Effect of age and season on respiratory mucosal immune marker profiles.

BACKGROUND: The upper respiratory tract is continuously exposed to microorganisms and noxious elements, leading to local immune responses and the secretion of immune markers. While several studies describe immune marker profiles in respiratory mucosal samples in defined patient cohorts, mucosal immune profiles from the general population during the different seasons are lacking. Such baseline profiles are essential to understand the effect of various exposures to the mucosal immune system throughout life. OBJECTIVE: We sought to establish baseline local upper respiratory mucosal immune profiles in the general population and assess these profiles with regard to age, sex, seasonality, and basic health and lifestyle factors. METHODS: We measured the concentrations of 35 immune markers involved in a broad range of immunological processes at the mucosa in nasopharyngeal swab samples from 951 individuals, aged 0 to 86 years, from a nationwide study. RESULTS: Clustering analysis showed that immune marker profiles clearly reflected immunological functions, such as tissue regeneration and antiviral responses. Immune marker concentrations changed strongly with seasonality and age, with the most profound changes occurring in the first 25 years of life; they were also associated with sex, body mass index, smoking, mild symptoms of airway infection, and chronic asthma and hay fever. CONCLUSION: Immunological analyses of noninvasive mucosal samples provide insight into mucosal immune responses to microbial and noxious element exposure in the general population. These data provide a baseline for future studies on respiratory mucosal immune responses and for the development of mucosal immune-based diagnostics.

Serological profiling of pneumococcal proteins reveals unique patterns of acquisition, maintenance and waning of antibodies throughout life.

Streptococcus pneumoniae is a leading cause of morbidity and mortality in children and older adults. Yet knowledge on the development of pneumococcal protein-specific antibody responses throughout life is limited. To investigate this, we measured serum IgG levels to 55 pneumococcal proteins in 11-month old infants (n=73), 24-month old children (n=101), parents (n=99), adults without children <6 years of age (n= 99) and older adults aged >60 years (n=100). Our findings revealed low IgG levels in infancy, with distinct development patterns peaking in adults. A decrease in levels was observed for 27 antigens towards older age. Adults and older adults had increased IgG levels during pneumococcal carriage and at increased exposure risk to S. pneumoniae. Carriage was a stronger predictor than exposure or age for antibody responses. These findings highlight the dynamic nature of naturally acquired humoral immunity to pneumococcal proteins throughout life, offering insights for age-targeted interventions.

Association between adverse events after COVID-19 vaccination and anti-SARS-CoV-2 antibody concentrations, the Netherlands, May 2021 to November 2022: a population-based prospective cohort study.

BackgroundNon-severe adverse events (AE) including pain at injection site or fever are common after COVID-19 vaccination.AimTo describe determinants of AE after COVID-19 vaccination and investigate the association between AE and pre- and post-vaccination antibody concentrations.MethodsParticipants of an ongoing prospective cohort study (VASCO) completed a questionnaire on AE within 2 months after vaccination and provided 6 monthly serum samples during May 2021-November 2022. Logistic regression analyses were performed to investigate AE determinants after mRNA vaccination, including pre-vaccination Ig antibody concentrations against the SARS-CoV-2 spike protein receptor binding domain. Multivariable linear regression was performed in SARS-CoV-2-naive participants to assess the association between AE and log-transformed antibody concentrations 3-8 weeks after mRNA vaccination.ResultsWe received 47,947 completed AE questionnaires by 28,032 participants. In 42% and 34% of questionnaires, injection site and systemic AE were reported, respectively. In 2.2% of questionnaires, participants sought medical attention. AE were reported more frequently by women, younger participants (< 60 years), participants with medical risk conditions and Spikevax recipients (vs Comirnaty). Higher pre-vaccination antibody concentrations were associated with higher incidence of systemic AE after the second and third dose, but not with injection site AE or AE for which medical attention was sought. Any AE after the third dose was associated with higher post-vaccination antibody concentrations (geometric mean concentration ratio: 1.38; 95% CI: 1.23-1.54).ConclusionsOur study suggests that high pre-vaccination antibody levels are associated with AE, and experiencing AE may be a marker for higher antibody response to vaccination.

