Chronic malaria exposure is associated with inhibitory markers on T cells that correlate with atypical memory and marginal zone-like B cells.

Chronic immune activation from persistent malaria infections can induce immunophenotypic changes associated with T-cell exhaustion. However, associations between T and B cells during chronic exposure remain undefined. We analyzed peripheral blood mononuclear cells from malaria-exposed pregnant women from Papua New Guinea and Spanish malaria-naïve individuals using flow cytometry to profile T-cell exhaustion markers phenotypically. T-cell lineage (CD3, CD4, and CD8), inhibitory (PD1, TIM3, LAG3, CTLA4, and 2B4), and senescence (CD28-) markers were assessed. Dimensionality reduction methods revealed increased PD1, TIM3, and LAG3 expression in malaria-exposed individuals. Manual gating confirmed significantly higher frequencies of PD1+CD4+ and CD4+, CD8+, and double-negative (DN) T cells expressing TIM3 in malaria-exposed individuals. Increased frequencies of T cells co-expressing multiple markers were also found in malaria-exposed individuals. T-cell data were analyzed with B-cell populations from a previous study where we reported an alteration of B-cell subsets, including increased frequencies of atypical memory B cells (aMBC) and reduction in marginal zone (MZ-like) B cells during malaria exposure. Frequencies of aMBC subsets and MZ-like B cells expressing CD95+ had significant positive correlations with CD28+PD1+TIM3+CD4+ and DN T cells and CD28+TIM3+2B4+CD8+ T cells. Frequencies of aMBC, known to associate with malaria anemia, were inversely correlated with hemoglobin levels in malaria-exposed women. Similarly, inverse correlations with hemoglobin levels were found for TIM3+CD8+ and CD28+PD1+TIM3+CD4+ T cells. Our findings provide further insights into the effects of chronic malaria exposure on circulating B- and T-cell populations, which could impact immunity and responses to vaccination.

Genotyping Hepatitis B virus by Next-Generation Sequencing: Detection of Mixed Infections and Analysis of Sequence Conservation.

Our aim was to develop an accurate, highly sensitive method for HBV genotype determination and detection of genotype mixtures. We examined the preS and 5' end of the HBV X gene (5X) regions of the HBV genome using next-generation sequencing (NGS). The 1852 haplotypes obtained were subjected to genotyping via the Distance-Based discrimination method (DB Rule) using two sets of 95 reference sequences of genotypes A-H. In clinical samples from 125 patients, the main genotypes were A, D, F and H in Caucasian, B and C in Asian and A and E in Sub-Saharan patients. Genotype mixtures were identified in 28 (22.40%) cases, and potential intergenotypic recombination was observed in 29 (23.20%) cases. Furthermore, we evaluated sequence conservation among haplotypes classified into genotypes A, C, D, and E by computing the information content. The preS haplotypes exhibited limited shared conserved regions, whereas the 5X haplotypes revealed two groups of conserved regions across the genotypes assessed. In conclusion, we developed an NGS-based HBV genotyping method utilizing the DB Rule for genotype classification. We identified two regions conserved across different genotypes at 5X, offering promising targets for RNA interference-based antiviral therapies.

Induction and decay of functional complement-fixing antibodies by the RTS,S malaria vaccine in children, and a negative impact of malaria exposure.

