levels were higher in UC/CD patients (P < 0.0001) than in controls, but no difference was observed between diseases. CONCLUSIONS: Increased infiltrating cell HLA-G expression associated with increased sHLA-G levels in CD/UC patients may reflect ongoing host strategies to suppress chronic inflammation.

Extravascular Dermal Trypanosomes in Suspected and Confirmed Cases of gambiense Human African Trypanosomiasis.

BACKGROUND: The diagnosis of gambiense human African trypanosomiasis (gHAT) typically involves 2 steps: a serological screen, followed by the detection of living trypanosome parasites in the blood or lymph node aspirate. Live parasites can, however, remain undetected in some seropositive individuals, who, we hypothesize, are infected with Trypanosoma brucei gambiense parasites in their extravascular dermis. METHODS: To test this hypothesis, we conducted a prospective observational cohort study in the gHAT focus of Forecariah, Republic of Guinea. Of the 5417 subjects serologically screened for gHAT, 66 were enrolled into our study and underwent a dermatological examination. At enrollment, 11 seronegative, 8 unconfirmed seropositive, and 18 confirmed seropositive individuals had blood samples and skin biopsies taken and examined for trypanosomes by molecular and immunohistological methods. RESULTS: In seropositive individuals, dermatological symptoms were significantly more frequent, relative to seronegative controls. T.b. gambiense parasites were present in the blood of all confirmed cases (n = 18) but not in unconfirmed seropositive individuals (n = 8). However, T. brucei parasites were detected in the extravascular dermis of all unconfirmed seropositive individuals and all confirmed cases. Skin biopsies of all treated cases and most seropositive untreated individuals progressively became negative for trypanosomes 6 and 20 months later. CONCLUSIONS: Our results highlight the skin as a potential reservoir for African trypanosomes, with implications for our understanding of this disease's epidemiology in the context of its planned elimination and underlining the skin as a novel target for gHAT diagnostics.

HLA-G 14 bp In/Del and +3142 C/G genotypes are differentially expressed between patients with grade IV gliomas and controls.

Aim: Human Leukocyte Antigen-G (HLA-G) is a non-classical class I molecule that is involved in maternal-fetal immunotolerance. In cancer, this molecule contributes to the tumor escape. The aim of this study was to evaluate the 14 bp In/Del and +3142 C > G polymorphisms of the HLA-G 3' UTR and its relation with plasma and tissue HLA-G expression in patients with grade IV (high-grade) and grade I/II (low-grade) gliomas and controls.Patients and methods: Peripheral blood and tumor biopsies were collected from 85 patients with gliomas and blood samples from 94 controls. Polymorphisms were analyzed from blood DNA. Soluble HLA-G (sHLA-G) was measured by ELISA in plasma of the subjects and the tissue expression by immunohistochemistry on patient's tissue.Results: Higher levels of sHLA-G were observed in grade IV gliomas patients than in controls (p < 0.0001). In grade IV patients, the heterozygous 14pb In/Del, +3142 C/G genotypes and Del/C*In/G haplotype were associated with higher sHLA-G levels (p < 0.0001) when compared with controls. GBM patients were stratified into high and low sHLA-G expression and an association was found between +3142 C allele and high sHLA-G plasmatic levels (p = 0.0095). Tissue HLA-G immunolabel was higher in high-grade than low-grade gliomas (p = 0.0033).Conclusion: This was the first study evaluating HLA-G 3' UTR polymorphisms and expression in patients with gliomas. The 14 bp In/Del and +3142 C/G genotypes and haplotypes showed high influence over sHLA-G expression, suggesting a heterozygous advantage in the tumor context and may contribute to a worse prognosis in glioma patients.

Hla-C genetic diversity and evolutionary insights in two samples from Brazil and Benin.