Antibody and T-Cell Responses 6 Months After Coronavirus Disease 2019 Messenger RNA-1273 Vaccination in Patients With Chronic Kidney Disease, on Dialysis, or Living With a Kidney Transplant.

BACKGROUND: The immune response to COVID-19 vaccination is inferior in kidney transplant recipients (KTRs) and to a lesser extent in patients on dialysis or with chronic kidney disease (CKD). We assessed the immune response 6 months after mRNA-1273 vaccination in kidney patients and compared this to controls. METHODS: A total of 152 participants with CKD stages G4/5 (eGFR <30 mL/min/1.73 m2), 145 participants on dialysis, 267 KTRs, and 181 controls were included. SARS-CoV-2 Spike S1 specific IgG antibodies were measured using fluorescent bead-based multiplex-immunoassay, neutralizing antibodies to ancestral, Delta, and Omicron (BA.1) variants by plaque reduction, and T-cell responses by interferon-γ release assay. RESULTS: At 6 months after vaccination, S1-specific antibodies were detected in 100% of controls, 98.7% of CKD G4/5 patients, 95.1% of dialysis patients, and 56.6% of KTRs. These figures were comparable to the response rates at 28 days, but antibody levels waned significantly. Neutralization of the ancestral and Delta variants was detected in most participants, whereas neutralization of Omicron was mostly absent. S-specific T-cell responses were detected at 6 months in 75.0% of controls, 69.4% of CKD G4/5 patients, 52.6% of dialysis patients, and 12.9% of KTRs. T-cell responses at 6 months were significantly lower than responses at 28 days. CONCLUSIONS: Although seropositivity rates at 6 months were comparable to rates at 28 days after vaccination, significantly decreased antibody levels and T-cell responses were observed. The combination of low antibody levels, reduced T-cell responses, and absent neutralization of the newly emerging variants indicates the need for additional boosts or alternative vaccination strategies in KTRs. CLINICAL TRIALS REGISTRATION: NCT04741386.

Effectiveness of bivalent mRNA booster vaccination against SARS-CoV-2 Omicron infection, the Netherlands, September to December 2022.

We used data of 32,542 prospective cohort study participants who previously received primary and one or two monovalent booster COVID-19 vaccinations. Between 26 September and 19 December 2022, relative effectiveness of bivalent original/Omicron BA.1 vaccination against self-reported Omicron SARS-CoV-2 infection was 31% in 18-59-year-olds and 14% in 60-85-year-olds. Protection of Omicron infection was higher than of bivalent vaccination without prior infection. Although bivalent booster vaccination increases protection against COVID-19 hospitalisations, we found limited added benefit in preventing SARS-CoV-2 infection.

Bordetella pertussis Induces Interferon Gamma Production by Natural Killer Cells, Resulting in Chemoattraction by Respiratory Epithelial Cells.

BACKGROUND: Whooping cough is caused by infection of the airways with Bordetella pertussis (Bp). As interferon gamma (IFN-γ) is essential for protective immunity against Bp, we investigated how IFN-γ is induced by Bp or the virulence antigens filamentous hemagglutinin adhesin, pertactin, or pertussis toxin, and how IFN-γ contributes to local immune responses in humans. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy donors and/or respiratory epithelial cells were stimulated with soluble antigens or inactivated intact Bp and the presence or absence of blocking antibodies or chemokines. Supernatants and cells were analyzed for IFN-γ and chemokine production, and lymphocyte migration was tested using epithelial supernatants. RESULTS: The soluble antigens failed to induce IFN-γ production, whereas inactivated Bp induced IFN-γ production. Natural killer (NK) cells were the main source of IFN-γ production, which was enhanced by interleukin 15. Epithelial-PBMC co-cultures showed robust IFN-γ-dependent CXCL9 and CXCL10 production by the epithelial cells following stimulation with IFN-γ and Bp. The epithelial-derived chemokines resulted in CXCR3-dependent recruitment of NK and T cells. CONCLUSIONS: Inactivated Bp, but not antigens, induced potent IFN-γ production by NK cells, resulting in chemoattraction of lymphocytes toward the respiratory epithelium. These data provide insight into the requirements for IFN-γ production and how IFN-γ enhances local immune responses to prevent Bp-mediated disease.

