Antibody signatures in Plasmodium falciparum and helminth infections: immune deviation, serological diagnosis and vaccine responses

[eng] INTRODUCTION: Some of the infectious agents that affect the largest proportion of the world population are Plasmodium falciparum and helminths such as soil-transmitted helminths (STH) and Schistosoma spp. The geographical distribution of these parasites has a great overlap, particularly in sub-Saharan Africa. This implies that coinfections with both types of parasites are very frequent. Control and, ultimately, elimination of these infections entails a coordination of efforts directed at several fronts, such as appropriate diagnosis and the development of effective vaccines that prevent these infections in first place. To achieve this, one of the key elements is to understand natural and protective immune responses to infectious diseases and the factors that deviate them. Antibodies play a central role in immunity to infectious diseases and vaccines. They provide valuable insights into historical exposure, current status and prospective susceptibility to infection and disease. Therefore, by studying antibody responses to these infections and to vaccines we can identify beneficial mechanisms to potentiate and negative ones to avoid. In this endeavor, it is crucial to consider the possible interference between organisms with different immunological requirements, such as P. falciparum and helminths, whether it is during current coinfections or as a result of repeated exposure. As an intracellular pathogen, the protective immune responses to P. falciparum are characterized initially by inflammatory cytokines with a type 1 helper T cell (TH1) profile and production of antibodies with cytophilic properties, mainly immunoglobulin (Ig) G1 and IgG3. In contrast, non-cytophilic IgG2 and IgG4 have been mainly associated with risk. In the case of helminths, the prototypical protective immune response is characterized by type 2 helper T cell (TH2) cytokines and the production of IgE antibodies. However, helminths have potent immunomodulatory effects, resulting in a switch to a regulatory helper T cell (TREG) phenotype, as evidenced by the dominance of regulatory cytokines in chronic infections and characterized by the production of IgG4. This regulatory response dampens TH1 and TH2 responses and it is thought to affect not only helminth but also bystander antigens and infections. HYPOTHESES: The primary hypothesis of this doctoral thesis is that immune deviation phenomena alter the quantity and quality of antibody responses, impacting naturally acquired immunity (NAI) to pathogens and the response to vaccines. Immune deviation might be caused by past exposure to and current coinfections with pathogens that induce different types of immune responses, such as P. falciparum and helminths. The secondary hypothesis is that antibody responses to parasite antigens are useful biomarkers for diagnosis and for measuring exposure in the context of infection surveillance, control and elimination strategies for parasites of global health importance. OBJECTIVES: The primary objective of this doctoral thesis is to characterize the phenomenon of immune deviation in terms of antibody responses in the context of past exposure and current coinfection with P. falciparum and helminths, and in the response to the RTS,S/AS01E malaria vaccine. The secondary objective is to evaluate the performance of P. falciparum- and helminth-specific IgG serology and total IgE responses as diagnostic and exposure markers.[spa] INTRODUCCIÓN: Algunos de los agentes infecciosos que afectan a la mayor proporción de la población mundial son Plasmodium falciparum y los helmintos como los helmintos transmitidos por el suelo (STH, por sus siglas en inglés) y Schistosoma spp. La distribución geográfica de estos parásitos tiene una gran superposición, particularmente en África subsahariana, lo cual hace que las coinfecciones con ambos tipos de parásitos sean muy frecuentes. El control y, en última instancia, la eliminación de estas infecciones conlleva una coordinación de esfuerzos dirigidos a varios frentes, como el diagnóstico adecuado y el desarrollo de vacunas eficaces que prevengan estas infecciones en primer lugar. Para lograrlo, uno de los elementos clave es comprender las respuestas inmunitarias naturales y protectoras frente a las enfermedades infecciosas y los factores que las desvían. Los anticuerpos juegan un papel central en la inmunidad a las enfermedades infecciosas y las vacunas. Proporcionan información valiosa sobre la exposición histórica, el estado actual y la susceptibilidad a prospectivas infecciones y enfermedades. Por lo tanto, al estudiar las respuestas de anticuerpos a estas infecciones y a las vacunas podemos identificar mecanismos beneficiosos a potenciar y negativos a evitar. En este esfuerzo, es crucial considerar la posible interferencia entre organismos con diferentes requisitos inmunológicos, como P. falciparum y helmintos, ya sea durante las coinfecciones actuales o como resultado de exposiciones repetidas. Como patógeno intracelular, las respuestas inmunitarias protectoras frente a P. falciparum se caracterizan inicialmente por citocinas inflamatorias con perfil de linfocitos T auxiliares tipo 1 (TH1) y la producción de anticuerpos con propiedades citofílicas, principalmente inmunoglobulina (Ig) G1 e IgG3. Por el contrario, las IgG2 e IgG4 no citofílicas se han asociado principalmente con el riesgo. En el caso de los helmintos, la respuesta inmunitaria protectora prototípica se caracteriza por citocinas de células T auxiliares de tipo 2 (TH2) y la producción de anticuerpos IgE. Sin embargo, los helmintos tienen potentes efectos inmunomoduladores, lo que resulta en un cambio a un fenotipo de células T auxiliares reguladoras (TREG), como lo demuestra el predominio de las citocinas reguladoras en infecciones crónicas y la producción de IgG4. Esta respuesta reguladora atenúa las respuestas TH1 y TH2 y se cree que afecta no solo a los antígenos de helmintos sino también a los de otras infecciones. HIPÓTESIS: La hipótesis principal de esta tesis doctoral es que los fenómenos de desviación inmunitaria alteran la cantidad y calidad de las respuestas de anticuerpos, afectando la inmunidad naturalmente adquirida (NAI, por sus siglas en ingés) a patógenos y la respuesta a vacunas. La desviación inmunitaria puede deberse a la exposición previa y a las coinfecciones actuales con patógenos que inducen diferentes tipos de respuestas inmunitarias, como P. falciparum y helmintos. La hipótesis secundaria es que las respuestas de anticuerpos a antígenos de parásitos son biomarcadores útiles para el diagnóstico y para medir la exposición en el contexto de la medicina del viajero y las estrategias de control y eliminación de enfermedades para parásitos de importancia para la salud mundial. OBJETIVOS: El objetivo principal de esta tesis doctoral es caracterizar el fenómeno de la desviación inmunitaria en términos de respuestas de anticuerpos en el contexto de exposición pasada y coinfección actual por P. falciparum y helmintos, y en la respuesta a la vacuna antipalúdica RTS,S/AS01E. El objetivo secundario es evaluar el rendimiento de la serología de IgG específica contra P. falciparum y helmintos y las respuestas de IgE total como marcadores de diagnóstico y exposición

