Systems vaccinology identifies clinical and immunological correlates of SARS-CoV-2 vaccine response in solid-organ transplant recipients (preprint)

Solid organ transplant (SOT) recipients are at enhanced risk of adverse outcomes following infectious challenges due to immunosuppressive treatment and additional comorbidities. Unfortunately, SOT recipients are also poor responders to the key medical intervention to preventing infection: vaccines. Here we performed a systems vaccinology study on a cohort of 59 kidney transplant recipients and 31 lung transplant recipients who received the mRNA Pfizer-BioNTech COVID-19 vaccine. Analyzing the immunological status of the patients prior to vaccination, we were able to identify multiple immunological associates of relatively improved vaccine responses following two or three doses of mRNA-based SARS-CoV-2 vaccine. These immunological associates predicted, with 95.0% and 93.3% accuracy, vaccine response after the second and third dose, respectively. Comparison of the immunological associates with vaccine response in SOT recipients revealed two distinct immune configurations: a non-classical configuration, distinct from the immune state of healthy subjects, associated with responses to two doses of mRNA vaccine and that could be mediated partly by the presence of double negative B cell subsets which are more prominently represented in responsive SOT recipients, and a "normalized" configuration, closer to the immune state of healthy subjects, associated with potent antibody responses to three doses of mRNA vaccine. These results suggest that immunosuppression in SOT recipients can result in distinct immune states associated with different trade-offs in vaccine responsiveness. Immune phenotyping of SOT recipients for immune constellation may be an effective approach for identifying patients most at risk of poor vaccine responses and susceptibility to vaccine-preventable diseases. One-sentence summarySOT recipients showed distinct immune states at baseline associated with different profiles of vaccine-associated immune response.

Prevalence of SARS-CoV-2 antibodies and associated factors in the adult population of Belgium: a general population cohort study between March 2021 and April 2022 (preprint)

Background This cohort study assessed seroprevalence trends of SARS-CoV-2 antibodies in the general Belgian population between March 2021 and April 2022, and explored factors associated with seropositivity among the vaccinated and unvaccinated population. Seroreversion and its potential determinants were also examined. Methods A random sample of the adult population in Belgium was invited to provide a saliva sample and to complete a survey questionnaire. Participants were followed up twice for a new saliva sample and updated information. Antibodies were assessed with a semi-quantitative measure of anti-RBD (Receptor Binding Domain) IgG ELISA. Seven time periods were defined for estimating SARS-CoV-2 antibody prevalence using post stratification weights to match the population distribution. Seroreversion was defined as passing from a positive to a negative antibody test from one data collection point to the next. Potential determinants of seropositivity were assessed through hierarchical multiple logistic regressions. Results In total 6,178 valid observations were obtained from 2,768 individuals. SARS-CoV-2 antibody prevalence increased from 25.1% in the beginning of the study period to 92.3% in the end. Among the vaccinated population, factors significantly associated with a higher seropositivity were being younger, having a bachelor diploma, living with others, having had a vaccine in the last 3 months and having received a nucleic-acid vaccine or a combination. Lower seropositivity rates were observed among vaccinated people with a neurological disease and transplant patients. Factors significantly associated with a higher seropositivity rate among the unvaccinated population were having non-O blood type and being non-smoker. Among fully vaccinated people the seroreversion rate was much lower (0.3%) among those who had received their latest vaccine in the last 3 months compared to those who had received their latest vaccine more than 3 months ago (2.7%). Conclusions The rapid increase in antibody seropositivity in the general adult population in Belgium during the study period was driven by the vaccination campaign which ran at full speed during this period. Factors associated with higher and lower seropositivity were identified among the vaccinated and unvaccinated people.