BACKGROUND: Leading malaria vaccine, RTS,S, is based on the circumsporozoite protein (CSP) of sporozoites. RTS,S confers partial protection against malaria in children, but efficacy wanes relatively quickly after primary immunization. Vaccine efficacy has some association with anti-CSP IgG; however, it is unclear how these antibodies function, and how functional antibodies are induced and maintained over time. Recent studies identified antibody-complement interactions as a potentially important immune mechanism against sporozoites. Here, we investigated whether RTS,S vaccine-induced antibodies could function by interacting with complement. METHODS: Serum samples were selected from children in a phase IIb trial of RTS,S/AS02A conducted at two study sites of high and low malaria transmission intensity in Manhiça, Mozambique. Samples following primary immunization and 5-year post-immunization follow-up time points were included. Vaccine-induced antibodies were characterized by isotype, subclass, and epitope specificity, and tested for the ability to fix and activate complement. We additionally developed statistical methods to model the decay and determinants of functional antibodies after vaccination. RESULTS: RTS,S vaccination induced anti-CSP antibodies that were mostly IgG1, with some IgG3, IgG2, and IgM. Complement-fixing antibodies were effectively induced by vaccination, and targeted the central repeat and C-terminal regions of CSP. Higher levels of complement-fixing antibodies were associated with IgG that equally recognized both the central repeat and C-terminal regions of CSP. Older age and higher malaria exposure were significantly associated with a poorer induction of functional antibodies. There was a marked decay in functional complement-fixing antibodies within months after vaccination, as well as decays in IgG subclasses and IgM. Statistical modeling suggested the decay in complement-fixing antibodies was mostly attributed to the waning of anti-CSP IgG1, and to a lesser extent IgG3. CONCLUSIONS: We demonstrate for the first time that RTS,S can induce complement-fixing antibodies in young malaria-exposed children. The short-lived nature of functional responses mirrors the declining vaccine efficacy of RTS,S over time. The negative influence of age and malaria exposure on functional antibodies has implications for understanding vaccine efficacy in different settings. These findings provide insights into the mechanisms and longevity of vaccine-induced immunity that will help inform the future development of highly efficacious and long-lasting malaria vaccines.

RTS,S/AS01E immunization increases antibody responses to vaccine-unrelated Plasmodium falciparum antigens associated with protection against clinical malaria in African children: a case-control study.

BACKGROUND: Vaccination and naturally acquired immunity against microbial pathogens may have complex interactions that influence disease outcomes. To date, only vaccine-specific immune responses have routinely been investigated in malaria vaccine trials conducted in endemic areas. We hypothesized that RTS,S/A01E immunization affects acquisition of antibodies to Plasmodium falciparum antigens not included in the vaccine and that such responses have an impact on overall malaria protective immunity. METHODS: We evaluated IgM and IgG responses to 38 P. falciparum proteins putatively involved in naturally acquired immunity to malaria in 195 young children participating in a case-control study nested within the African phase 3 clinical trial of RTS,S/AS01E (MAL055 NCT00866619) in two sites of different transmission intensity (Kintampo high and Manhiça moderate/low). We measured antibody levels by quantitative suspension array technology and applied regression models, multimarker analysis, and machine learning techniques to analyze factors affecting their levels and correlates of protection. RESULTS: RTS,S/AS01E immunization decreased antibody responses to parasite antigens considered as markers of exposure (MSP142, AMA1) and levels correlated with risk of clinical malaria over 1-year follow-up. In addition, we show for the first time that RTS,S vaccination increased IgG levels to a specific group of pre-erythrocytic and blood-stage antigens (MSP5, MSP1 block 2, RH4.2, EBA140, and SSP2/TRAP) which levels correlated with protection against clinical malaria (odds ratio [95% confidence interval] 0.53 [0.3-0.93], p = 0.03, for MSP1; 0.52 [0.26-0.98], p = 0.05, for SSP2) in multivariable logistic regression analyses. CONCLUSIONS: Increased antibody responses to specific P. falciparum antigens in subjects immunized with this partially efficacious vaccine upon natural infection may contribute to overall protective immunity against malaria. Inclusion of such antigens in multivalent constructs could result in more efficacious second-generation multistage vaccines.

Changing plasma cytokine, chemokine and growth factor profiles upon differing malaria transmission intensities.

