Multiplex Antibody Analysis of IgM, IgA and IgG to SARS-CoV-2 in Saliva and Serum From Infected Children and Their Close Contacts.

COVID-19 affects children to a lesser extent than adults but they can still get infected and transmit SARS-CoV-2 to their contacts. Field deployable non-invasive sensitive diagnostic techniques are needed to evaluate the infectivity dynamics of SARS-CoV-2 in pediatric populations and guide public health interventions, particularly if this population is not fully vaccinated. We evaluated the utility of high-throughput Luminex assays to quantify saliva IgM, IgA and IgG antibodies against five SARS-CoV-2 spike (S) and nucleocapsid (N) antigens in a contacts and infectivity longitudinal study in 122 individuals (52 children and 70 adults). We compared saliva versus serum/plasma samples in infected children and adults diagnosed by weekly RT-PCR over 35 days (n=62), and those who consistently tested negative over the same follow up period (n=60), in the Summer of 2020 in Barcelona, Spain. Saliva antibody levels in SARS-CoV-2 RT-PCR positive individuals were significantly higher than in negative individuals and correlated with those measured in sera/plasmas. Asymptomatic infected individuals had higher levels of anti-S IgG than symptomatic individuals, suggesting a protective anti-disease role for antibodies. Higher anti-S IgG and IgM levels in serum/plasma and saliva, respectively, in infected children compared to infected adults could also be related to stronger clinical immunity in them. Among infected children, males had higher levels of saliva IgG to N and RBD than females. Despite overall correlation, individual clustering analysis suggested that responses that may not be detected in blood could be patent in saliva, and vice versa. In conclusion, measurement of SARS-CoV-2-specific saliva antibodies should be considered as a complementary non-invasive assay to serum/plasma to determine COVID-19 prevalence and transmission in pediatric populations before and after vaccination campaigns.

Determinants of early antibody responses to COVID-19 mRNA vaccines in a cohort of exposed and naïve healthcare workers.

BACKGROUND: Two doses of mRNA vaccination have shown >94% efficacy at preventing COVID-19 mostly in naïve adults, but it is not clear if the second dose is needed to maximize effectiveness in those previously exposed to SARS-CoV-2 and what other factors affect responsiveness. METHODS: We measured IgA, IgG and IgM levels against SARS-CoV-2 spike (S) and nucleocapsid (N) antigens from the wild-type and S from the Alpha, Beta and Gamma variants of concern, after BNT162b2 (Pfizer/BioNTech) or mRNA-1273 (Moderna) vaccination in a cohort of health care workers (N=578). Neutralizing capacity and antibody avidity were evaluated. Data were analyzed in relation to COVID-19 history, comorbidities, vaccine doses, brand and adverse events. FINDINGS: Vaccination induced robust IgA and IgG levels against all S antigens. Neutralization capacity and S IgA and IgG levels were higher in mRNA-1273 vaccinees, previously SARS-CoV-2 exposed, particularly if symptomatic, and in those experiencing systemic adverse effects (p<0·05). A second dose in pre-exposed did not increase antibody levels. Smoking and comorbidities were associated with 43% (95% CI, 19-59) and 45% (95% CI, 63-18) lower neutralization, respectively, and 35% (95% CI, 3-57%) and 55% (95% CI, 33-70%) lower antibody levels, respectively. Among fully vaccinated, 6·3% breakthroughs were detected up to 189 days post-vaccination. Among pre-exposed non-vaccinated, 90% were IgG seropositive more than 300 days post-infection. INTERPRETATION: Our data support administering a single-dose in pre-exposed healthy individuals as primary vaccination. However, heterogeneity of responses suggests that personalized recommendations may be necessary depending on COVID-19 history and life-style. Higher mRNA-1273 immunogenicity would be beneficial for those expected to respond worse to vaccination and in face of variants that escape immunity such as Omicron. Persistence of antibody levels in pre-exposed unvaccinated indicates maintenance of immunity up to one year. FUNDING: This work was supported by Institut de Salut Global de Barcelona (ISGlobal) internal funds, in-kind contributions from Hospital Clínic de Barcelona, the Fundació Privada Daniel Bravo Andreu, and European Institute of Innovation and Technology (EIT) Health (grant number 20877), supported by the European Institute of Innovation and Technology, a body of the European Union receiving support from the H2020 Research and Innovation Programme. We acknowledge support from the Spanish Ministry of Science and Innovation and State Research Agency through the "Centro de Excelencia Severo Ochoa 2019-2023" Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. L. I. work was supported by PID2019-110810RB-I00 grant from the Spanish Ministry of Science & Innovation. Development of SARS-CoV-2 reagents was partially supported by the National Institute of Allergy and Infectious Diseases Centers of Excellence for Influenza Research and Surveillance (contract number HHSN272201400008C). The funders had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.

