T helper cells (Th) are central to mucosal IgA induction and key targets for modulation by vaccine adjuvants. To improve understanding of cellular mechanisms underlying mucosal vaccine-induced immunity, we analyzed antigen-specific peripheral blood Th responses elicited by the oral enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX, administered with or without the double mutant heat-labile toxin (dmLT) adjuvant. ETVAX, consisting of inactivated E. coli overexpressing colonization factors CFA/I, CS3, CS5, and CS6 with a heat-labile toxin B-subunit toxoid, was given orally in two doses to adult volunteers, either alone or with 10 or 25 µg dmLT. Antigen-specific Th-associated cytokine responses were assessed in stimulated peripheral blood mononuclear cells isolated from 15 to 18 individuals/group using ELISA and electrochemiluminescence assays. ETVAX predominantly induced Th1 (IFN-γ) and Th17 (IL-17A) responses, with minimal Th2-associated cytokines. Responses were markedly reduced after CD4+ T-cell depletion, supporting a Th cell origin. The strongest responses targeted LTB and CS3, with IFN-γ responses detected in 60–80% and IL-17A in 40–60% across all vaccinees. Responses to CFA/I, CS5 and CS6 were generally weaker. Exploratory comparisons suggested broader IFN-γ responses and more consistent IFN-γ and IL-17A responses to lower-dose antigens, particularly CS6, in recipients receiving vaccine plus 10 µg dmLT. These trends paralleled IgA antibody-secreting cell response patterns, with significantly enhanced IgA responses to CS6 in the vaccine plus 10 µg dmLT group. In conclusion, ETVAX induces antigen-specific Th1- and Th17-type responses in peripheral blood, supporting a role for cellular immunity in mucosal responses to oral ETEC vaccines.

BACKGROUND:Cholera and typhoid fever are often co-endemic, making vaccine co-administration practical. However, due to lack of immunogenicity data, current guidelines advise against co-administration of the oral inactivated whole-cell recombinant cholera toxin B-subunit vaccine (WC-rCTB) and the oral live Salmonella Typhi Ty21a vaccine. METHODS:Healthy adults (18-65 years) were randomized 1:1:1 to receive WC-rCTB with Ty21a (group Ch + Ty), WC-rCTB alone (group Ch) or Ty21a alone (group Ty). Peripheral blood mononuclear cells (PBMCs) were isolated on Days 0, 5 and 7 from all, plus on Days 12 and 14 from WC-rCTB recipients, to assess antibody-secreting cells (ASCs) specific to rCTB and to typhoidal O9,12-structures by enzyme-linked immunosorbent spot (ELISPOT) assay. Vibriocidal antibodies were assessed, and anti-rCTB IgA/IgG and anti-S. Typhi lipopolysaccharide (LPS) IgA/IgG/IgM were measured by enzyme-linked immunosorbent assay (ELISA) in Day 0 and 28 ± 3 serum samples. Adverse events (AEs) were recorded during one month. RESULTS:The final study population included 63 volunteers, 21 per group. A non-significant trend towards stronger rCTB-specific ASC (IgA + IgG + IgM) peak responses was observed in group Ch + Ty compared to group Ch (geometric mean, GM 94 vs 32 ASC/106 PBMC, P = 0.096). Serum anti-rCTB IgA and IgG fold rises (post-vaccination vs pre-vaccination) were higher in group Ch + Ty than in group Ch (IgA P = 0.039, IgG P = 0.028), whereas vibriocidal fold rises were comparable between the two groups (P = 0.847). ASC (IgA + IgG + IgM) peak responses to typhoidal O9,12-structures were comparable between groups Ch + Ty and Ty (GM 183 vs. 210 ASC/106 PBMC, P = 0.684). Serum anti-S. Typhi LPS IgA, IgG and IgM fold rises were also similar across Ch + Ty and Ty groups (all P-values ≥0.145). AEs were comparable in single and co-administration groups. CONCLUSIONS:Co-administration of the oral cholera and typhoid vaccines demonstrated favourable safety and robust immunogenicity for both vaccines, supporting their simultaneous use without spacing precautions.