BACKGROUND: Malaria epidemiological and immunological data suggest that parasite tolerance wanes in the absence of continuous exposure to the parasite, potentially enhancing pathogenesis. The expansion of control interventions and elimination campaigns raises the necessity to better understand the host factors leading to susceptibility or tolerance that are affected by rapid changes in malaria transmission intensity (MTI). Mediators of cellular immune responses are responsible for the symptoms and pathological alterations during disease and are expected to change rapidly upon malaria exposure or cessation. METHODS: The plasma concentrations of 30 cytokine, chemokine and growth factors in individuals of all ages from a malaria endemic area of southern Mozambique were compared between 2 years of different MTI: 2010 (lower, n = 234) and 2013 (higher, n = 143). The effect of the year on the correlations between cytokines, chemokines and growth factors and IgGs to Plasmodium falciparum (markers of exposure) was explored. The effects of age, sex, neighbourhood and parasitaemia on analyte levels and their interactions with year were also assessed. RESULTS: An inverse correlation of several cellular immune mediators with malarial antibodies in 2013, and a lack of correlation or even a positive correlation in 2010 were observed. Most cytokines, chemokines and growth factors, regardless of their immune function, had higher concentrations in 2010 compared with 2013 in P. falciparum-infected and uninfected subjects. Age and neighbourhood showed an effect on analyte concentrations. CONCLUSIONS: The results show a different regulation of the cellular immune response in 2010 vs 2013 which could be related to a loss of immune-tolerance after a decline in MTI in 2010 and previous years, and a rapid re-establishment of tolerance as a consequence of more continuous exposure as MTI began increasing in 2012. Cellular immune mediators warrant further investigation as possible surrogates of MTI-associated host susceptibility or tolerance.

Immune reactivity to Trypanosoma cruzi chimeric proteins for Chagas disease diagnosis in immigrants living in a non-endemic setting.

BACKGROUND: Chronic Chagas Disease (CD) diagnosis is based on serological methods employing crude, semipurified or recombinant antigens, which may result in low sensitivity or cross-reactivity. To reduce these restrictions, we developed a strategy involving use of molecules containing repetitive fragments of Trypanosoma cruzi conserved proteins. Diagnostic performance of IBMP-8.1 and IBMP-8.4 chimeric antigens (Molecular Biology Institute of Paraná - IBMP in Portuguese acronym) was assessed to diagnose T. cruzi-infected and non-infected immigrants living in Barcelona (Spain), a non-endemic setting for Chagas disease. METHODS: Reactivity of IBMP-8.1 and IBMP-8.4 was assessed using an in-house automated ELISA with 347 positive and 331 negative individuals to Chagas disease. Antigenic cross-reactivity was measured with sera samples from pregnant women with Toxoplasma gondii (n = 98) and Zika virus (n = 75) antibodies. RESULTS: The area under the curve values was 1 and 0.99 for the IBMP-8.1 and IBMP-8.4 proteins, respectively, demonstrating excellent diagnostic accuracy. The reactivity index was higher for IBMP-8.1 than IBMP-8.4 in positive samples and no significant difference in reactivity index was observed in negative samples. Sensitivity ranged from 99.4% for IBMP-8.1 to 99.1% for IBMP-8.4 and was not statistically different. Specificity for IBMP-8.1 reached 100 and 99.7% for IBMP-8.4, both nearly 100% accurate. No antigenic cross-reactivity was observed and reactivity index was similar to that for negative Chagas disease individuals. CONCLUSIONS: Our results showed an outstanding performance of IBMP-8.1 and IBMP-8.4 chimeric antigens by ELISA and suggest both chimeric antigens could also be used for Chagas disease diagnosis in immigrants living in non-endemic settings.

Baseline exposure, antibody subclass, and hepatitis B response differentially affect malaria protective immunity following RTS,S/AS01E vaccination in African children.