Predictive model for BNT162b2 vaccine response in cancer patients based on cytokines and growth factors (preprint)

Background: Patients with cancer, especially haematological cancer, are at increased risk for breakthrough COVID-19 infection. However, so far, a predictive biomarker that can assess compromised vaccine-induced anti-SARS-CoV-2 immunity in cancer patients has not been proposed. Methods: Here, we employed machine learning approaches to identify a biomarker signature based on blood cytokine and growth factors linked to vaccine response from 199 cancer patients receiving BNT162b2 vaccine. Results: We show that C-reactive protein (CRP; general marker of inflammation), interleukin (IL)-15 (a pro-inflammatory cytokine), IL-18 (interferon-gamma inducing factor), and placental growth factor (an angiogenic cytokine) can correctly classify patients with a diminished vaccine response assessed at day 49 with >80% accuracy. Amongst these, CRP showed the highest predictive value for poor response to vaccine administration. Importantly, this unique signature of vaccine response was present at different studied timepoints both before and after vaccination and was not majorly affected by different anti-cancer treatments. Conclusion: While we propose a blood-based signature of cytokines and growth factors that can be employed in identifying cancer patients at continued risk of COVID-19, our data also importantly suggest that such a signature could reflect the inherent make-up of some cancer patients who are also refractive to immunotherapy.

Humoral and cellular immune correlates of protection against COVID-19 in kidney transplant recipients (preprint)

As solid organ recipients are at high risk of severe COVID-19 and respond poorly to primary SARS-CoV-2 mRNA vaccination, they have been prioritized for booster vaccination. However, an immunological correlate of protection has not been identified in this vulnerable population. We conducted a prospective monocentric cohort study of 65 kidney transplant recipients who received three doses of SARS-CoV-2 BNT162b2 mRNA vaccination. Associations between symptomatic breakthrough infection (BTI) and vaccine responses, patient demographic and clinical characteristics were explored. Symptomatic COVID-19 was diagnosed in 32% of kidney transplant recipients during a period of six months after the administration of the third vaccine dose. During this period, SARS-CoV-2 delta and omicron were the dominant variants in the general population. Univariate analyzes identified avidity of SARS-CoV-2 receptor binding domain (RBD) binding IgG, neutralizing antibodies and SARS-CoV-2 S2 domain-specific IFN-gamma responses as correlates of protection against BTI. Some demographic and clinical parameters correlated with vaccine responses, but none correlated with the risk of BTI. In multivariate analysis, the risk of BTI was best predicted by neutralizing antibody and S2-specific IFN-gamma responses, adjusting for age, graft function and mycophenolate mofetil use. In conclusion, both antibody and T cell responses predict the risk of BTI in kidney transplant recipients who received three doses of SARS-CoV-2 mRNA vaccine. T cell responses may help compensate for the suboptimal antibody response to vaccination in this vulnerable population.

The prevalence, incidence and longevity of antibodies against SARS-CoV-2 among primary healthcare providers in Belgium: a prospective cohort study with 12 months of follow-up (preprint)

Objectives: To estimate the prevalence, incidence, and longevity of antibodies against SARS-CoV-2 among primary healthcare providers (PHCPs). Design: Prospective cohort study with 12 months of follow-up. Setting: Primary care in Belgium Participants: Any general practitioner (GP) working in primary care in Belgium and any other PHCP from the same GP practice who physically manages (examines, tests, treats) patients were eligible. A convenience sample of 3,648 eligible PHCPs from 2,001 GP practices registered for this study (3,044 and 604 to start in December 2020 and January 2021, respectively). 3,390 PHCPs (92,9%) participated in their first testing timepoint (2,820 and 565, respectively) and 2,557 PHCPs (70,1%) in the last testing timepoint (December 2021). Interventions: Participants were asked to perform a rapid serological test (RST) targeting IgM and IgG against the receptor binding domain (RBD) of SARS-CoV-2 and to complete an online questionnaire at each of maximum 8 testing timepoints. Primary and secondary outcome measures: The prevalence, incidence, and longevity of antibodies against SARS-CoV-2 both after natural infection and after vaccination. Results: Among all participants, 67% were women and 77% GPs. Median age was 43 years. The seroprevalence in December 2020 (before vaccination availability) was 15.1% (95% CI: 13.5% to 16.6%), increased to 84.2% (95% CI: 82.9% to 85.5%) in March 2021 (after vaccination availability) and reached 93.9% (95% CI: 92.9% to 94.9%) in December 2021 (during booster vaccination availability and fourth (delta variant dominant) covid wave). Among not (yet) vaccinated participants the first monthly incidence of antibodies against SARS-CoV-2 was estimated to be 2.91% (95% CI: 1.80% to 4.01%). The longevity of antibodies is higher in PHCPs with self-reported COVID-19 infection. Conclusions: This study confirms that occupational health measures provided sufficient protection when managing patients. High uptake of vaccination resulted in high seroprevalence of SARS-CoV-2 antibodies in PHCPs in Belgium. Longevity of antibodies was supported by booster vaccination and virus circulation. Registration: Trial registration number: NCT04779424