A scientific consultation with experts in cholera immunology, vaccinology, and public health was convened in June 2025 to review the current understanding of gut mucosal immunology in relation to cholera and cholera vaccination and to identify critical knowledge gaps and urgent research needs. Areas for discussion included protective mechanisms, correlates of protection (CoPs), and strategies to enhance oral cholera vaccine (OCV) performance, especially in young children.Key recommendations included advancing immunological research, including standardization of mucosal immune response assays in humans, to better understand gut mucosal responses across age groups and to define improved CoPs. Modeling was highlighted as an essential tool to evaluate innovative strategies for optimizing the use of existing OCV targeted to specific contexts, e.g. alternative strategies in outbreaks; national/regional immunization days in endemic settings; and integration with other health interventions. Age-descending studies for evaluation of adjuvants (e.g. dmLT) to enhance cholera vaccine efficacy and durability, especially in children, were recommended, and under strict ethical control also the use of Controlled Human Infection Models (CHIMs) in cholera-endemic settings for evaluation of immune responses and vaccine candidates.The insights from the meeting provide a roadmap for both immediate and longer-term improvements in cholera vaccination impact and disease control.

Abstract Immunoglobulins (Ig) are essential for protection against invading pneumococci but are not routinely analyzed in patients with invasive pneumococcal disease (IPD). In this prospective study, we assessed the prevalence of monoclonal Ig (M protein) and analyzed Ig concentrations in sera from 156 adult IPD patients (median age 70 years). Patients were sampled during acute infection ( n = 153) and in the convalescence phase 2–4 months after acute infection ( n = 76). Sixty-four sex- and age-matched individuals without IPD served as controls. During infection, M protein was detected in 22% (31/141) of patients without previously known hematological malignancy as compared with 5% of the controls ( p = 0.002). Seven of these patients were subsequently diagnosed with a hematological malignancy, including multiple myeloma, Waldenstrom macroglobulinemia, and mantle cell lymphoma. Another 12 patients were diagnosed with monoclonal gammopathy of undetermined significance (MGUS). Convalescence levels of IgA, IgG2, or IgG4 were below the reference intervals in 16–20% of patients and 0–2% of controls. Three patients were diagnosed with a primary Ig deficiency, and seven patients started Ig replacement therapy. Our findings suggest that assessment of M protein and Ig levels could be considered in adults with IPD, as an episode of IPD may unmask a previously undiagnosed B-cell malignancy or immunodeficiency.

BACKGROUND:Long-term prospective data on antibody and T-cell responses beyond the third COVID-19 mRNA vaccine dose, particularly in relation to prior SARS-CoV-2 infections and breakthrough infections, remain limited. METHODS:Health care workers (HCWs) vaccinated with BNT162b2 were enrolled in January 2021. Blood samples were collected before and one month after each of four vaccine doses through December 2022. IgG antibodies against the spike receptor-binding domain (RBD) and the nucleocapsid (N) proteins were analysed, and T-cell responses (IFN-γ, IL-2, TNF-α) were measured after spike peptide stimulation. Neutralising antibodies (NAbs) against Omicron BA.1 were assessed in a subset (n = 61). RBD-IgG and cytokine levels were compared between infected and infection-naïve individuals, adjusted for age and sex. RESULTS:Among 108 HCWs, 32% were infected before vaccination. Breakthrough infections were rare before dose 3 but increased with the emergence of Omicron, affecting 77% of the participants until study end. RBD-IgG levels were consistently significantly higher in individuals with hybrid immunity than in infection-naïve participants, except one month after dose 3. When breakthrough infections were excluded, this difference only persisted through dose 3. IFN-γ responses largely mirrored RBD-IgG, while IL-2 responses were less affected by repeat doses and TNF-α responses were highly variable. CONCLUSIONS:Individuals with hybrid immunity had significantly higher RBD-IgG levels than infection-naïve subjects up to 23 months post-vaccination when including breakthrough infections, while elevated antibody responses were limited to the first year in those infected pre-vaccination. Differences were more pronounced for antibody than T-cell responses when comparing individuals with and without breakthrough infections.