Ambient Air Pollution in Relation to SARS-CoV-2 Infection, Antibody Response, and COVID-19 Disease: A Cohort Study in Catalonia, Spain (COVICAT Study).

BACKGROUND: Emerging evidence links ambient air pollution with coronavirus 2019 (COVID-19) disease, an association that is methodologically challenging to investigate. OBJECTIVES: We examined the association between long-term exposure to air pollution with SARS-CoV-2 infection measured through antibody response, level of antibody response among those infected, and COVID-19 disease. METHODS: We contacted 9,605 adult participants from a population-based cohort study in Catalonia between June and November 2020; most participants were between 40 and 65 years of age. We drew blood samples from 4,103 participants and measured immunoglobulin M (IgM), IgA, and IgG antibodies against five viral target antigens to establish infection to the virus and levels of antibody response among those infected. We defined COVID-19 disease using self-reported hospital admission, prior positive diagnostic test, or more than three self-reported COVID-19 symptoms after contact with a COVID-19 case. We estimated prepandemic (2018-2019) exposure to fine particulate matter [PM with an aerodynamic diameter of ≤2.5µm (PM2.5)], nitrogen dioxide (NO2), black carbon (BC), and ozone (O3) at the residential address using hybrid land-use regression models. We calculated log-binomial risk ratios (RRs), adjusting for individual- and area-level covariates. RESULTS: Among those tested for SARS-CoV-2 antibodies, 743 (18.1%) were seropositive. Air pollution levels were not statistically significantly associated with SARS-CoV-2 infection: Adjusted RRs per interquartile range were 1.07 (95% CI: 0.97, 1.18) for NO2, 1.04 (95% CI: 0.94, 1.14) for PM2.5, 1.00 (95% CI: 0.92, 1.09) for BC, and 0.97 (95% CI: 0.89, 1.06) for O3. Among infected participants, exposure to NO2 and PM2.5 were positively associated with IgG levels for all viral target antigens. Among all participants, 481 (5.0%) had COVID-19 disease. Air pollution levels were associated with COVID-19 disease: adjusted RRs=1.14 (95% CI: 1.00, 1.29) for NO2 and 1.17 (95% CI: 1.03, 1.32) for PM2.5. Exposure to O3 was associated with a slightly decreased risk (RR=0.92; 95% CI: 0.83, 1.03). Associations of air pollution with COVID-19 disease were more pronounced for severe COVID-19, with RRs=1.26 (95% CI: 0.89, 1.79) for NO2 and 1.51 (95% CI: 1.06, 2.16) for PM2.5. DISCUSSION: Exposure to air pollution was associated with a higher risk of COVID-19 disease and level of antibody response among infected but not with SARS-CoV-2 infection. https://doi.org/10.1289/EHP9726.

Antibody conversion rates to SARS-CoV-2 in saliva from children attending summer schools in Barcelona, Spain.

BACKGROUND: Surveillance tools to estimate viral transmission dynamics in young populations are essential to guide recommendations for school opening and management during viral epidemics. Ideally, sensitive techniques are required to detect low viral load exposures among asymptomatic children. We aimed to estimate SARS-CoV-2 infection rates in children and adult populations in a school-like environment during the initial COVID-19 pandemic waves using an antibody-based field-deployable and non-invasive approach. METHODS: Saliva antibody conversion defined as ≥ 4-fold increase in IgM, IgA, and/or IgG levels to five SARS-CoV-2 antigens including spike and nucleocapsid constructs was evaluated in 1509 children and 396 adults by high-throughput Luminex assays in samples collected weekly in 22 summer schools and 2 pre-schools in 27 venues in Barcelona, Spain, from June 29th to July 31st, 2020. RESULTS: Saliva antibody conversion between two visits over a 5-week period was 3.22% (49/1518) or 2.36% if accounting for potentially cross-reactive antibodies, six times higher than the cumulative infection rate (0.53%) assessed by weekly saliva RT-PCR screening. IgG conversion was higher in adults (2.94%, 11/374) than children (1.31%, 15/1144) (p=0.035), IgG and IgA levels moderately increased with age, and antibodies were higher in females. Most antibody converters increased both IgG and IgA antibodies but some augmented either IgG or IgA, with a faster decay over time for IgA than IgG. Nucleocapsid rather than spike was the main antigen target. Anti-spike antibodies were significantly higher in individuals not reporting symptoms than symptomatic individuals, suggesting a protective role against COVID-19. CONCLUSION: Saliva antibody profiling including three isotypes and multiplexing antigens is a useful and user-friendlier tool for screening pediatric populations to detect low viral load exposures among children, particularly while they are not vaccinated and vulnerable to highly contagious variants, and to recommend public health policies during pandemics.