Human leukocyte antigen-C (HLA-C) is a classical HLA class I molecule that binds and presents peptides to cytotoxic T lymphocytes in the cell surface. HLA-C has a dual function because it also interacts with Killer-cell immunoglobulin-like receptors (KIR) receptors expressed in natural killer and T cells, modulating their activity. The structure and diversity of the HLA-C regulatory regions, as well as the relationship among variants along the HLA-C locus, are poorly addressed, and few population-based studies explored the HLA-C variability in the entire gene in different population samples. Here we present a molecular and bioinformatics method to evaluate the entire HLA-C diversity, including regulatory sequences. Then, we applied this method to survey the HLA-C diversity in two population samples with different demographic histories, one highly admixed from Brazil with major European contribution, and one from Benin with major African contribution. The HLA-C promoter and 3'UTR were very polymorphic with the presence of few, but highly divergent haplotypes. These segments also present conserved sequences that are shared among different primate species. Nucleotide diversity was higher in other segments rather than exons 2 and 3, particularly around exon 5 and the second half of the 3'UTR region. We detected evidence of balancing selection on the entire HLA-C locus and positive selection in the HLA-C leader peptide, for both populations. HLA-C motifs previously associated with KIR interaction and expression regulation are similar between both populations. Each allele group is associated with specific regulatory sequences, reflecting the high linkage disequilibrium along the entire HLA-C locus in both populations.

Plasma levels of soluble HLA-G and cytokines in papillary thyroid carcinoma before and after thyroidectomy.

BACKGROUND: Increasing evidence shows that chronic inflammation plays an important role in thyroid tumorigenesis. Cytokines as central mediators in inflammatory microenvironment can present both pro-tumour and anti-tumour effects and cytokine release may be influenced by soluble HLA-G (sHLA-G), an immune checkpoint molecule whose expression can also be induced by certain cytokines. AIM: To understand the role of these soluble factors in papillary thyroid cancer (PTC). METHODS: We evaluated plasma levels of sHLA-G and of 13 cytokines using ELISA and flow cytometry, respectively, in PTC patients at two time points: pre- and post-thyroidectomy; and control subjects. RESULTS: Compared with controls, IL-6 levels were increased, while IL-1ß, IFN-α and TGF-ß1 levels were decreased in pre-thyroidectomy PTC patients. IFN-α and TGF-ß1 efficiently discriminated patients from controls and were associated with extrathyroidal extension and lymph node metastasis, respectively. In addition, TNF and IL-13 were associated with male gender, lymph node metastasis and Hashimoto thyroiditis, and sHLA-G with tumour invasion. Compared with pre-thyroidectomy, IL-4, IL-10, TNF, IFN-α and TGF-ß1 levels were increased in post-thyroidectomy. CONCLUSION: There are significant changes in the cytokine profile after surgical removal of the thyroid tumour, and IFN-α e TGF-ß1 showed to be promising cytokines for discriminating PTC patients from controls. We also found that different cytokines are associated with clinicohistopathological characteristics of PTC related to poor prognosis, suggesting that cytokines seem to play an important role in PTC development and management.

Analysis of HLA-G protein expression in leprosy.

The aim of this study was to evaluate the expression of human leukocyte antigen G (HLA-G) in leprosy. Biopsy and serum samples were collected from 18 patients presenting with leprosy and from healthy controls. Samples were analyzed using immunohistochemistry and ELISA techniques. HLA-G expression was observed in biopsy samples of all patients. The healthy control samples were consistently negative for HLA-G expression. Control plasma samples displayed significantly higher HLA-G expression than those from the patients (p < 0.01). These results are the first demonstration of the expression of HLA-G in leprosy.

First genome-wide association study of non-severe malaria in two birth cohorts in Benin.

Recent research efforts to identify genes involved in malaria susceptibility using genome-wide approaches have focused on severe malaria. Here, we present the first GWAS on non-severe malaria designed to identify genetic variants involved in innate immunity or innate resistance mechanisms. Our study was performed on two cohorts of infants from southern Benin (525 and 250 individuals used as discovery and replication cohorts, respectively) closely followed from birth to 18-24 months of age, with an assessment of a space- and time-dependent environmental risk of exposure. Both the recurrence of mild malaria attacks and the recurrence of malaria infections as a whole (symptomatic and asymptomatic) were considered. Post-GWAS functional analyses were performed using positional, eQTL, and chromatin interaction mapping to identify the genes underlying association signals. Our study highlights a role of PTPRT, a tyrosine phosphatase receptor involved in STAT3 pathway, in the protection against both mild malaria attacks and malaria infections (p = 9.70 × 10-8 and p = 1.78 × 10-7, respectively, in the discovery cohort). Strong statistical support was also found for a role of MYLK4 (meta-analysis, p = 5.29 × 10-8 with malaria attacks), and for several other genes, whose biological functions are relevant in malaria infection. Results shows that GWAS on non-severe malaria can successfully identify new candidate genes and inform physiological mechanisms underlying natural protection against malaria.