Decreased Antibody Response After Severe Acute Respiratory Syndrome Coronavirus 2 Vaccination in Patients With Down Syndrome.

The risk of a severe course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in adults with Down syndrome is increased, resulting in an up to 10-fold increase in mortality, in particular in those >40 years of age. After primary SARS-CoV-2 vaccination, the higher risks remain. In this prospective observational cohort study, SARS-CoV-2 spike S1-specific antibody responses after routine SARS-CoV-2 vaccination (BNT162b2, messenger RNA [mRNA]-1273, or ChAdOx1) in adults with Down syndrome and healthy controls were compared. Adults with Down syndrome showed lower antibody concentrations after 2 mRNA vaccinations or after 2 ChAdOx1 vaccinations. After 2 mRNA vaccinations, lower antibody concentrations were seen with increasing age. CLINICAL TRIALS REGISTRATION: NCT05145348.

Analysing the protection from respiratory tract infections and allergic diseases early in life by human milk components: the PRIMA birth cohort.

BACKGROUND: Many studies support the protective effect of breastfeeding on respiratory tract infections. Although infant formulas have been developed to provide adequate nutritional solutions, many components in human milk contributing to the protection of newborns and aiding immune development still need to be identified. In this paper we present the methodology of the "Protecting against Respiratory tract lnfections through human Milk Analysis" (PRIMA) cohort, which is an observational, prospective and multi-centre birth cohort aiming to identify novel functions of components in human milk that are protective against respiratory tract infections and allergic diseases early in life. METHODS: For the PRIMA human milk cohort we aim to recruit 1000 mother-child pairs in the first month postpartum. At one week, one, three, and six months after birth, fresh human milk samples will be collected and processed. In order to identify protective components, the level of pathogen specific antibodies, T cell composition, Human milk oligosaccharides, as well as extracellular vesicles (EVs) will be analysed, in the milk samples in relation to clinical data which are collected using two-weekly parental questionnaires. The primary outcome of this study is the number of parent-reported medically attended respiratory infections. Secondary outcomes that will be measured are physician diagnosed (respiratory) infections and allergies during the first year of life. DISCUSSION: The PRIMA human milk cohort will be a large prospective healthy birth cohort in which we will use an integrated, multidisciplinary approach to identify the longitudinal effect human milk components that play a role in preventing (respiratory) infections and allergies during the first year of life. Ultimately, we believe that this study will provide novel insights into immunomodulatory components in human milk. This may allow for optimizing formula feeding for all non-breastfed infants.

The estimated disease burden of acute COVID-19 in the Netherlands in 2020, in disability-adjusted life-years.