Infection induced SARS-CoV-2 seroprevalence and heterogeneity of antibody responses in a general population cohort study in Catalonia Spain.

Sparse data exist on the complex natural immunity to SARS-CoV-2 at the population level. We applied a well-validated multiplex serology test in 5000 participants of a general population study in Catalonia in blood samples collected from end June to mid November 2020. Based on responses to fifteen isotype-antigen combinations, we detected a seroprevalence of 18.1% in adults (n = 4740), and modeled extrapolation to the general population of Catalonia indicated a 15.3% seroprevalence. Antibodies persisted up to 9 months after infection. Immune profiling of infected individuals revealed that with increasing severity of infection (asymptomatic, 1-3 symptoms, ≥ 4 symptoms, admitted to hospital/ICU), seroresponses were more robust and rich with a shift towards IgG over IgA and anti-spike over anti-nucleocapsid responses. Among seropositive participants, lower antibody levels were observed for those ≥ 60 years vs < 60 years old and smokers vs non-smokers. Overweight/obese participants vs normal weight had higher antibody levels. Adolescents (13-15 years old) (n = 260) showed a seroprevalence of 11.5%, were less likely to be tested seropositive compared to their parents and had dominant anti-spike rather than anti-nucleocapsid IgG responses. Our study provides an unbiased estimate of SARS-CoV-2 seroprevalence in Catalonia and new evidence on the durability and heterogeneity of post-infection immunity.

Seven-month kinetics of SARS-CoV-2 antibodies and role of pre-existing antibodies to human coronaviruses.

Unraveling the long-term kinetics of antibodies to SARS-CoV-2 and the individual characteristics influencing it, including the impact of pre-existing antibodies to human coronaviruses causing common cold (HCoVs), is essential to understand protective immunity to COVID-19 and devise effective surveillance strategies. IgM, IgA and IgG levels against six SARS-CoV-2 antigens and the nucleocapsid antigen of the four HCoV (229E, NL63, OC43 and HKU1) were quantified by Luminex, and antibody neutralization capacity was assessed by flow cytometry, in a cohort of health care workers followed up to 7 months (N = 578). Seroprevalence increases over time from 13.5% (month 0) and 15.6% (month 1) to 16.4% (month 6). Levels of antibodies, including those with neutralizing capacity, are stable over time, except IgG to nucleocapsid antigen and IgM levels that wane. After the peak response, anti-spike antibody levels increase from ~150 days post-symptom onset in all individuals (73% for IgG), in the absence of any evidence of re-exposure. IgG and IgA to HCoV are significantly higher in asymptomatic than symptomatic seropositive individuals. Thus, pre-existing cross-reactive HCoVs antibodies could have a protective effect against SARS-CoV-2 infection and COVID-19 disease.

SARS-CoV-2 Seroprevalence and Antibody Kinetics Among Health Care Workers in a Spanish Hospital After 3 Months of Follow-up.