INTRODUCTION:Patients with non-small cell lung cancer (NSCLC) faced an elevated risk for severe COVID-19 outcomes and were prioritized for early vaccination. However, immune responses to SARS-CoV-2 vaccines remain poorly characterized in this group, especially in patients receiving immune checkpoint inhibitor PD-1/PDL-1 (ICI) therapy. MATERIALS AND METHODS:In this prospective observational study, we evaluated humoral and cellular immune responses to COVID-19 vaccination in 20 NSCLC patients receiving ICI therapy and compared to responses in 38 controls. Patients received two doses of either BNT162b2 (Pfizer-BioNTech) or ChAdOx1-S (AstraZeneca) vaccines followed by a third dose of BNT162b2 while controls received three doses of BNT162b2. Serum IgG to the receptor-binding domain (RBD) of the spike protein and T-cell cytokine responses (IFN-γ, IL-2 and TNF) induced by spike peptide stimulation of whole blood were assessed at multiple time points. RESULTS AND DISCUSSION:After two doses, patients exhibited lower and more variable IgG responses than controls, but a third dose raised antibody levels to match those observed in controls. The largest fold-increase in antibody concentrations after the third dose occurred in patients receiving BNT162b2 after two doses of ChAdOx1-S. T-cell responses were significantly lower in patients after two doses but improved following the third dose. Antibody and IFN-γ responses were significantly correlated both after the second and third doses in patients, suggesting coordinated humoral and cellular immunity. No associations were found between immune responses and patient age. These findings highlight the immunogenicity of COVID-19 vaccines in NSCLC patients on ICI therapy and support that booster vaccinations are needed to enhance immunity in this vulnerable group.

BACKGROUND:Perfluoroalkyl substances (PFAS) have been associated with impaired antibody levels after childhood vaccinations and immunosuppressive effects in animals. However, the in vivo effects of PFAS on antigen specific human T cell responses have not been investigated in adults. In Ronneby, Sweden, the drinking water of one of the water works was previously highly contaminated with primarily perfluorohexane sulfonic acid (PFHxS) and perfluorooctane sulfonic acid (PFOS). The COVID-19 vaccination scheme presented the possibility to assess antigen specific T cell function after vaccination in adults with high PFAS serum levels. OBJECTIVES:To investigate the relationship between PFAS exposure and T cell responses after COVID-19 vaccination in a population with varied PFAS exposure. METHODS:116 COVID-19 naïve individuals from Ronneby and a background exposed group were included from the PFAS Immune Response After COVID-19 Vaccination cohort (PIRVACoV). All participants received two doses of Spikevax® (Moderna) vaccine. Blood T cells were stimulated with overlapping peptides based on the SARS-CoV-2 spike protein and their production of the cytokines IFN-γ, IL-2, and TNF were measured. The general immune response was assessed by measurement of phytohemagglutinin stimulated cytokines and total immunoglobulin serum levels. Adjusted mixed linear regressions were fitted against measured, address-based and prenatal PFAS exposure indices. RESULTS:PFAS median serum levels differed greatly between participants ever having had contaminated drinking water at home (PFOS 47 ng/mL, 5th to 95th percentile 6-221 ng/mL) and the background group (PFOS 4 ng/mL, 2-9 ng/mL). PFAS exposure was not associated with SARS-CoV-2 specific T cell cytokine responses (e.g., measured PFOS to IFN-γ: +3% per interquartile range PFOS, 95% confidence interval: 10, 17), nor general immune response. CONCLUSIONS:This study indicates, in concordance with the PIRVACoV antibody study and other antibody PFAS/COVID-19 studies, that PFAS exposed, healthy adults mount adequate immune responses to mRNA COVID-19 vaccination. EudraCT-number: 2021-000842-16.