The impact of COVID-19 on population health is recognised as being substantial, yet few studies have attempted to quantify to what extent infection causes mild or moderate symptoms only, requires hospital and/or ICU admission, results in prolonged and chronic illness, or leads to premature death. We aimed to quantify the total disease burden of acute COVID-19 in the Netherlands in 2020 using the disability-adjusted life-years (DALY) measure, and to investigate how burden varies between age-groups and occupations. Using standard methods and diverse data sources (mandatory notifications, population-level seroprevalence, hospital and ICU admissions, registered COVID-19 deaths, and the literature), we estimated years of life lost (YLL), years lived with disability, DALY and DALY per 100,000 population due to COVID-19, excluding post-acute sequelae, stratified by 5-year age-group and occupation category. The total disease burden due to acute COVID-19 was 286,100 (95% CI: 281,700-290,500) DALY, and the per-capita burden was 1640 (95% CI: 1620-1670) DALY/100,000, of which 99.4% consisted of YLL. The per-capita burden increased steeply with age, starting from 60 to 64 years, with relatively little burden estimated for persons under 50 years old. SARS-CoV-2 infection and associated premature mortality was responsible for a considerable direct health burden in the Netherlands, despite extensive public health measures. DALY were much higher than for other high-burden infectious diseases, but lower than estimated for coronary heart disease. These findings are valuable for informing public health decision-makers regarding the expected COVID-19 health burden among population subgroups, and the possible gains from targeted preventative interventions.

Antibody Responses to Respiratory Syncytial Virus: A Cross-Sectional Serosurveillance Study in the Dutch Population Focusing on Infants Younger Than 2 Years.

BACKGROUND: Respiratory syncytial virus (RSV) generally causes mild disease but can cause severe infections in (premature) infants and elderly adults. Here, we studied RSV-specific antibody concentrations throughout life with emphasis on infants and chronic obstructive pulmonary disease (COPD) patients. METHODS: Sera (N = 2655) from 2 nationwide cross-sectional studies in the Netherlands including individuals aged 0-90 years were analyzed for IgG and IgA antibodies to RSV prefusion F, postfusion F, N, Ga, and Gb proteins and for antibody avidity in 42 COPD patients. RESULTS: Maternal IgG concentrations declined to age 10-12 months. After the first year of life, approximately 40% of children lacked infection-induced IgA antibodies and may therefore be uninfected. All Dutch children showed serological evidence of RSV infection by age 3 years. Antibody concentrations reached a plateau by age 5-9 years and remains constant throughout life. COPD patients had similar levels and avidity of RSV-specific IgG antibodies compared with age-matched healthy controls. CONCLUSIONS: RSV-IgG antibody patterns throughout life can be used to estimate the degree of immunity acquisition to RSV and to identify groups at increased risk of infection. Seroprevalence of IgA could be a proxy to determine RSV infection in children younger than 1 year.

mRNA-1273 COVID-19 vaccination in patients receiving chemotherapy, immunotherapy, or chemoimmunotherapy for solid tumours: a prospective, multicentre, non-inferiority trial.