Plasmodium falciparum merozoite surface antigen-specific cytophilic IgG and control of malaria infection in a Beninese birth cohort.

BACKGROUND: Substantial evidence indicates that cytophilic IgG responses to Plasmodium falciparum merozoite antigens play a role in protection from malaria. The specific targets mediating immunity remain unclear. Evaluating antibody responses in infants naturally-exposed to malaria will allow to better understand the establishment of anti-malarial immunity and to contribute to a vaccine development by identifying the most appropriate merozoite candidate antigens. METHODS: The study was based on parasitological and clinical active follow-up of infants from birth to 18 months of age conducted in the Tori Bossito area of southern Benin. For 399 infants, plasma levels of cytophilic IgG antibodies with specificity for five asexual stage malaria vaccine candidate antigens were determined by ELISA in infants' peripheral blood at 6, 9, 12 and 15 months of age. Multivariate mixed logistic model was used to investigate the association between antibody levels and anti-malarial protection in the trimester following the IgG quantification. Moreover, the concentrations of merozoite antigen-specific IgG were compared between a group of infants apparently able to control asymptomatic malaria infection (CAIG) and a group of infants with no control of malaria infection (Control group (NCIG)). Protective effect of antibodies was also assessed after 15 months of malaria exposure with a Cox regression model adjusted on environmental risk. RESULTS: Cytophilic IgG responses to AMA1, MSP1, MSP2-3D7, MSP2-FC27, MSP3 and GLURP R2 were associated with increasing malarial infection risk in univariate analysis. The multivariate mixed model showed that IgG1 and IgG3 to AMA1 were associated with an increased risk of malarial infection. However infants from CAIG (n = 53) had significantly higher AMA1-, MSP2-FC27-, MSP3-specific IgG1 and AMA1-, MSP1-, MSP2-FC27-, MSP3 and GLURP-R2-specific IgG3 than those from NCIG (n = 183). The latter IgG responses were not associated with protection against clinical malaria in the whole cohort when protective effect is assessed after 15 months of malaria exposition. CONCLUSION: In this cohort, merozoite antigen-specific cytophilic IgG levels represent a marker of malaria exposure in infants from 6 to 18 months of age. However, infants with resolution of asymptomatic infection (CAIG) seem to have acquired naturally immunity against P. falciparum. This observation is encouraging in the context of the development of multitarget P. falciparum vaccines.

High level of soluble human leukocyte antigen (HLA)-G at beginning of pregnancy as predictor of risk of malaria during infancy.

Placental malaria has been associated with an immune tolerance phenomenon and a higher susceptibility to malaria infection during infancy. HLA-G is involved in fetal maternal immune tolerance by inhibiting maternal immunity. During infections HLA-G can be involved in immune escape of pathogens by creating a tolerogenic environment. Recent studies have shown an association between the risk of malaria and HLA-G at both genetic and protein levels. Moreover, women with placental malaria have a higher probability of giving birth to children exhibiting high sHLA-G, independently of their own level during pregnancy. Our aim was to explore the association between the level of maternal soluble HLA-G and the risk of malaria infection in their newborns. Here, 400 pregnant women and their children were actively followed-up during 24 months. The results show a significant association between the level of sHLA-G at the first antenatal visit and the time to first malaria infection during infancy adjusted to the risk of exposure to vector bites (aHR = 1.02, 95%CI [1.01-1.03], p = 0.014). The level of sHLA-G is a significant predictor of the occurrence of malaria infection during infancy consistent with the hypothesis that mother sHLA-G could be a biomarker of malaria susceptibility in children.

HLA-G expression during hookworm infection in pregnant women.