BackgroundInfection with enterotoxigenic Escherichia coli (ETEC) gives rise to IgA antibodies against both the heat labile toxin (LT) and colonization factors (CFs), which are considered to synergistically protect against ETEC diarrhea. Since the development of ETEC-specific long lived plasma cells and memory B cells is likely to be dependent on T helper (Th) cells, we investigated if natural ETEC diarrhea elicits ETEC-specific Th cells and their relation to IgA responses.MethodsTh cell subsets were analyzed in adult Bangladeshi patients hospitalized due to ETEC diarrhea by flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) isolated from blood collected day 2, 7, 30 and 90 after hospitalization as well as in healthy controls. The LT- and CF-specific Th responses were determined by analysis of IL-17A and IFN-γ in antigen stimulated PBMC cultures using ELISA. ETEC-specific IgA secreted by circulating antibody secreting cells (plasmablasts) were analyzed by using the antibodies in lymphocyte supernatants (ALS) ELISA-based method and plasma IgA was also measured by ELISA.ResultsETEC patients mounted significant ALS and plasma IgA responses against LTB and CFs on day 7 after hospitalization. ETEC patients had significantly elevated proportions of memory Th cells with a Th17 phenotype (CCR6+CXCR3-) in blood compared to controls, while frequencies of Th1 (CCR6-CXCR3+) or Th2 (CCR6-CXCR3-) cells were not increased. Antigen stimulation of PBMCs revealed IL-17A responses to LT, most clearly observed after stimulation with double mutant heat labile toxin (dmLT), but also with LT B subunit (LTB), and to CS6 in samples from patients with LT+ or CS6+ ETEC bacteria. Some individuals also mounted IFN-γ responses to dmLT and LTB. Levels of LTB specific IgA antibodies in ALS, but not plasma samples correlated with both IL-17A (r=0.5, p=0.02) and IFN-γ (r=0.6, p=0.01) responses to dmLT.ConclusionsOur results show that ETEC diarrhea induces T cell responses, which are predominantly of the Th17 type. The correlations between IL-17A and IFN-g and intestine-derived plasmablast responses support that Th responses may contribute to the development of protective IgA responses against ETEC infection. These observations provide important insights into T cell responses that need to be considered in the evaluation of advanced ETEC vaccine candidates.

ObjectivesImmunotherapy by blocking programmed death protein-1 (PD-1) or programmed death protein-ligand1 (PD-L1) with antibodies (PD-1 blockade) has revolutionized treatment options for patients with non-small cell lung cancer (NSCLC). However, the benefit of immunotherapy is limited to a subset of patients. This study aimed to investigate the value of combining immune and genetic variables analyzed within 3–4 weeks after the start of PD-1 blockade therapy to predict long-term clinical response.Materials and methodologyBlood collected from patients with NSCLC were analyzed for changes in the frequency and concentration of immune cells using a clinical flow cytometry assay. Next-generation sequencing (NGS) was performed on DNA extracted from archival tumor biopsies of the same patients. Patients were categorized as clinical responders or non-responders based on the 9 months’ assessment after the start of therapy.ResultsWe report a significant increase in the post-treatment frequency of activated effector memory CD4+ and CD8+ T-cells compared with pre-treatment levels in the blood. Baseline frequencies of B cells but not NK cells, T cells, or regulatory T cells were associated with the clinical response to PD-1 blockade. NGS of tumor tissues identified pathogenic or likely pathogenic mutations in tumor protein P53, Kirsten rat sarcoma virus, Kelch-like ECH-associated protein 1, neurogenic locus notch homolog protein 1, and serine/threonine kinase 11, primarily in the responder group. Finally, multivariate analysis of combined immune and genetic factors but neither alone, could discriminate between responders and non-responders.ConclusionCombined analyses of select immune cell subsets and genetic mutations could predict early clinical responses to immunotherapy in patients with NSCLC and after validation, can guide clinical precision medicine efforts.