BACKGROUND: Patients with cancer have an increased risk of complications from SARS-CoV-2 infection. Vaccination to prevent COVID-19 is recommended, but data on the immunogenicity and safety of COVID-19 vaccines for patients with solid tumours receiving systemic cancer treatment are scarce. Therefore, we aimed to assess the impact of immunotherapy, chemotherapy, and chemoimmunotherapy on the immunogenicity and safety of the mRNA-1273 (Moderna Biotech, Madrid, Spain) COVID-19 vaccine as part of the Vaccination Against COVID in Cancer (VOICE) trial. METHODS: This prospective, multicentre, non-inferiority trial was done across three centres in the Netherlands. Individuals aged 18 years or older with a life expectancy of more than 12 months were enrolled into four cohorts: individuals without cancer (cohort A [control cohort]), and patients with solid tumours, regardless of stage and histology, treated with immunotherapy (cohort B), chemotherapy (cohort C), or chemoimmunotherapy (cohort D). Participants received two mRNA-1273 vaccinations of 100 µg in 0·5 mL intramuscularly, 28 days apart. The primary endpoint, analysed per protocol (excluding patients with a positive baseline sample [>10 binding antibody units (BAU)/mL], indicating previous SARS-CoV-2 infection), was defined as the SARS-CoV-2 spike S1-specific IgG serum antibody response (ie, SARS-CoV-2-binding antibody concentration of >10 BAU/mL) 28 days after the second vaccination. For the primary endpoint analysis, a non-inferiority design with a margin of 10% was used. We also assessed adverse events in all patients who received at least one vaccination, and recorded solicited adverse events in participants who received at least one vaccination but excluding those who already had seroconversion (>10 BAU/mL) at baseline. This study is ongoing and is registered with ClinicalTrials.gov, NCT04715438. FINDINGS: Between Feb 17 and March 12, 2021, 791 participants were enrolled and followed up for a median of 122 days (IQR 118 to 128). A SARS-CoV-2-binding antibody response was found in 240 (100%; 95% CI 98 to 100) of 240 evaluable participants in cohort A, 130 (99%; 96 to >99) of 131 evaluable patients in cohort B, 223 (97%; 94 to 99) of 229 evaluable patients in cohort C, and 143 (100%; 97 to 100) of 143 evaluable patients in cohort D. The SARS-CoV-2-binding antibody response in each patient cohort was non-inferior compared with cohort A. No new safety signals were observed. Grade 3 or worse serious adverse events occurred in no participants in cohort A, three (2%) of 137 patients in cohort B, six (2%) of 244 patients in cohort C, and one (1%) of 163 patients in cohort D, with four events (two of fever, and one each of diarrhoea and febrile neutropenia) potentially related to the vaccination. There were no vaccine-related deaths. INTERPRETATION: Most patients with cancer develop, while receiving chemotherapy, immunotherapy, or both for a solid tumour, an adequate antibody response to vaccination with the mRNA-1273 COVID-19 vaccine. The vaccine is also safe in these patients. The minority of patients with an inadequate response after two vaccinations might benefit from a third vaccination. FUNDING: ZonMw, The Netherlands Organisation for Health Research and Development.

Associations Between Measures of Social Distancing and Severe Acute Respiratory Syndrome Coronavirus 2 Seropositivity: A Nationwide Population-based Study in the Netherlands.

This large, nationwide, population-based, seroepidemiological study provides evidence of the effectiveness of physical distancing (>1.5 m) and indoor group size reductions in reducing severe acute respiratory syndrome coronavirus 2 infection. Additionally, young adults may play an important role in viral spread, contrary to children up until age 12 years with whom close contact is permitted. CLINICAL TRIALS REGISTRATION: NTR8473.

Persistence of Antibodies to Severe Acute Respiratory Syndrome Coronavirus 2 in Relation to Symptoms in a Nationwide Prospective Study.

BACKGROUND: Assessing the duration of immunity following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a first priority to gauge the degree of protection following infection. Such knowledge is lacking, especially in the general population. Here, we studied changes in immunoglobulin isotype seropositivity and immunoglobulin G (IgG) binding strength of SARS-CoV-2-specific serum antibodies up to 7 months following onset of symptoms in a nationwide sample. METHODS: Participants from a prospective representative serological study in the Netherlands were included based on IgG seroconversion to the spike S1 protein of SARS-CoV-2 (N = 353), with up to 3 consecutive serum samples per seroconverted participant (N = 738). Immunoglobulin M (IgM), immunoglobulin A (IgA), and IgG antibody concentrations to S1, and increase in IgG avidity in relation to time since onset of disease symptoms, were determined. RESULTS: While SARS-CoV-2-specific IgM and IgA antibodies declined rapidly after the first month after disease onset, specific IgG was still present in 92% (95% confidence interval [CI], 89%-95%) of the participants after 7 months. The estimated 2-fold decrease of IgG antibodies was 158 days (95% CI, 136-189 days). Concentrations were sustained better in persons reporting significant symptoms compared to asymptomatic persons or those with mild upper respiratory complaints only. Similarly, avidity of IgG antibodies for symptomatic persons showed a steeper increase over time compared with persons with mild or no symptoms (P = .022). CONCLUSIONS: SARS-CoV-2-specific IgG antibodies persist and show increasing avidity over time, indicative of underlying immune maturation. These data support development of immune memory against SARS-CoV-2, providing insight into protection of the general unvaccinated part of the population. CLINICAL TRIALS REGISTRATION: NL8473 (the Dutch trial registry).