Interleukin-25 induces resistance against intestinal trematodes

Echinostoma caproni is an intestinal trematode that has been extensively used as an experimental model to investigate the factors determining the resistance to intestinal helminths or the development of chronic infections. ICR mice are permissive hosts for E. caproni in which chronic infections are developed, concomitantly with local Th1 responses, elevated levels of local IFN-γ, inflammation and antibody responses. However, mice develop partial resistance to homologous challenge infections after cure of a primary infection, which converts this subject into an adequate model for the study of the mechanisms generating resistance against intestinal helminths. The purpose of the present study was to compare the immune response induced in primary and secondary infections to elucidate the factors determining the different outcome of the infection in each type of infection. The results obtained indicate that susceptibility is determined by the lack of IL-25 expression in response to primary infection. In contrast, infection in an environment with elevated levels of IL-25, as occurs in challenge infection, results in a Th2 phenotype impairing parasite survival. This was confirmed by treatment of naïve mice with exogenous IL-25 and subsequent infection. Changes induced in goblet cell populations and mucin glycosylation could be implicated in resistance to infection

A balanced pro-inflammatory and regulatory cytokine signature in young African children is associated with lower risk of clinical malaria

Background: The effect of timing of exposure to first Plasmodium falciparum infections during early childhood on the induction of innate and adaptive cytokine responses and their contribution to the development of clinical malaria immunity is not well established. Methods: As part of a double-blind randomized placebo-controlled trial in Mozambique using monthly chemoprophylaxis with sulfadoxine-pyrimethamine plus artesunate to selectively control timing of malaria exposure during infancy, peripheral blood mononuclear cells collected at ages 2.5, 5.5, 10.5, 15 and 24 months were stimulated ex vivo with parasite schizont and erythrocyte lysates. Cytokine mRNA expressed in cell pellets and proteins secreted in supernatants were quantified by real time quantitative PCR and multiplex flow cytometry, respectively. Children were followed up for clinical malaria from birth until 4 years of age. Results: Higher pro-inflammatory (IL-1, IL-6, TNF) and regulatory (IL-10) cytokine concentrations during the second year of life were associated with reduced incidence of clinical malaria up to 4 years of age, adjusting by chemoprophylaxis and prior malaria exposure. Significantly lower concentrations of antigen-specific TH1 (IL-2, IL-12, IFN-) and TH2 (IL-4, IL-5) cytokines by 2 years of age were measured in children under chemoprophylaxis compared to children receiving placebo (p<0.03). Conclusions: Selective chemoprophylaxis altering early natural exposure to malaria blood stage antigens during infancy had a significant effect on TH lymphocyte cytokine production more than one year later. Importantly, a balanced pro-inflammatory and anti-inflammatory cytokine signature probably by innate cells around age 2 years was associated with protective clinical immunity during childhood

Plasmodium falciparum and helminth coinfections Increase IgE and parasite-specific IgG responses