BACKGROUND: The RTS,S/AS01E vaccine provides partial protection against malaria in African children, but immune responses have only been partially characterized and do not reliably predict protective efficacy. We aimed to evaluate comprehensively the immunogenicity of the vaccine at peak response, the factors affecting it, and the antibodies associated with protection against clinical malaria in young African children participating in the multicenter phase 3 trial for licensure. METHODS: We measured total IgM, IgG, and IgG1-4 subclass antibodies to three constructs of the Plasmodium falciparum circumsporozoite protein (CSP) and hepatitis B surface antigen (HBsAg) that are part of the RTS,S vaccine, by quantitative suspension array technology. Plasma and serum samples were analyzed in 195 infants and children from two sites in Ghana (Kintampo) and Mozambique (Manhiça) with different transmission intensities using a case-control study design. We applied regression models and machine learning techniques to analyze immunogenicity, correlates of protection, and factors affecting them. RESULTS: RTS,S/AS01E induced IgM and IgG, predominantly IgG1 and IgG3, but also IgG2 and IgG4, subclass responses. Age, site, previous malaria episodes, and baseline characteristics including antibodies to CSP and other antigens reflecting malaria exposure and maternal IgGs, nutritional status, and hemoglobin concentration, significantly affected vaccine immunogenicity. We identified distinct signatures of malaria protection and risk in RTS,S/AS01E but not in comparator vaccinees. IgG2 and IgG4 responses to RTS,S antigens post-vaccination, and anti-CSP and anti-P. falciparum antibody levels pre-vaccination, were associated with malaria risk over 1-year follow-up. In contrast, antibody responses to HBsAg (all isotypes, subclasses, and timepoints) and post-vaccination IgG1 and IgG3 to CSP C-terminus and NANP were associated with protection. Age and site affected the relative contribution of responses in the correlates identified. CONCLUSIONS: Cytophilic IgG responses to the C-terminal and NANP repeat regions of CSP and anti-HBsAg antibodies induced by RTS,S/AS01E vaccination were associated with malaria protection. In contrast, higher malaria exposure at baseline and non-cytophilic IgG responses to CSP were associated with disease risk. Data provide new correlates of vaccine success and failure in African children and reveal key insights into the mode of action that can guide development of more efficacious next-generation vaccines.

Development of a high-throughput flexible quantitative suspension array assay for IgG against multiple Plasmodium falciparum antigens.

BACKGROUND: Antibody responses to Plasmodium falciparum play a critical role in disease control. Finding reliable IgG biomarkers of protection is complicated by a parasite proteome of over 5000 proteins, some with polymorphisms. Studies of anti-malarial naturally acquired and vaccine immunity would benefit from a standard high-throughput immunoassay to measure multiple antibodies. A multiplex quantitative suspension assay to measure antigen-specific IgGs was developed and its precision (reproducibility and repeatability), dynamic range, limits of detection and quantification, and non-specific binding to different P. falciparum proteins tested. A set of 288 human plasma samples from a malaria-endemic region were analysed twice by two different operators. Another set of samples from 9 malaria-naïve and 10 malaria-exposed individuals were repetitively assayed during 22 consecutive days. Positive controls, negative controls, blanks and microspheres coated with bovine serum albumin were included in all assays. RESULTS: The multiplex quantitative suspension assay demonstrated low non-specific signal and good estimates of precision and reproducibility between operators. The overall mean of non-specific binding measured in 288 plasma samples was 32.83 to ± 44.81 median fluorescence intensity (MFI). Repeatability was 7.66% ± 15.89 between triplicates for all antigens and samples, being lower in samples from malaria-exposed than malaria-naïve individuals. No evidence of significantly different variance across days in MFI or arbitrary units (AU)/mL was found, assuming homogeneity of variance between days of analysis. Intra-class correlation coefficient between 22 days of analysis was 0.98 (0.97-0.98) for MFI units and 0.9 (0.87-0.93) for AU/mL. Reproducibility between operators for all samples and antigens had an overall adjusted correlation of 0.929 for MFI and 0.836 for AU/mL. CONCLUSIONS: This high-throughput multiplex immunoassay is simple and highly reproducible. This represents an asset for malaria vaccine studies involving CSP-specific antibodies and selected antigens for sero-epidemiological purposes. Measuring a multiplex antigen panel in a single reaction will help to assess not only vaccine immunogenicity but also potential malaria vaccine effects on naturally acquired immune responses. This will accelerate the identification of immune correlates of protection, down-selection of vaccine formulations, antigen discovery and guide second-generation vaccine design.

Optimization of incubation conditions of Plasmodium falciparum antibody multiplex assays to measure IgG, IgG1-4, IgM and IgE using standard and customized reference pools for sero-epidemiological and vaccine studies.