INTRODUCTION: HLA-G plays a key role on immune tolerance. Pathogens can induce soluble HLA-G (sHLA-G) production to down-regulate the host immune response, creating a tolerogenic environment favorable for their dissemination. To our knowledge, no study has yet been conducted to assess the relationship between sHLA-G and geohelminth infections. METHODS: The study was conducted in Allada, Southeastern Benin, from 2011-2014. The study population encompassed 400 pregnant women, included before the end of the 28th week of gestation and followed-up until delivery. At two antenatal care visits and at delivery, stool and blood samples were collected. Helminths were diagnosed by means of the Kato-Katz concentration technique. We used quantile regression to analyze the association between helminth infections and sHLA-G levels during pregnancy. RESULTS: sHLA-G levels gradually increased during pregnancy and reached maximal levels at delivery. Prevalence of helminth infections was low, with a majority of hookworm infections. We found significantly more hookworm-infected women above the 80th quantile (Q80) of the distribution of the mean sHLA-G level (p < 0.03, multivariate quantile regression). Considering only women above the Q80 percentile, the mean sHLA-G level was significantly higher in hookworm-infected compared to uninfected women (p = 0.04). CONCLUSION: High levels of sHLA-G were associated with hookworm infection in pregnant women. This result is consistent with the potential involvement of sHLA-G in immune tolerance induced by helminths during pregnancy.

The association of HLA-G polymorphisms and the synergistic effect of sMICA and sHLA-G with chronic kidney disease and allograft acceptance.

The HLA-G and MICA genes are stimulated under inflammatory conditions and code for soluble (sMICA and sHLA-G) or membrane-bound molecules that exhibit immunomodulatory properties. It is still unclear whether they would have a synergistic or antagonistic effect on the immunomodulation of the inflammatory response, such as in chronic kidney disease (CKD), contributing to a better prognosis after the kidney transplantation. In this study, we went from genetic to plasma analysis, first evaluating the polymorphism of MICA, NKG2D and HLA-G in a cohort from Southern Brazil, subdivided in a control group of individuals (n = 75), patients with CKD (n = 94), and kidney-transplant (KT) patients (n = 64). MICA, NKG2D and HLA-G genotyping was performed by polymerase chain reaction with specific oligonucleotide probes, Taqman and Sanger sequencing, respectively. Levels of soluble forms of MICA and HLA-G were measured in plasma with ELISA. Case-control analysis showed that the individuals with haplotype HLA-G*01:01/UTR-4 have a lower susceptibility to develop chronic kidney disease (OR = 0.480; p = 0.032). Concerning the group of kidney-transplant patients, the HLA-G genotypes +3010 GC (rs1710) and +3142 GC (rs1063320) were associated with higher risk for allograft rejection (OR = 5.357; p = 0.013 and OR = 5.357, p = 0.013, respectively). Nevertheless, the genotype +3010 GG (OR = 0.136; p = 0.041) was associated with kidney allograft acceptance, suggesting that it is a protection factor for rejection. In addition, the phenotypic analysis revealed higher levels of sHLA-G (p = 0.003) and sMICA (p < 0.001) in plasma were associated with the development of CKD. For patients who were already under chronic pathological stress and underwent a kidney transplant, a high sMICA (p = 0.001) in pre-transplant proved to favor immunomodulation and allograft acceptance. Even so, the association of genetic factors with differential levels of soluble molecules were not evidenced, we displayed a synergistic effect of sMICA and sHLA-G in response to inflammation. This increase was observed in CKD patients, that when undergo transplantation, had this previous amount of immunoregulatory molecules as a positive factor for the allograft acceptance.

HLA-G, -E and -F regulatory and coding region variability and haplotypes in the Beninese Toffin population sample.