Safety and immunogenicity of a reduced dose of the BNT162b2 mRNA COVID-19 vaccine (REDU-VAC): a single blind, randomized, non-inferiority trial (preprint)

Background The use of fractional dose regimens of COVID-19 vaccines has the potential to accelerate vaccination rates in low-income countries. Dose-finding studies of the mRNA vaccine BNT162b2 (Pfizer-BioNTech) have suggested that a fractional dose induces comparable antibody responses to the full, licensed dose in people below 55 years old. Here, we report the safety and immunogenicity of a fractional dose regimen of the BNT162b2 vaccine. Methods REDU-VAC is a participant-blinded, randomised, phase 4, multicentre, non-inferiority study investigating safety, reactogenicity and immunogenicity of BNT162b2. Adults aged between 18 and 55 years, without uncontrolled co-morbidities, either previously infected or infection naive, were eligible and recruited at five sites across Belgium. Participants were randomly assigned to receive 20ug/20ug (fractional dose) or 30ug/30ug (full dose) of BNT162b2, administered intra-muscularly at a three-week interval. The primary endpoint was the geometric mean ratio (GMR) of serum SARS-CoV-2 anti-RBD IgG titres at 28 days post second dose between the reduced and the full dose regimens. The reduced dose was considered non-inferior to the full dose if the lower limit of the two-sided 95% CI of the GMR was greater than 0.67. The primary analysis was done on the per-protocol population, including infection naive participants only. Findings Between April 19 and April 23, 2021, 145 participants were enrolled in the study and randomized, of whom 141 were vaccinated and reached the primary endpoint. Participants were mostly female (69.5%), of European origin (95%), with a mean age of 40.4 years (SD 7.9). At 28 days post second dose, the geometric mean titre (GMT) of SARS-CoV-2 anti-RBD IgG of the reduced dose regimen (1,705 BAU/mL) was not non-inferior to the full dose regimen (2,387 BAU/mL), with a GMR of 0.714 (two-sided 95% CI 0.540-0.944). No serious adverse events occurred. Conclusions While non-inferiority of the reduced dose regimen was not demonstrated, the SARS-CoV-2 anti-RBD IgG titre was only moderately lower than that of the full dose regimen and, importantly, still markedly higher than the reported antibody response to the licensed adenoviral vector vaccines. These data suggest that reduced doses of the BNT162b2 mRNA vaccine may offer additional benefit as compared to the vaccines currently in use in most low and middle-income countries, warranting larger immunogenicity and effectiveness trials. The trial is registered at ClinicalTrials.gov (NCT04852861).

Three doses of the BNT162b2 vaccine confer neutralising antibody capacity against the SARS-CoV-2 B.1.1.529 (Omicron) variant of concern (preprint)

We report the levels of neutralising antibodies against Wuhan, Delta and Omicron variants in healthy individuals pre-infected or not with SARS-CoV-2 and immunized with three doses of the BNT162b2 vaccine. Our observations support the rapid administration of a booster vaccine dose to prevent infection and disease caused by Omicron.

PREVALENCE AND INCIDENCE OF ANTIBODIES AGAINST SARS-COV-2 AMONG PRIMARY HEALTHCARE PROVIDERS IN BELGIUM DURING ONE YEAR OF THE COVID-19 EPIDEMIC: PROSPECTIVE COHORT STUDY PROTOCOL (preprint)