Coinfection with Plasmodium falciparum and helminths may impact the immune response to these parasites because they induce different immune profiles. We studied the effects of coinfections on the antibody profile in a cohort of 715 Mozambican children and adults using the Luminex technology with a panel of 16 antigens from P. falciparum and 11 antigens from helminths (Ascaris lumbricoides, hookworm, Trichuris trichiura, Strongyloides stercoralis, and Schistosoma spp.) and measured antigen-specific IgG and total IgE responses. We compared the antibody profile between groups defined by P. falciparum and helminth previous exposure (based on serology) and/or current infection (determined by microscopy and/or qPCR). In multivariable regression models adjusted by demographic, socioeconomic, water, and sanitation variables, individuals exposed/infected with P. falciparum and helminths had significantly higher total IgE and antigen-specific IgG levels, magnitude (sum of all levels) and breadth of response to both types of parasites compared to individuals exposed/infected with only one type of parasite (P ≤ 0.05). There was a positive association between exposure/infection with P. falciparum and exposure/infection with helminths or the number of helminth species, and vice versa (P ≤ 0.001). In addition, children coexposed/coinfected tended (P = 0.062) to have higher P. falciparum parasitemia than those single exposed/infected. Our results suggest that an increase in the antibody responses in coexposed/coinfected individuals may reflect higher exposure and be due to a more permissive immune environment to infection in the host. IMPORTANCE Coinfection with Plasmodium falciparum and helminths may impact the immune response to these parasites because they induce different immune profiles. We compared the antibody profile between groups of Mozambican individuals defined by P. falciparum and helminth previous exposure and/or current infection. Our results show a significant increase in antibody responses in individuals coexposed/coinfected with P. falciparum and helminths in comparison with individuals exposed/infected with only one of these parasites, and suggest that this increase is due to a more permissive immune environment to infection in the host. Importantly, this study takes previous exposure into account, which is particularly relevant in endemic areas where continuous infections imprint and shape the immune system. Deciphering the implications of coinfections deserves attention because accounting for the real interactions that occur in nature could improve the design of integrated disease control strategies

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

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

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

Maternal and neonatal immune response to SARS-CoV-2, IgG transplacental transfer and cytokine profile

SARS-CoV-2 infected pregnant women are at increased risk of severe COVID-19 than non-pregnant women and have a higher risk of adverse pregnancy outcomes like intrauterine/fetal distress and preterm birth. However, little is known about the impact of SARS-CoV-2 infection on maternal and neonatal immunological profiles. In this study, we investigated the inflammatory and humoral responses to SARS-CoV-2 in maternal and cord blood paired samples. Thirty-six pregnant women were recruited at delivery at Hospital Sant Joan de Déu, Barcelona, Spain, between April-August 2020, before having COVID-19 available vaccines. Maternal and pregnancy variables, as well as perinatal outcomes, were recorded in questionnaires. Nasopharyngeal swabs and maternal and cord blood samples were collected for SARS-CoV-2 detection by rRT-PCR and serology, respectively. We measured IgM, IgG and IgA levels to 6 SARS-CoV-2 antigens (spike [S], S1, S2, receptor-binding domain [RBD], nucleocapsid [N] full-length and C-terminus), IgG to N from 4 human coronaviruses (OC43, HKU1, 229E and NL63), and the concentrations of 30 cytokines, chemokines and growth factors by Luminex. Mothers were classified as infected or non-infected based on the rRT-PCR and serology results. Sixty-four % of pregnant women were infected with SARS-CoV-2 (positive by rRT-PCR during the third trimester and/or serology just after delivery). None of the newborns tested positive for rRT-PCR. SARS-CoV-2 infected mothers had increased levels of virus-specific antibodies and several cytokines. Those with symptoms had higher cytokine levels. IFN-α was increased in cord blood from infected mothers, and in cord blood of symptomatic mothers, EGF, FGF, IL-17 and IL-15 were increased, whereas RANTES was decreased. Maternal IgG and cytokine levels showed positive correlations with their counterparts in cord blood. rRT-PCR positive mothers showed lower transfer of SARS-CoV-2-specific IgGs, with a stronger effect when infection was closer to delivery. SARS-CoV-2 infected mothers carrying a male fetus had higher antibody levels and higher EGF, IL-15 and IL-7 concentrations. Our results show that SARS-CoV-2 infection during the third trimester of pregnancy induces a robust antibody and cytokine response at delivery and causes a significant reduction of the SARS-CoV-2-specific IgGs transplacental transfer, with a stronger negative effect when the infection is closer to delivery.This work was supported by the Fundació Privada Daniel Bravo Andreu. RR had the support of the Health Department, Catalan Government (PERIS SLT017/20/000224). LI work was supported by the PID2019-110810RB-I00 grant from the Spanish Ministry of Science & Innovation. 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

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.We are indebted to the Biobanc de l’Hospital Infantil Sant Joan de Déu per a la Investigació for the sample and data procurement. Some serum/plasma samples were tested against RBD protein produced in lentivirus by the P. Santamaria lab, IDIBAPS, and Jordi Chi contributed for production of N proteins (through PID2019-110810RB-I00 grant to LI)