BACKGROUND: The quantitative suspension array technology (qSAT) is a useful platform for malaria immune marker discovery. However, a major challenge for large sero-epidemiological and malaria vaccine studies is the comparability across laboratories, which requires the access to standardized control reagents for assay optimization, to monitor performance and improve reproducibility. Here, the Plasmodium falciparum antibody reactivities of the newly available WHO reference reagent for anti-malaria human plasma (10/198) and of additional customized positive controls were examined with seven in-house qSAT multiplex assays measuring IgG, IgG1-4 subclasses, IgM and IgE against a panel of 40 antigens. The different positive controls were tested at different incubation times and temperatures (4 °C overnight, 37 °C 2 h, room temperature 1 h) to select the optimal conditions. RESULTS: Overall, the WHO reference reagent had low IgG2, IgG4, IgM and IgE, and also low anti-CSP antibody levels, thus this reagent was enriched with plasmas from RTS,S-vaccinated volunteers to be used as standard for CSP-based vaccine studies. For the IgM assay, another customized plasma pool prepared with samples from malaria primo-infected adults with adequate IgM levels proved to be more adequate as a positive control. The range and magnitude of IgG and IgG1-4 responses were highest when the WHO reference reagent was incubated with antigen-coupled beads at 4 °C overnight. IgG levels measured in the negative control did not vary between incubations at 37 °C 2 h and 4 °C overnight, indicating no difference in unspecific binding. CONCLUSIONS: With this study, the immunogenicity profile of the WHO reference reagent, including seven immunoglobulin isotypes and subclasses, and more P. falciparum antigens, also those included in the leading RTS,S malaria vaccine, was better characterized. Overall, incubation of samples at 4 °C overnight rendered the best performance for antibody measurements against the antigens tested. Although the WHO reference reagent performed well to measure IgG to the majority of the common P. falciparum blood stage antigens tested, customized pools may need to be used as positive controls depending on the antigens (e.g. pre-erythrocytic proteins of low natural immunogenicity) and isotypes/subclasses (e.g. IgM) under study.

Pregnancy and malaria exposure are associated with changes in the B cell pool and in plasma eotaxin levels.

Pregnancy triggers immunological changes aimed to tolerate the fetus, but its impact on B lymphocytes is poorly understood. In addition, exposure to the Plasmodium parasite is associated with altered distribution of peripheral memory B cell (MBC) subsets. To study the combined impact of high malaria exposure and pregnancy in B cell subpopulations, we analyzed PBMCs from pregnant and nonpregnant individuals from a malaria-nonendemic country (Spain) and from a high malaria-endemic country (Papua New Guinea). In the malaria-naive cohorts, pregnancy was associated with a significant expansion of all switched (IgD(-)) MBC and a decrease of naive B cells. Malaria-exposed women had more atypical MBC and fewer marginal zone-like MBC, and their levels correlated with both Plasmodium vivax- and Plasmodium falciparum-specific plasma IgG levels. Classical but not atypical MBC were increased in P. falciparum infections. Moreover, active atypical MBC positively correlated with proinflammatory cytokine plasma concentrations and had lower surface IgG levels than the average. Decreased plasma eotaxin (CCL11) levels were associated with pregnancy and malaria exposure and also correlated with B cell subset frequencies. Additionally, active atypical and active classical MBC expressed higher levels of eotaxin receptor CCR3 than the other B cell subsets, suggesting a chemotactic effect of eotaxin on these B cell subsets. These findings are important to understand immunity to infections like malaria that result in negative outcomes for both the mother and the newborn and may have important implications on vaccine development.

Prevalence of Hepatitis C Virus Infection, Genotypes and Subtypes in Migrants from Pakistan in Barcelona, Spain.

Background: Hepatitis C virus (HCV) is a major cause of chronic liver infection with 71 million people infected worldwide. Pakistan has the second highest prevalence of HCV infection and more than half (52%) of Pakistani living in Spain reside in Barcelona. The aim of this study was to analyse the seroprevalence and viraemic rate and determine the genotypes and subtypes of HCV among Pakistanis living in the southern metropolitan area of Barcelona. Methods: We included all Pakistani patients seeking primary healthcare in the southern metropolitan area of Barcelona from August 2011 to July 2014. Serum samples were screened for HCV antibodies. HCV viral load was determined by reverse transcription polymerase chain reaction and genotypes and subtypes were performed using Versant HCV Genotype and/or deep-sequencing. Screening for hepatitis B virus (HBV) was also carried out. Results: Among 5877 Pakistani patients, 565 (9.61%) were screened for anti-HCV antibodies, with 68 (12.04%) being positive. The viral load was determined in 65, with 31 presenting active infection and the viraemic rate was 47.69% (95% confidence interval 36.02-59.62). HCV genotyping and subtyping were performed in 24 individuals. Most infections corresponded to HCV genotype 3 (91.67%), and high resolution HCV subtyping was performed in 18 samples, 16 of which presented subtype 3a. One subject presented HBV coinfection with undetectable HBV DNA. During the study period, we identified a possible case of HCV vertical transmission followed by spontaneous viraemia clearance in a chronically infected mother with a C/T IL28B genetic polymorphism. Conclusion: These results suggest that general HCV screening protocols in patients from high prevalence countries, such as Pakistan, would be helpful to identify and treat active HCV infections. This could avoid further transmission and contribute to building targeted health policies for micro-elimination of HCV infection in specific communities.