HLA-G/E/F genes exhibit immunomodulatory properties and are expressed in placenta. Little attention has been devoted to the study of these genes in sub-Saharan African populations, which are yet the most diverse. To fill this gap, we evaluated the complete gene variability, approximately 5.1 kb for HLA-G (n = 149), 7.7 kb for HLA-E (n = 150) and 6.2 kb for HLA-F (n = 152) in the remote Beninese Toffin population, using massive parallel sequencing. Overall, 96, 37 and 68 variable sites were detected along the entire HLA-G, -E and -F, respectively, arranged into region-specific haplotypes; i.e., promoter haplotypes (16, 19, and 15 respectively), coding haplotypes (19, 15, and 29 respectively), 3' untranslated region (3'UTR) haplotypes (12, 7 and 2, respectively) and extended haplotypes (33, 31 and 32 respectively). All promoter/coding/3'UTR haplotypes followed the patterns already described in worldwide populations. HLA-E was the most conserved, exhibiting mainly two full-length encoded-molecules (E*01:01 and E*01:03), followed by HLA-F, three full-length proteins (F*01:01, F*01:02 and F*01:03) and HLA-G, four proteins: three full-length (G*01:01, G*01:03 and G*01:04) and one truncated (G*01:05N). Although HLA-G/E/F alleles in the Toffin population were the most frequently observed worldwide, the frequencies of the coding haplotypes were closely similar to those described for other African populations (Guinea-Conakry and Burkina-Faso), when compared to non-African ones (Brazilian), indicating that variable sites along these genes were present in Africa before human dispersion.

Associations between an IgG3 polymorphism in the binding domain for FcRn, transplacental transfer of malaria-specific IgG3, and protection against Plasmodium falciparum malaria during infancy: A birth cohort study in Benin.

BACKGROUND: Transplacental transfer of maternal immunoglobulin G (IgG) to the fetus helps to protect against malaria and other infections in infancy. Recent studies have emphasized the important role of malaria-specific IgG3 in malaria immunity, and its transfer may reduce the risk of malaria in infancy. Human IgGs are actively transferred across the placenta by binding the neonatal Fc receptor (FcRn) expressed within the endosomes of the syncytiotrophoblastic membrane. Histidine at position 435 (H435) provides for optimal Fc-IgG binding. In contrast to other IgG subclasses, IgG3 is highly polymorphic and usually contains an arginine at position 435, which reduces its binding affinity to FcRn in vitro. The reduced binding to FcRn is associated with reduced transplacental transfer and reduced half-life of IgG3 in vivo. Some haplotypes of IgG3 have histidine at position 435. This study examines the hypotheses that the IgG3-H435 variant promotes increased transplacental transfer of malaria-specific antibodies and a prolonged IgG3 half-life in infants and that its presence correlates with protection against clinical malaria during infancy. METHODS AND FINDINGS: In Benin, 497 mother-infant pairs were included in a longitudinal birth cohort. Both maternal and cord serum samples were assayed for levels of IgG1 and IgG3 specific for MSP119, MSP2 (both allelic families, 3D7 and FC27), MSP3, GLURP (both regions, R0 and R2), and AMA1 antigens of Plasmodium falciparum. Cord:maternal ratios were calculated. The maternal IgG3 gene was sequenced to identify the IgG3-H435 polymorphism. A multivariate logistic regression was used to examine the association between maternal IgG3-H435 polymorphism and transplacental transfer of IgG3, adjusting for hypergammaglobulinemia, maternal malaria, and infant malaria exposure. Twenty-four percent of Beninese women living in an area highly endemic for malaria had the IgG3-H435 allele (377 women homozygous for the IgG3-R435 allele, 117 women heterozygous for the IgG3-R/H alleles, and 3 women homozygous for the IgG3-H435 allele). Women with the IgG3-H435 allele had a 78% (95% CI 17%, 170%, p = 0.007) increased transplacental transfer of GLURP-R2 IgG3 compared to those without the IgG3-H435 allele. Furthermore, in infants born to mothers with the IgG3-H435 variant, a 28% longer IgG3 half-life was noted (95% CI 4%, 59%, p = 0.02) compared to infants born to mothers homozygous for the IgG3-R435 allele. Similar findings were observed for AMA1, MSP2-3D7, MSP3, GLURP-R0, and GLURP-R2 but not for MSP119 and MSP2-FC27. Infants born to women with IgG3-H435 had a 32% lower risk of symptomatic malaria during infancy (incidence rate ratio [IRR] = 0.68 [95% CI 0.51, 0.91], p = 0.01) compared to infants born to mothers homozygous for IgG3-R435. We did not find a lower risk of asymptomatic malaria in infants born to women with or without IgG3-H435. Limitations of the study were the inability to determine (i) the actual amount of IgG3-H435 relative to IgG-R435 in serum samples and (ii) the proportion of malaria-specific IgG produced by infants versus acquired from their mothers. CONCLUSIONS: An arginine-to-histidine replacement at residue 435 in the binding domain of IgG3 to FcRn increases the transplacental transfer and half-life of malaria-specific IgG3 in young infants and is associated with reduced risk of clinical malaria during infancy. The IgG3-H435 allele may be under positive selection, given its relatively high frequency in malaria endemic areas.