Abstract Background No licensed human vaccines are available against enterotoxigenic Escherichia coli (ETEC), a major diarrhoeal pathogen affecting children in low- and middle-income countries and foreign travellers alike. ETVAX®, a multivalent oral whole-cell vaccine containing four inactivated ETEC strains and the heat-labile enterotoxin B subunit (LTB), has proved promising in Phase 1 and Phase 1/ 2 studies. Methods We conducted a Phase 2b double-blinded, randomized, placebo-controlled trial amongst Finnish travellers to Benin, West Africa. This report presents study design and safety and immunogenicity data. Volunteers aged 18–65 years were randomized 1:1 to receive ETVAX® or placebo. They visited Benin for 12 days, provided stool and blood samples and completed adverse event (AE) forms. IgA and IgG antibodies to LTB and O78 lipopolysaccharide (LPS) were measured by electrochemiluminescence. Results The AEs did not differ significantly between vaccine (n = 374) and placebo (n = 375) recipients. Of the solicited AEs, loose stools/diarrhoea (26.7/25.9%) and stomach ache (23.0/20.0%) were reported most commonly. Of all possibly/probably vaccine-related AEs, the most frequent were gastrointestinal symptoms (54.0/48.8%) and nervous system disorders (20.3/25.1%). Serious AEs were recorded for 4.3/5.6%, all unlikely to be vaccine related. Amongst the ETVAX® recipients, LTB-specific IgA antibodies increased 22-fold. For the 370/372 vaccine/placebo recipients, the frequency of ≥2-fold increases against LTB was 81/2.4%, and against O78 LPS 69/2.7%. The majority of ETVAX® recipients (93%) responded to either LTB or O78. Conclusions This Phase 2b trial is the largest on ETVAX® undertaken amongst travellers to date. ETVAX® showed an excellent safety profile and proved strongly immunogenic, which encourages the further development of this vaccine.

It was shown earlier that immune responses against cholera toxin (CT) as well as Vibrio cholerae lipopolysaccharide (LPS) or whole bacterial cells (WC) were protective and that these different antibody specificities co-operated synergistically for protection against experimental cholera. Similarly, antibodies against the heat-labile toxin (LT) and major colonization factors (CFs) of enterotoxingenic Escherichia coli (ETEC) co-operated synergistically for protection against LT-producing ETEC expressing homologous CFs. Studies in humans revealed that repeated oral antigen administration was optimal in inducing intestinal immune responses. Based on these findings oral inactivated vaccines consisting of toxin antigen and whole cells, i.e. the licensed recombinant cholera B subunit (rCTB)-WC cholera vaccine Dukoral®, and candidate ETEC vaccines have been developed. In different trials the rCTB-WC cholera vaccine has provided very high (85-100%) short term protection, which was significantly higher than that induced by the WC component alone, whereas rCTB-WC and WC alone provided comparable (50-60%), long term protection. An oral ETEC vaccine consisting of rCTB and formalin-inactivated E. coli bacteria expressing major CFs was shown to be safe and immunogenic in adults and children in different countries. The vaccine also induced significant protection against non-mild ETEC diarrhoea, i.e. diarrhoea interfering with daily activity in American travellers but not against ETEC diarrhoea in young children in Egypt. Against this background, a modified ETEC vaccine consisting of recombinant E. coli strains overexpressing the major CFs and a more LT like hybrid toxoid (LCTBA) has been developed. This vaccine will be tested soon alone and together with a mucosal adjuvant, i.e. dmLT, in clinical trials.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea in children in developing countries and in travelers. WHO has affirmed ETEC as a priority vaccine target, but there is no licensed ETEC vaccine available yet. We here describe recent, promising developments of different live, inactivated, and subunit ETEC candidate vaccines expressing or containing nontoxic enterotoxin and/or colonization factor antigens with a focus on oral vaccines. Many of the ETEC candidate vaccines have been tested in clinical trials for safety and immunogenicity and some of them also for protective efficacy in field trials or in challenge studies.