What Accounts for Physical Activity during Pregnancy? A Study on the Sociodemographic Predictors of Self-Reported and Objectively Assessed Physical Activity during the 1st and 2nd Trimesters of Pregnancy.

Physical activity (PA) during pregnancy has positive health implications for both mother and child. However, current literature indicates that not all pregnant women meet the international recommendations for PA (at least 150 min/week of moderate-to-vigorous PA). The main objective of this study was to assess PA levels among pregnant women in the city of Donostia-San Sebastian and identify their main sociodemographic predictors. We recruited 441 women in the 12th week of pregnancy from the local public obstetric health services. Women wore an accelerometer for one week during two separate time points (1st and 2nd trimesters of pregnancy) and completed a questionnaire assessing several sociodemographic variables as well as self-reported PA. With this information, we estimated women's overall PA levels during both time points. The fulfillment of PA recommendations raised up to 77% and 85% during the first and second trimesters, respectively. We found that a higher number of children and a greater preference for exercise positively predicted light-to-moderate PA, being the most consistent predictors. The availability of a greater number of cars negatively predicted moderate-to-vigorous PA.

Erratum: Author Correction: Antibody responses to the RTS,S/AS01E vaccine and Plasmodium falciparum antigens after a booster dose within the phase 3 trial in Mozambique.

[This corrects the article DOI: 10.1038/s41541-020-0192-7.].

Antibody responses to the RTS,S/AS01E vaccine and Plasmodium falciparum antigens after a booster dose within the phase 3 trial in Mozambique.

The RTS,S/AS01E vaccine has shown consistent but partial vaccine efficacy in a pediatric phase 3 clinical trial using a 3-dose immunization schedule. A fourth-dose 18 months after the primary vaccination was shown to restore the waning efficacy. However, only total IgG against the immunodominant malaria vaccine epitope has been analyzed following the booster. To better characterize the magnitude, nature, and longevity of the immune response to the booster, we measured levels of total IgM, IgG, and IgG1-4 subclasses against three constructs of the circumsporozoite protein (CSP) and the hepatitis B surface antigen (HBsAg, also present in RTS,S) by quantitative suspension array technology in 50 subjects in the phase 3 trial in Manhiça, Mozambique. To explore the impact of vaccination on naturally acquired immune responses, we measured antibodies to P. falciparum antigens not included in RTS,S. We found increased IgG, IgG1, IgG3 and IgG4, but not IgG2 nor IgM, levels against vaccine antigens 1 month after the fourth dose. Overall, antibody responses to the booster dose were lower than the initial peak response to primary immunization and children had higher IgG and IgG1 levels than infants. Higher anti-Rh5 IgG and IgG1-4 levels were detected after the booster dose, suggesting that RTS,S partial protection could increase some blood stage antibody responses. Our work shows that the response to the RTS,S/AS01E booster dose is different from the primary vaccine immune response and highlights the dynamic changes in subclass antibody patterns upon the vaccine booster and with acquisition of adaptive immunity to malaria.

Corrigendum: Chronic Exposure to Malaria Is Associated with Inhibitory and Activation Markers on Atypical Memory B Cells and Marginal Zone-Like B Cells.

[This corrects the article DOI: 10.3389/fimmu.2017.00966.].