BACKGROUND: At the COVID-19 spring 2020 pandemic peak in Spain, prevalence of SARS-CoV-2 infection in a cohort of 578 randomly selected health care workers (HCWs) from Hospital Clínic de Barcelona was 11.2%. METHODS: A follow-up survey 1 month later (April-May 2020) measured infection by rRT-PCR and IgM, IgA, and IgG to the receptor-binding domain of the spike protein by Luminex. Antibody kinetics, including IgG subclasses, was assessed until month 3. RESULTS: At month 1, the prevalence of infection measured by rRT-PCR and serology was 14.9% (84/565) and seroprevalence 14.5% (82/565). We found 25 (5%) new infections in 501 participants without previous evidence of infection. IgM, IgG, and IgA levels declined in 3 months (antibody decay rates 0.15 [95% CI, .11-.19], 0.66 [95% CI, .54-.82], and 0.12 [95% CI, .09-.16], respectively), and 68.33% of HCWs had seroreverted for IgM, 3.08% for IgG, and 24.29% for IgA. The most frequent subclass responses were IgG1 (highest levels) and IgG2, followed by IgG3, and only IgA1 but no IgA2 was detected. CONCLUSIONS: Continuous and improved surveillance of SARS-CoV-2 infections in HCWs remains critical, particularly in high-risk groups. The observed fast decay of IgA and IgM levels has implications for seroprevalence studies using these isotypes.

Seroprevalence of antibodies against SARS-CoV-2 among health care workers in a large Spanish reference hospital.

Health care workers (HCW) are a high-risk population to acquire SARS-CoV-2 infection from patients or other fellow HCW. This study aims at estimating the seroprevalence against SARS-CoV-2 in a random sample of HCW from a large hospital in Spain. Of the 578 participants recruited from 28 March to 9 April 2020, 54 (9.3%, 95% CI: 7.1-12.0) were seropositive for IgM and/or IgG and/or IgA against SARS-CoV-2. The cumulative prevalence of SARS-CoV-2 infection (presence of antibodies or past or current positive rRT-PCR) was 11.2% (65/578, 95% CI: 8.8-14.1). Among those with evidence of past or current infection, 40.0% (26/65) had not been previously diagnosed with COVID-19. Here we report a relatively low seroprevalence of antibodies among HCW at the peak of the COVID-19 epidemic in Spain. A large proportion of HCW with past or present infection had not been previously diagnosed with COVID-19, which calls for active periodic rRT-PCR testing in hospital settings.

Cytokine profiling in immigrants with clinical malaria after extended periods of interrupted exposure to Plasmodium falciparum.

Immunity to malaria is believed to wane with time in the absence of exposure to Plasmodium falciparum infection, but immunoepidemiological data on longevity of immunity remain controversial. We quantified serum cytokines and chemokines by suspension array technology as potential biomarkers for durability of immunity in immigrants with clinical malaria after years without parasite exposure. These were compared to serum/plasma profiles in naïve adults (travelers) and semi-immune adults under continuous exposure, with malaria, along with immigrant and traveler patients without malaria. Immigrants had higher levels of IL-2, IL-5 and IL-8 compared to semi-immune adults with malaria (P≤0.0200). Time since immigration correlated with increased IL-2 (rho=0.2738P=0.0495) and IFN-γ (rho=0.3044P=0.0282). However, immigrants did not show as high IFN-γ concentrations as travelers during a first malaria episode (P<0.0001). Immigrants and travelers with malaria had higher levels of IFN-γ, IL-6, and IL-10 (P<0.0100) than patients with other diseases, and IL-8 and IL-1ß were elevated in immigrants with malaria (P<0.0500). Therefore, malaria patients had a characteristic strong pro-inflammatory/Th1 signature. Upon loss of exposure, control of pro-inflammatory responses and tolerance to P. falciparum appeared to be reduced. Understanding the mechanisms to maintain non-pathogenic effector responses is important to develop new malaria control strategies.

High antibody responses against Plasmodium falciparum in immigrants after extended periods of interrupted exposure to malaria.