Diarrheal infections remain a major global health problem by causing up to 1.7 billion disease episodes and 700,000 deaths annually as well as malnutrition and cognitive disturbances, mainly in children in low- and middle-income countries. Travelers are also at high risk for being infected by enteric pathogens. There are only licensed vaccines for human use against three enteric pathogens, Salmonella enterica serovar Typhi (S. Typhi), Vibrio cholerae, and rotavirus. Enteric pathogens differ in the way they cause infection and disease. This influences what type of immune responses they elicit and how oral-mucosal or parenteral vaccination may protect. After briefly discussing these aspects for different bacterial enteric pathogens, this chapter focuses on reviewing current vaccine development efforts against enterotoxigenic Escherichia coli (ETEC) and Shigella, and more briefly it also comments on the vaccine development situation against Campylobacter, enterohemorrhagic/Shiga toxin–producing E. coli (EHEC/STEC), and non-typhoidal Salmonella (NTS) infections.

Antigen-specific class-switched antibodies are detected at the same time or even before IgM in serum of non-vaccinated individuals infected with SARS-CoV-2. These derive from the first wave of plasmablasts formed. Hence, the phenotype and specificity of plasmablasts can reveal information about early B-cell activation. Here we have analyzed B cells and plasmablasts circulating in blood of COVID-19 patients not previously exposed to SARS-CoV-2 during and after disease. We find that during infection with the original Wuhan strain, plasmablasts in blood produce IgA1, IgG1, and IgM, and that most express CCR10 and integrin β1, only some integrin β7, while the majority lack CCR9. Plasmablast-secreted antibodies are reactive to the spike (S) and nucleocapsid (N) proteins of the Wuhan strain as well as later variants of concern, but also bind S proteins from endemic and non-circulating betacoronaviruses. In contrast, after recovery, antibodies produced from memory B cells target variants of SARS-CoV-2 and SARS-CoV-1 but compared to previously non-infected individuals do not show increased binding to endemic coronaviruses. This suggests that the early antibody response to a large extent stems from pre-existing cross-reactive class-switched memory B cells, and that although newly formed memory cells target the novel SARS-CoV-2 virus the numbers of broadly cross-reactive memory B cells do not increase extensively. The observations give insight into the role of pre-existing memory B cells in early antibody responses to novel pathogens and may explain why class-switched antibodies are detected early in the serum of COVID-19 patients.

We tested an oral enterotoxigenic Escherichia coli (ETEC) vaccine, ETVAX, consisting of inactivated E. coli overexpressing the most prevalent ETEC colonization factors (CFs) and a toxoid (LCTBA), in Bangladeshi children for capacity to induce mucosal and plasma immune responses against O78 lipopolysaccharide (LPS) expressed on the vaccine strains. The vaccine was given +/- double-mutant heat-labile toxin (dmLT) adjuvant. We evaluated the impact of dmLT on anti-O78 LPS immune responses and whether such responses can predict responses against the CFs as a marker for vaccine "take". Two fractionated doses of ETVAX +/- different amounts of dmLT were administered biweekly to groups of children 24-59 (n = 125), 12-23 (n = 97) and 6-11 (n = 158) months of age. Immune responses were evaluated in antibody in lymphocyte supernatants (ALS), fecal extracts and plasma. ALS IgA responses against O78 LPS were induced in 44-49% of the children aged 12-59 months. The magnitudes of the ALS responses were significantly higher in children receiving a half-dose (5 x 10(10) bacteria) of ETVAX +/- dmLT than in placebo recipients. <10% of the vaccinees aged 6-11 months mounted ALS responses against O78 LPS. However, 49% of the infants developed fecal secretory IgA responses which were significantly more frequent in those receiving a quarter-dose (2.5 x 10(10) bacteria) of vaccine + dmLT (62%) compared to a quarter-dose alone (36%). Plasma IgA antibody responses were induced in 80% of older children and 36% of infants. The frequencies of O78 LPS responses in plasma and feces were comparable or higher than against the vaccine CFs in infants. Our findings show that ETVAX induced mucosal and systemic immune responses against O78 LPS in all age groups and that dmLT improved intestinal immune responses among infants. These observations may have implications for more successful use of other oral vaccines based on O antigens in children. (C) 2021 Published by Elsevier Ltd.