Estimating the asymptomatic proportion of SARS-CoV-2 infection in the general population: Analysis of nationwide serosurvey data in the Netherlands.

BACKGROUND: The proportion of SARS-CoV-2 positive persons who are asymptomatic-and whether this proportion is age-dependent-are still open research questions. Because an unknown proportion of reported symptoms among SARS-CoV-2 positives will be attributable to another infection or affliction, the observed, or 'crude' proportion without symptoms may underestimate the proportion of persons without symptoms that are caused by SARS-CoV-2 infection. METHODS: Based on two rounds of a large population-based serological study comprising test results on seropositivity and self-reported symptom history conducted in April/May and June/July 2020 in the Netherlands (n = 7517), we estimated the proportion of reported symptoms among those persons infected with SARS-CoV-2 that is attributable to this infection, where the set of relevant symptoms fulfills the ECDC case definition of COVID-19, using inferential methods for the attributable risk (AR). Generalised additive regression modelling was used to estimate the age-dependent relative risk (RR) of reported symptoms, and the AR and asymptomatic proportion (AP) were calculated from the fitted RR. RESULTS: Using age-aggregated data, the 'crude' AP was 37% but the model-estimated AP was 65% (95% CI 63-68%). The estimated AP varied with age, from 74% (95% CI 65-90%) for < 20 years, to 61% (95% CI 57-65%) for the 50-59 years age-group. CONCLUSION: Whereas the 'crude' AP represents a lower bound for the proportion of persons infected with SARS-CoV-2 without COVID-19 symptoms, the AP as estimated via an attributable risk approach represents an upper bound. Age-specific AP estimates can inform the implementation of public health actions such as targetted virological testing and therefore enhance containment strategies.

Asthma-Associated Long TSLP Inhibits the Production of IgA.

Thymic stromal lymphopoietin (TSLP) contributes to asthmatic disease. The concentrations of protective IgA may be reduced in the respiratory tract of asthma patients. We investigated how homeostatic short TSLP (shTSLP) and asthma-associated long TSLP (loTSLP) regulate IgA production. B cells from healthy donors were stimulated in the presence or absence of shTSLP or loTSLP; the concentrations of IgA, IgM, IgE, and IgG antibodies were determined in cell culture supernatants; and B cells were analyzed by flow cytometry. LoTSLP, but not shTSLP, suppressed the secretion of IgA but not of IgE. The type 2 cytokine IL-4, which in addition to loTSLP contributes to asthmatic disease, did not affect the production of IgA or the frequency of IgA+ B cells. Instead, IL-4 increased IgG production, especially of the subclasses IgG2 and IgG4. LoTSLP inhibited IgA secretion by sorted memory B cells but not by naïve B cells. Although loTSLP inhibited IgA production, the vitamin A metabolite retinoic acid promoted the secretion of IgA, also in the presence of loTSLP, suggesting that vitamin A may promote IgA production in asthma. Our data demonstrate that asthma-associated loTSLP negatively regulates the secretion of IgA, which may negatively impact the surveillance of mucosal surfaces in asthma.

SARS-CoV-2-Specific Antibody Detection for Seroepidemiology: A Multiplex Analysis Approach Accounting for Accurate Seroprevalence.