Soluble human leukocyte antigen -G during pregnancy and infancy in Benin: Mother/child resemblance and association with the risk of malaria infection and low birth weight.

Human leukocyte antigen (HLA) G is a tolerogenic molecule involved in the maternal-fetal immune tolerance phenomenon. Its expression during some infectious diseases leading to immune evasion has been established. A first study conducted in Benin has shown that the production of soluble HLA-G (sHLA-G) during the first months of life is strongly correlated with the maternal level at delivery and associated with low birth weight and malaria. However sHLA-G measurements during pregnancy were not available for mothers and furthermore, to date the evolution of sHLA-G in pregnancy is not documented in African populations. To extend these previous findings, between January 2010 and June 2013, 400 pregnant women of a malaria preventive trial and their newborns were followed up in Benin until the age of 2 years. Soluble HLA-G was measured 3 times during pregnancy and repeatedly during the 2 years follow-up to explore how sHLA-G evolved and the factors associated. During pregnancy, plasma levels of sHLA-G remained stable and increased significantly at delivery (p<0.001). Multigravid women seemed to have the highest levels (p = 0.039). In infants, the level was highest in cord blood and decreased before stabilizing after 18 months (p<0.001). For children, a high level of sHLA-G was associated with malaria infection during the follow-up (p = 0.02) and low birth weight (p = 0.06). The mean level of sHLA-G during infancy was strongly correlated with the mother's level during pregnancy (<0.001), and not only at delivery. Moreover, mothers with placental malaria infection had a higher probability of giving birth to a child with a high level of sHLA-g (p = 0.006). High sHLA-G levels during pregnancy might be associated with immune tolerance related to placental malaria. Further studies are needed but this study provides a first insight concerning the potential role of sHLA-G as a biomarker of weakness for newborns and infants.

Acquisition of natural humoral immunity to P. falciparum in early life in Benin: impact of clinical, environmental and host factors.

To our knowledge, effects of age, placental malaria infection, infections during follow-up, nutritional habits, sickle-cell trait and individual exposure to Anopheles bites were never explored together in a study focusing on the acquisition of malaria antibody responses among infants living in endemic areas.Five hundred and sixty-seven Beninese infants were weekly followed-up from birth to 18 months of age. Immunoglobulin G (IgG), IgG1 and IgG3 specific for 5 malaria antigens were measured every 3 months. A linear mixed model was used to analyze the effect of each variable on the acquisition of antimalarial antibodies in 6-to18-month old infants in univariate and multivariate analyses. Placental malaria, nutrition intakes and sickle-cell trait did not influence the infant antibody levels to P. falciparum antigens. In contrary, age, malaria antibody levels at birth, previous and present malaria infections as well as exposure to Anopheles bites were significantly associated with the natural acquisition of malaria antibodies in 6-to18-month old Beninese infants. This study highlighted inescapable factors to consider simultaneously in an immuno-epidemiological study or a vaccine trial in early life.

Multivariate Longitudinal Analysis with Bivariate Correlation Test.

In the context of multivariate multilevel data analysis, this paper focuses on the multivariate linear mixed-effects model, including all the correlations between the random effects when the dimensional residual terms are assumed uncorrelated. Using the EM algorithm, we suggest more general expressions of the model's parameters estimators. These estimators can be used in the framework of the multivariate longitudinal data analysis as well as in the more general context of the analysis of multivariate multilevel data. By using a likelihood ratio test, we test the significance of the correlations between the random effects of two dependent variables of the model, in order to investigate whether or not it is useful to model these dependent variables jointly. Simulation studies are done to assess both the parameter recovery performance of the EM estimators and the power of the test. Using two empirical data sets which are of longitudinal multivariate type and multivariate multilevel type, respectively, the usefulness of the test is illustrated.