Background & Aims: Per- and polyfluoroalkyl substances (PFAS) are widely used, environmentally ubiquitous chemicals. Inhabitants in Ronneby, Sweden, have had long-term, moderate to high PFAS-exposure through drinking water contaminated by firefighting foams. PFAS has been associated with decreased vaccine-induced antibody responses in children. Effects in adults have been poorly investigated, in part owing to lack of well-designed trials of primary immunizations in PFAS exposed populations. The novelty of vaccination against COVID-19 and the associated clinical trials, offers a unique opportunity to investigate if PFAS decrease vaccine-induced antibody response in adults. Methods: Healthy, COVID-19 unvaccinated adults, 20-60 years old, from Ronneby (n=319; Perfluorooctanesulfonic acid (PFOS) median 46 ng/mL, range 1-543) and referents from neighboring Karlshamn (n=41; PFOS median 4 mg/mL, range 1-36), were vaccinated with two doses of the mRNA vaccine Spikevax® (Moderna). Seven PFAS were measured in serum before vaccination. Serum IgG antibodies against the spike antigen were measured before vaccination, and at five weeks (n=350) and six months (n=329) after the second vaccination. Regression analyses of antibodies were fitted against serum PFAS levels, quartile groups, smooth splines and between exposed and reference areas, adjusting for sex, age, smoking and previous SARS-CoV-2-infection. Results: Participants from Ronneby and referents had similar, high antibody levels (100% positive) five weeks and six months post vaccination, with waning levels over time. Regression models showed no clear pattern between antibody levels at five weeks or six months after vaccination and PFAS exposure. Results were replicated using different exposure indices, covariate adjustments and sensitivity analyses, and consistent for seven different PFAS compounds. Conclusions: In this clinical trial, with a wide PFAS exposure range and a low dropout rate, we assessed no indication that moderate to high PFAS exposure adversely affected antibody production in adults after COVID-19 mRNA vaccination. Keywords: PFAS, Immunotoxicity, Antibody, COVID-19 Vaccination

Understanding persistence and evolution of B cell clones after COVID-19 infection and vaccination is crucial for predicting responses against emerging viral variants and optimizing vaccines. Here, we collected longitudinal samples from patients with severe COVID-19 every third to seventh day during hospitalization and every third month after recovery. We profiled their antigen-specific immune cell dynamics by combining single-cell RNA-Seq, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), and B cell receptor–Seq (BCR-Seq) with oligo-tagged antigen baits. While the proportion of Spike receptor binding domain–specific memory B cells (MBC) increased from 3 months after infection, the other Spike- and Nucleocapsid-specific B cells remained constant. All patients showed ongoing class switching and sustained affinity maturation of antigen-specific cells, and affinity maturation was not significantly increased early after vaccine. B cell analysis revealed a polyclonal response with limited clonal expansion; nevertheless, some clones detected during hospitalization, as plasmablasts, persisted for up to 1 year, as MBC. Monoclonal antibodies derived from persistent B cell families increased their binding and neutralization breadth and started recognizing viral variants by 3 months after infection. Overall, our findings provide important insights into the clonal evolution and dynamics of antigen-specific B cell responses in longitudinally sampled patients infected with COVID-19.

Pregnancy might impact immunity after SARS-CoV-2 infection and/or vaccination. We describe the first case of reinfection with SARS-CoV-2 during a pregnancy. While the mother lacked detectable antibodies 2 months after the first infection, both mother and baby had IgG antibodies at delivery. Infection did not cause any adverse pregnancy outcome.