Differential Patterns of IgG Subclass Responses to Plasmodium falciparum Antigens in Relation to Malaria Protection and RTS,S Vaccination.

Naturally acquired immunity (NAI) to Plasmodium falciparum malaria is mainly mediated by IgG antibodies but the subclasses, epitope targets and effector functions have not been unequivocally defined. Dissecting the type and specificity of antibody responses mediating NAI is a key step toward developing more effective vaccines to control the disease. We investigated the role of IgG subclasses to malaria antigens in protection against disease and the factors that affect their levels, including vaccination with RTS,S/AS01E. We analyzed plasma and serum samples at baseline and 1 month after primary vaccination with RTS,S or comparator in African children and infants participating in a phase 3 trial in two sites of different malaria transmission intensity: Kintampo in Ghana and Manhiça in Mozambique. We used quantitative suspension array technology (qSAT) to measure IgG1-4 responses to 35 P. falciparum pre-erythrocytic and blood stage antigens. Our results show that the pattern of IgG response is predominantly IgG1 or IgG3, with lower levels of IgG2 and IgG4. Age, site and RTS,S vaccination significantly affected antibody subclass levels to different antigens and susceptibility to clinical malaria. Univariable and multivariable analysis showed associations with protection mainly for cytophilic IgG3 levels to selected antigens, followed by IgG1 levels and, unexpectedly, also with IgG4 levels, mainly to antigens that increased upon RTS,S vaccination such as MSP5 and MSP1 block 2, among others. In contrast, IgG2 was associated with malaria risk. Stratified analysis in RTS,S vaccinees pointed to novel associations of IgG4 responses with immunity mainly involving pre-erythrocytic antigens upon RTS,S vaccination. Multi-marker analysis revealed a significant contribution of IgG3 responses to malaria protection and IgG2 responses to malaria risk. We propose that the pattern of cytophilic and non-cytophilic IgG antibodies is antigen-dependent and more complex than initially thought, and that mechanisms of both types of subclasses could be involved in protection. Our data also suggests that RTS,S efficacy is significantly affected by NAI, and indicates that RTS,S vaccination significantly alters NAI.

Concentration and avidity of antibodies to different circumsporozoite epitopes correlate with RTS,S/AS01E malaria vaccine efficacy.

RTS,S/AS01E has been tested in a phase 3 malaria vaccine study with partial efficacy in African children and infants. In a cohort of 1028 subjects from one low (Bagomoyo) and two high (Nanoro, Kintampo) malaria transmission sites, we analysed IgG plasma/serum concentration and avidity to CSP (NANP-repeat and C-terminal domains) after a 3-dose vaccination against time to clinical malaria events during 12-months. Here we report that RTS,S/AS01E induces substantial increases in IgG levels from pre- to post-vaccination (p < 0.001), higher in NANP than C-terminus (2855 vs 1297 proportional change between means), and higher concentrations and avidities in children than infants (p < 0.001). Baseline CSP IgG levels are elevated in malaria cases than controls (p < 0.001). Both, IgG magnitude to NANP (hazard ratio [95% confidence interval] 0.61 [0.48-0.76]) and avidity to C-terminus (0.07 [0.05-0.90]) post-vaccination are significantly associated with vaccine efficacy. IgG avidity to the C-terminus emerges as a significant contributor to RTS,S/AS01E-mediated protection.

Antibody responses to α-Gal in African children vary with age and site and are associated with malaria protection.

Naturally-acquired antibody responses to malaria parasites are not only directed to protein antigens but also to carbohydrates on the surface of Plasmodium protozoa. Immunoglobulin M responses to α-galactose (α-Gal) (Galα1-3Galß1-4GlcNAc-R)-containing glycoconjugates have been associated with protection from P. falciparum infection and, as a result, these molecules are under consideration as vaccine targets; however there are limited field studies in endemic populations. We assessed a wide breadth of isotype and subclass antibody response to α-Gal in children from Mozambique (South East Africa) and Ghana (West Africa) by quantitative suspension array technology. We showed that anti-α-Gal IgM, IgG and IgG1-4 levels vary mainly depending on the age of the child, and also differ in magnitude in the two sites. At an individual level, the intensity of malaria exposure to P. falciparum and maternally-transferred antibodies affected the magnitude of α-Gal responses. There was evidence for a possible protective role of anti-α-Gal IgG3 and IgG4 antibodies. However, the most consistent findings were that the magnitude of IgM responses to α-Gal was associated with protection against clinical malaria over a one-year follow up period, especially in the first months of life, while IgG levels correlated with malaria risk.