BACKGROUND: Malaria immunity is commonly believed to wane in the absence of Plasmodium falciparum exposure, based on limited epidemiological data and short-lived antibody responses in some longitudinal studies in endemic areas. METHODS: A cross-sectional study was conducted among sub-Saharan African adults residing in Spain for 1 up to 38 years (immigrants) with clinical malaria (n=55) or without malaria (n=37), naïve adults (travelers) with a first clinical malaria episode (n=20) and life-long malaria exposed adults from Mozambique (semi-immune adults) without malaria (n=27) or with clinical malaria (n=50). Blood samples were collected and IgG levels against the erythrocytic antigens AMA-1 and MSP-142 (3D7 and FVO strains), EBA-175 and DBL-α were determined by Luminex. IgG levels against antigens on the surface of infected erythrocytes (IEs) were measured by flow cytometry. RESULTS: Immigrants without malaria had lower IgG levels than healthy semi-immune adults regardless of the antigen tested (P≤0.026), but no correlation was found between IgG levels and time since migration. Upon reinfection, immigrants with malaria had higher levels of IgG against all antigens than immigrants without malaria. However, the magnitude of the response compared to semi-immune adults with malaria depended on the antigen tested. Thus, immigrants had higher IgG levels against AMA-1 and MSP-142 (P≤0.015), similar levels against EBA-175 and DBL-α, and lower levels against IEs (P≤0.016). Immigrants had higher IgG levels against all antigens tested compared to travelers (P≤0.001), both with malaria. CONCLUSIONS: Upon cessation of malaria exposure, IgG responses to malaria-specific antigens were maintained to a large extent, although the conservation and the magnitude of the recall response depended on the nature of the antigen. Studies on immigrant populations can shed light on the factors that determine the duration of malaria specific antibody responses and its effect on protection, with important implications for future vaccine design and public health control measures.

Cytokine and antibody responses to Plasmodium falciparum in naïve individuals during a first malaria episode: effect of age and malaria exposure.

Age- and exposure-dependent immune responses during a malaria episode may be key to understanding the role of these factors in the acquisition of immunity to malaria. Plasma/serum samples collected from naïve Mozambican children (n=48), European adults (naïve travelers, n=22; expatriates with few prior malaria exposures, n=15) and Mozambican adults with long-life malaria exposure (n=99) during and after a malaria episode were analyzed for IgG against merozoite proteins by Luminex and against infected erythrocytes by flow cytometry. Cytokines and chemokines were analyzed in plasmas/sera by suspension array technology. No differences were detected between children and adults with a primary infection, with the exception of higher IgG levels against 3D7 MSP-1(42) (P=0.030) and a P. falciparum isolate (P=0.002), as well as higher IL-12 (P=0.020) in children compared to other groups. Compared to malaria-exposed adults, children, travelers and expatriates had higher concentrations of IFN-γ (P ≤ 0.0090), IL-2 (P ≤ 0.0379) and IL-8 (P ≤ 0.0233). Children also had higher IL-12 (P=0.0001), IL-4 (P=0.003), IL-1ß (P=0.024) and TNF (P=0.006) levels compared to malaria-exposed adults. Although IL-12 was elevated in children, overall the data do not support a role of age in immune responses to a first malaria episode. A T(H)1/pro-inflammatory response was the hallmark of non-immune subjects.

Low antibodies against Plasmodium falciparum and imbalanced pro-inflammatory cytokines are associated with severe malaria in Mozambican children: a case-control study.

BACKGROUND: The factors involved in the progression from Plasmodium falciparum infection to severe malaria (SM) are still incompletely understood. Altered antibody and cellular immunity against P. falciparum might contribute to increase the risk of developing SM. METHODS: To identify immune responses associated with SM, a sex- and age-matched case-control study was carried out in 134 Mozambican children with SM (cerebral malaria, severe anaemia, acidosis and/or respiratory distress, prostration, hypoglycaemia, multiple seizures) or uncomplicated malaria (UM). IgG and IgM against P. falciparum lysate, merozoite antigens (MSP-119, AMA-1 and EBA-175), a Duffy binding like (DBL)-α rosetting domain and antigens on the surface of infected erythrocytes were measured by ELISA or flow cytometry. Plasma concentrations of IL-12p70, IL-2, IFN-γ, IL-4, IL-5, IL-10, IL-8, IL-6, IL-1ß, TNF, TNF-ß and TGF-ß1 were measured using fluorescent bead immunoassays. Data was analysed using McNemar's and Signtest. RESULTS: Compared to UM, matched children with SM had reduced levels of IgG against DBLα (P < 0.001), IgM against MSP-119 (P = 0.050) and AMA-1 (P = 0.047), TGF-ß1 (P < 0.001) and IL-12 (P = 0.039). In addition, levels of IgG against P. falciparum lysate and IL-6 concentrations were increased (P = 0.004 and P = 0.047, respectively). Anti-DBLα IgG was the only antibody response associated to reduced parasite densities in a multivariate regression model (P = 0.026). CONCLUSIONS: The lower levels of antibodies found in children with SM compared to children with UM were not attributable to lower exposure to P. falciparum in the SM group. IgM against P. falciparum and specific IgG against a rosetting PfEMP1 domain may play a role in the control of SM, whereas an imbalanced pro-inflammatory cytokine response may exacerbate the severity of infection. A high overlap in symptoms together with a limited sample size of different SM clinical groups reduced the power to identify immunological correlates for particular forms of SM.