BACKGROUND: The COVID-19 pandemic necessitates better understanding of the kinetics of antibody production induced by infection with SARS-CoV-2. We aimed to develop a high-throughput multiplex assay to detect antibodies to SARS-CoV-2 to assess immunity to the virus in the general population. METHODS: Spike protein subunits S1 and receptor binding domain, and nucleoprotein were coupled to microspheres. Sera collected before emergence of SARS-CoV-2 (n = 224) and of non-SARS-CoV-2 influenza-like illness (n = 184), and laboratory-confirmed cases of SARS-CoV-2 infection (n = 115) with various severities of COVID-19 were tested for SARS-CoV-2-specific IgG concentrations. RESULTS: Our assay discriminated SARS-CoV-2-induced antibodies and those induced by other viruses. The assay specificity was 95.1%-99.0% with sensitivity 83.6%-95.7%. By merging the test results for all 3 antigens a specificity of 100% was achieved with a sensitivity of at least 90%. Hospitalized COVID-19 patients developed higher IgG concentrations and the rate of IgG production increased faster compared to nonhospitalized cases. CONCLUSIONS: The bead-based serological assay for quantitation of SARS-CoV-2-specific antibodies proved to be robust and can be conducted in many laboratories. We demonstrated that testing of antibodies against multiple antigens increases sensitivity and specificity compared to single-antigen-specific IgG determination.

BAFF augments IgA2 and IL-10 production by TLR7/8 stimulated total peripheral blood B cells.

Class-switching of B cells to IgA can be induced via both T-cell-dependent and T-cell-independent mechanisms. IgA is most predominantly produced mucosally and is important for combating infections and allergies. In contrast to mice, humans have two forms of IgA; IgA1 and IgA2 with diverse tissue distribution. In early life, IgA levels might be sub-optimal especially during the fall season when bacterial and viral infections are more common. Therefore, we investigated using human B cells whether T-cell-independent factors -promoting cell survival, class switching and immunoglobulin secretion- BAFF, APRIL, IL-10 and retinoic acid can boost IgA production in the context of viral or bacterial infection. To this end total and naive peripheral blood B cells were stimulated with these factors for 6 days in the presence or absence of TLR7/8 agonist R848 (mimicking viral infection) or TLR9 agonist CpG-ODN (mimicking bacterial infection). We show that BAFF significantly augments IgA2 production in TLR7/8 stimulated mature, but not naïve B cells. In addition, BAFF augments IL-10 production and viability in TLR7/8 and TLR9 stimulated mature B cells. These data warrant further investigation of its role in immune regulation both in the periphery and mucosal tissues in early life or during disease.

Regulation of Rac1 and Reactive Oxygen Species Production in Response to Infection of Gastrointestinal Epithelia.

Generation of reactive oxygen species (ROS) during infection is an immediate host defense leading to microbial killing. APE1 is a multifunctional protein induced by ROS and after induction, protects against ROS-mediated DNA damage. Rac1 and NAPDH oxidase (Nox1) are important contributors of ROS generation following infection and associated with gastrointestinal epithelial injury. The purpose of this study was to determine if APE1 regulates the function of Rac1 and Nox1 during oxidative stress. Gastric or colonic epithelial cells (wild-type or with suppressed APE1) were infected with Helicobacter pylori or Salmonella enterica and assessed for Rac1 and NADPH oxidase-dependent superoxide production. Rac1 and APE1 interactions were measured by co-immunoprecipitation, confocal microscopy and proximity ligation assay (PLA) in cell lines or in biopsy specimens. Significantly greater levels of ROS were produced by APE1-deficient human gastric and colonic cell lines and primary gastric epithelial cells compared to control cells after infection with either gastric or enteric pathogens. H. pylori activated Rac1 and Nox1 in all cell types, but activation was higher in APE1 suppressed cells. APE1 overexpression decreased H. pylori-induced ROS generation, Rac1 activation, and Nox1 expression. We determined that the effects of APE1 were mediated through its N-terminal lysine residues interacting with Rac1, leading to inhibition of Nox1 expression and ROS generation. APE1 is a negative regulator of oxidative stress in the gastrointestinal epithelium during bacterial infection by modulating Rac1 and Nox1. Our results implicate APE1 in novel molecular interactions that regulate early stress responses elicited by microbial infections.