Analysis of factors affecting the variability of a quantitative suspension bead array assay measuring IgG to multiple Plasmodium antigens.

Reducing variability of quantitative suspension array assays is key for multi-center and large sero-epidemiological studies. To maximize precision and robustness of an in-house IgG multiplex assay, we analyzed the effect of several conditions on variability to find the best combination. The following assay conditions were studied through a fractional factorial design: antigen-bead coupling (stock vs. several), sample predilution (stock vs. daily), temperature of incubation of sample with antigen-bead (22°C vs. 37°C), plate washing (manual vs. automatic) and operator expertise (expert vs. apprentice). IgG levels against seven P. falciparum antigens with heterogeneous immunogenicities were measured in test samples, in a positive control and in blanks. We assessed the variability and MFI quantification range associated to each combination of conditions, and their interactions, and evaluated the minimum number of samples and blank replicates to achieve good replicability. Results showed that antigen immunogenicity and sample seroreactivity defined the optimal dilution to assess the effect of assay conditions on variability. We found that a unique antigen-bead coupling, samples prediluted daily, incubation at 22°C, and automatic washing, had lower variability. However, variability increased when performing several couplings and incubating at 22°C vs. 37°C. In addition, no effect of temperature was seen with a unique coupling. The expertise of the operator had no effect on assay variability but reduced the MFI quantification range. Finally, differences between sample replicates were minimal, and two blanks were sufficient to capture assay variability, as suggested by the constant Intraclass Correlation Coefficient of three and two blanks. To conclude, a single coupling was the variable that most consistently reduced assay variability, being clearly advisable. In addition, we suggest having more sample dilutions instead of replicates to increase the likelihood of sample MFIs falling in the linear part of the antigen-specific curve, thus increasing precision.

Chronic Exposure to Malaria Is Associated with Inhibitory and Activation Markers on Atypical Memory B Cells and Marginal Zone-Like B Cells.

In persistent infections that are accompanied by chronic immune activation, such as human immunodeficiency virus, hepatitis C virus, and malaria, there is an increased frequency of a phenotypically distinct subset of memory B cells lacking the classic memory marker CD27 and showing a reduced capacity to produce antibodies. However, critical knowledge gaps remain on specific B cell changes and immune adaptation in chronic infections. We hypothesized that expansion of atypical memory B cells (aMBCs) and reduction of activated peripheral marginal zone (MZ)-like B cells in constantly exposed individuals might be accompanied by phenotypic changes that would confer a tolerogenic profile, helping to establish tolerance to infections. To better understand malaria-associated phenotypic abnormalities on B cells, we analyzed peripheral blood mononuclear cells from 55 pregnant women living in a malaria-endemic area of Papua Nueva Guinea and 9 Spanish malaria-naïve individuals using four 11-color flow cytometry panels. We assessed the expression of markers of B cell specificity (IgG and IgM), activation (CD40, CD80, CD86, b220, TACI, and CD150), inhibition (PD1, CD95, and CD71), and migration (CCR3, CXCR3, and CD62l). We found higher frequencies of active and resting aMBC and marked reduction of MZ-like B cells, although changes in absolute cell counts could not be assessed. Highly exposed women had higher PD1+-, CD95+-, CD40+-, CD71+-, and CD80+-activated aMBC frequencies than non-exposed subjects. Malaria exposure increased frequencies of b220 and proapoptotic markers PD1 and CD95, and decreased expression of the activation marker TACI on MZ-like B cells. The increased frequencies of inhibitory and apoptotic markers on activated aMBCs and MZ-like B cells in malaria-exposed adults suggest an immune-homeostatic mechanism for maintaining B cell development and function while simultaneously downregulating hyperreactive B cells. This mechanism would keep the B cell activation threshold high enough to control infection but impaired enough to tolerate it, preventing systemic inflammation.