Intermittent preventive treatment with sulfadoxine-pyrimethamine does not modify plasma cytokines and chemokines or intracellular cytokine responses to Plasmodium falciparum in Mozambican children.

BACKGROUND: Cytokines and chemokines are key mediators of anti-malarial immunity. We evaluated whether Intermittent Preventive Treatment in infants with Sulfadoxine-Pyrimethamine (IPTi-SP) had an effect on the acquisition of these cellular immune responses in Mozambican children. Multiple cytokines and chemokines were quantified in plasma by luminex, and antigen-specific cytokine production in whole blood was determined by intracellular cytokine staining and flow cytometry, at ages 5, 9, 12 and 24 months. RESULTS: IPTi-SP did not significantly affect the proportion of CD3+ cells producing IFN-γ, IL-4 or IL-10. Overall, plasma cytokine or chemokine concentrations did not differ between treatment groups. Th1 and pro-inflammatory responses were higher than Th2 and anti-inflammatory responses, respectively, and IFN-γ:IL-4 ratios were higher for placebo than for SP recipients. Levels of cytokines and chemokines varied according to age, declining from 5 to 9 months. Plasma concentrations of IL-10, IL-12 and IL-13 were associated with current infection or prior malaria episodes. Higher frequencies of IFN-γ and IL-10 producing CD3+ cells and elevated IL-10, IFN-γ, MCP-1 and IL-13 in plasma were individually associated with increased malaria incidence, at different time points. When all markers were analyzed together, only higher IL-17 at 12 months was associated with lower incidence of malaria up to 24 months. CONCLUSIONS: Our work has confirmed that IPTi-SP does not negatively affect the development of cellular immune response during early childhood. This study has also provided new insights as to how these cytokine responses are acquired upon age and exposure to P. falciparum, as well as their associations with malaria susceptibility. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00209795.

Age-dependent IgG subclass responses to Plasmodium falciparum EBA-175 are differentially associated with incidence of malaria in Mozambican children.

Plasmodium falciparum blood-stage antigens such as merozoite surface protein 1 (MSP-1), apical membrane antigen 1 (AMA-1), and the 175-kDa erythrocyte binding antigen (EBA-175) are considered important targets of naturally acquired immunity to malaria. However, it is not clear whether antibodies to these antigens are effectors in protection against clinical disease or mere markers of exposure. In the context of a randomized, placebo-controlled trial of intermittent preventive treatment in infants conducted between 2002 and 2004, antibody responses to Plasmodium falciparum blood-stage antigens in a cohort of 302 Mozambican children were evaluated by immunofluorescence antibody test and enzyme-linked immunosorbent assay at 5, 9, 12, and 24 months of age. We found that IgG subclass responses to EBA-175 were differentially associated with the incidence of malaria in the follow-up period. A double amount of cytophilic IgG1 or IgG3 was associated with a significant decrease in the incidence of malaria (incidence rate ratio [IRR] = 0.49, 95% confidence interval [CI] = 0.25 to 0.97, and P = 0.026 and IRR = 0.44, CI = 0.19 to 0.98, and P = 0.037, respectively), while a double amount of noncytophilic IgG4 was significantly correlated with an increased incidence of malaria (IRR = 3.07, CI = 1.08 to 8.78, P = 0.020). No significant associations between antibodies to the 19-kDa fragment of MSP-1 (MSP-1(19)) or AMA-1 and incidence of malaria were found. Age, previous episodes of malaria, present infection, and neighborhood of residence were the main factors influencing levels of antibodies to all merozoite antigens. Deeper understanding of the acquisition of antibodies against vaccine target antigens in early infancy is crucial for the rational development and deployment of malaria control tools in this vulnerable population.

Comparison of commercial kits to measure cytokine responses to Plasmodium falciparum by multiplex microsphere suspension array technology.

BACKGROUND: Multiplex cytokine profiling systems are useful tools for investigating correlates of protective immunity. Several Luminex and flow cytometry methods are commercially available but there is limited information on the relative performance of different kits. A series of comparison experiments were carried out to determine the most appropriate method for our subsequent studies. METHODS: Two Luminex methods were compared, the Bio-Rad human 17-plex panel and the Invitrogen (formerly BioSource) human cytokine 10-plex kit, and two flow cytometry methods, the Becton Dickinson Human Th1/Th2 Cytokine Kit (CBA) and the Bender MedSystems Human Th1/Th2 11plex FlowCytomix Multiplex Kit. All kits were tested for the measurement of cytokines in supernatants collected from human leukocytes stimulated with viable Plasmodium falciparum infected red blood cells (iRBC) or P. falciparum schizont lysates. RESULTS: Data indicated that the kits differed in sensitivity and reproducibility depending on the cytokine, and detected different quantities of some cytokines. The Bio-Rad 17-plex kit was able to detect more positive responses than the Invitrogen 10-plex kit. However, only when detecting IL-1, IL-6 or TNF did the two Luminex based methods correlate with one another. In this study, the flow cytometry based techniques were less variable and correlated better with one another. The two flow cytometry based kits showed significant correlation when detecting IFN-γ, IL-2, TNF, IL-10 and IL-6, but overall the BD kit detected more positive responses than the Bender MedSystems kit. CONCLUSIONS: The microsphere suspension array technologies tested differed in reproducibility and the absolute quantity of cytokine detected. Sample volume, the number of cytokines measured, and the time and cost of the assays also differed. These data provide an accurate assessment of the four techniques, which will allow individual researchers to select the tool most suited for their study population.

Four year immunogenicity of the RTS,S/AS02(A) malaria vaccine in Mozambican children during a phase IIb trial.

Previous studies with the malaria vaccine RTS,S/AS02(A) in young children in a malaria endemic area of Mozambique have shown it to have a promising safety profile and to reduce the risk of Plasmodium falciparum infection and disease. In this study, we assessed the antibody responses to the P. falciparum and hepatitis B components of the RTS,S/AS02(A) vaccine over a 45 months surveillance period in a large phase IIb trial which included 2022 children aged 1-4 years at recruitment. The RTS,S/AS02(A) vaccine induced high anti-circumsporozoite antibody levels with at least 96% of children remaining seropositive during the entire follow-up period. IgG titers decayed over the first 6 months of follow-up to about 25% of the initial level, but still remained 30-fold higher until month 45 compared to controls. Children with higher levels of naturally acquired immunity at baseline, assessed by blood stage indirect fluorescent antibody test, had slightly higher anti-circumsporozoite levels, after adjusting for the effect of age. The RTS,S/AS02(A) vaccine also induced high levels of anti-hepatitis B surface antigen antibodies (seroprotection >97%). RTS,S/AS02(A) vaccine is immunogenic and induces long-lasting anti-circumsporozoite antibodies, persisting at least 42 months after immunization. These antibodies may play a role in protection against malaria.

IgG against Plasmodium falciparum variant surface antigens and growth inhibitory antibodies in Mozambican children receiving intermittent preventive treatment with sulfadoxine-pyrimethamine.

This study aimed to evaluate whether intermittent preventive treatment in infants with sulfadoxine-pyrimethamine (IPTi-SP) had an effect on the acquisition of IgG against Plasmodium falciparum variant surface antigens (VSA) and growth-inhibitory antibodies in Manhiça, Mozambique. In addition, we assessed factors affecting the magnitude of these responses and the association between antibody levels and protection against malaria. IgG to VSA expressed by MOZ2, R29 and E8B parasite isolates were measured in plasma samples collected at 5, 9, 12 and 24 months of age by flow cytometry. Growth-inhibitory antibodies in dialyzed plasmas using GFP-D10 parasites were measured by flow cytometry at 12 and 24 months. IPTi-SP did not significantly modify the levels of IgG against VSA nor the growth-inhibitory capacity of antibodies up to 2 years of age. Age but not previous episodes of malaria influenced the magnitude of these responses. In addition, anti-VSA IgG levels were 7% higher in children with current P. falciparum infection and were associated with neighborhood of residence. Children aged 24 months had 10% less parasite growth than those aged 12 months (95% CI 0.88-0.93, P<0.0001). Growth-inhibitory antibodies correlated with levels of IgG against AMA-1, when evaluating the 10% (R(2)=0.444, P=0.049) and 20% (R(2)=0.230, P=0.037) highest inhibitory samples. None of the responses were associated with subsequent risk of malaria. In conclusion, IPTi-SP does not negatively affect the development of antibody responses thought to be major contributors to the acquisition of immunity to malaria in infancy.

Clinical and epidemiological features of 33 imported Strongyloides stercoralis infections.

Strongyloides stercoralis has a unique ability to replicate in the human host and lead to chronic infection that can persist for several decades. Thirty-three patients (10 travellers and 23 immigrants) with imported S. stercoralis infection were studied and clinical and epidemiological characteristics described. Only 16 patients (48.5%) reported symptoms, mainly of the gastrointestinal tract. Eosinophilia was present in 21 (63.6%) patients. Seven patients (21.2%) had an immunocompromising condition. Patients were classified into chronic asymptomatic infection (17/33, 51.5%), chronic symptomatic infection (11/33, 33.3%) and hyperinfection (5/33, 15.2%). Four of the latter (80%) had an immunocompromising condition. Strongyloides stercoralis infection should be considered in immigrants and travellers with eosinophilia or compatible symptoms coming from endemic areas. Diagnosis should always be sought in immunocompromised hosts.

Functional and immunological characterization of a Duffy binding-like alpha domain from Plasmodium falciparum erythrocyte membrane protein 1 that mediates rosetting.

The Duffy binding-like (DBL) domains are common adhesion modules present in Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variants, which are responsible for immune evasion and cytoadherence. Knowledge about how immune responses are acquired against polymorphic DBL domains of PfEMP1 can aid in the development of vaccines for malaria. A recombinant DBLalpha domain, encoded by R29 var1, which binds complement receptor 1 to mediate rosetting by the P. falciparum laboratory strain R29, was expressed in Escherichia coli, renatured by oxidative refolding to its native form, and purified to homogeneity. Antibody levels in 704 plasmas obtained from residents of areas of different levels of malaria endemicity in Orissa (India) and Manhiça (Mozambique) were assessed by enzyme-linked immunosorbent assay. The refolded DBLalpha domain was pure, homogeneous, and functional in that it bound human erythrocytes with specificity and was capable of inhibiting rosetting. The proportion of individuals who had measurable anti-DBLalpha immunoglobulin G responses was low in areas of low malaria endemicity in Orissa (6.7%) but high in areas of high endemicity in Orissa (87.5%) and Manhiça (74.5%). Seroprevalence and antibody levels against the recombinant protein increased with the age of inhabitants from areas with high transmission rates (P < 0.001). Half of the children in these areas had seroconverted by the age of 5 years. These findings suggest that in spite of the extreme polymorphism of PfEMP1 DBLalpha domains, the acquisition of specific antibodies is rapid and age related and reflects the reduced risk of malaria in areas with high transmission rates. Further studies are required to elucidate the role of these antibodies in protection from malaria.

Sub-microscopic infections and long-term recrudescence of Plasmodium falciparum in Mozambican pregnant women.

BACKGROUND: Control of malaria in pregnancy remains a public health challenge. Improvements in its correct diagnosis and the adequacy of protocols to evaluate anti-malarial drug efficacy in pregnancy, are essential to achieve this goal. METHODS: The presence of Plasmodium falciparum was assessed by real-time (RT) PCR in 284 blood samples from pregnant women with clinical complaints suggestive of malaria, attending the maternity clinic of a Mozambican rural hospital. Parasite recrudescences in 33 consecutive paired episodes during the same pregnancy were identified by msp1 and msp2 genotyping. RESULTS: Prevalence of parasitaemia by microscopy was 5.3% (15/284) and 23.2% (66/284) by RT-PCR. Sensitivity of microscopy, compared to RT-PCR detection, was 22.7%. Risk of maternal anaemia was higher in PCR-positive women than in PCR-negative women (odds ratio [OR] = 1.92, 95% confidence interval [CI] 1.09-3.36). Genotyping confirmed that recrudescence after malaria treatment occurred in 7 (21%) out of 33 pregnant women with consecutive episodes during the same pregnancy (time range between recrudescent episodes: 14 to 187 days). CONCLUSION: More accurate and sensitive diagnostic indicators of malaria infection in pregnancy are needed to improve malaria control. Longer follow-up periods than the standard in vivo drug efficacy protocol should be used to assess anti-malarial drug efficacy in pregnancy.