Introduction National severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence data provides essential information about population exposure to the virus and helps predict the future course of the epidemic. Early cohort studies have suggested declines in levels of antibodies in individuals, associated with, for example, illness severity, age and co-morbidities. This protocol focuses on the seroprevalence among primary health care providers (PHCPs) in Belgium. They manage the vast majority of COVID-19 patients in addition to other patients and therefore play an essential role in the efficient organisation of health care. Currently, evidence is lacking on 1. how many PHCPs get infected with SARS-CoV-2 in Belgium, 2. the rate at which this happens, 3. their clinical spectrum, 4. their risk factors, 5. the effectiveness of the measures to prevent infection and 6. the accuracy of the serology-based point-of-care test in a primary care setting. Methods and analysis This study will be set up as a prospective cohort study. General practitioners (GPs) and other PHCPs (working in a GP practice) will be recruited via professional networks and professional media outlets to register online to participate. Registered GPs and other PHCPs will be asked at each testing point (n=9) to perform a capillary blood sample antibody point-of-care test (OrientGene) and complete an online questionnaire. The primary outcomes are the prevalence and incidence of antibodies against SARS-CoV-2 in PHCPs during a 12-month follow-up period. Secondary outcomes include the longevity of antibodies against SARS-CoV-2. Ethics and dissemination: Ethical approval has been granted by the Ethics Committee of the University Hospital Antwerp/University of Antwerp (Belgian registration number: 3002020000237). Alongside journal publications, dissemination activities include the publication of monthly reports to be shared with the participants and the general population through the publicly available website of the Belgian health authorities (Sciensano).

Poor antibody response to BioNTech/Pfizer COVID-19 vaccination in SARS-CoV-2 naïve residents of nursing homes (preprint)

Background Residents of nursing homes (NH) are at high risk of COVID-19 related morbidity and death and may respond poorly to vaccination because of old age and frequent comorbidities. Methods Forty residents and forty staff members either naive or previously infected with SARS-CoV-2 were recruited in two NH in Belgium before immunization with two doses of 30g BNT162b2 mRNA vaccine at day 0 and day 21. Binding antibodies (Ab) to SARS-CoV-2 receptor binding domain (RBD), spike domains S1 and S2, RBD Ab avidity, and neutralizing Ab against SARS-CoV-2 wild type and B.1.351 variant were assessed at days 0, 21, 28, and 49. Results SARS-CoV-2 naive residents had lower Ab responses to BNT162b2 mRNA vaccination than naive staff. These poor responses involved lower levels of IgG to all domains of the vaccine antigen, lower avidity of RBD IgG, and lower levels of Ab neutralizing the vaccine strain. No naive resident had detectable neutralizing Ab to the B.1.351 variant. High and comparable Ab responses were observed in residents and staff previously infected with SARS-CoV-2. Clustering analysis revealed that poor vaccine responders not only included naive residents but also naive staff, emphasizing the heterogeneity of responses to mRNA vaccination in the general population. Conclusions The poor Ab responses to mRNA vaccination observed in infection naive residents and in some naive staff members of NH suggest suboptimal protection against breakthrough infection, especially with variants of concern. Adapted vaccination regimens may be needed to provide optimal protection against COVID-19 to vulnerable populations.

Outlining the Prior Infection with SARS-CoV-2 study (PICOV) - preliminary findings on symptoms in nursing home residents and staff. (preprint)

Background: COVID-19 has presented itself as one of the most important health concerns of 2020. The geriatric population is arguably hit the hardest by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The ”Prior Infection with SARS-COV-2” (PICOV) study investigates both residents and staff members from nursing homes. The primary aim of the study is to compare the time to occurrence of an influenza-like illness (ILI) or acute respiratory infection (ARI) between participants with a confirmed past SARS-CoV-2 infection and those without an infection. This paper details the study design, sampling scheme, biological measurements, and population characteristics at baseline.Methods: In 26 Belgian nursing homes, all eligible residents and staff members with or without a past SARS-CoV-2 infection (ratio 40/60) were invited to participate. Consent was obtained from 1,375 participants and 1,226 completed the baseline questionnaire. Prevalence of symptoms during a prior SARS-CoV-2 infection was compared between residents and staff members with χ2 statistics.Results: Nursing home residents (both with and without a prior SARS-CoV-2 infection) systematically reported fewer symptoms than staff members. Moreover, results from prior nasopharyngeal RT-qPCR and baseline serology show that antibody development after a SARS-CoV-2 infection differs between residents and staff members.Conclusions: We can postulate that disease development and symptoms is different between a geriatric and younger population. Therefore, the occurrence and severity of a future ILI and/or ARI might vary from resident to staff.

Age-dependent seroprevalence of SARS-CoV-2 antibodies in school-aged children from areas with low and high community transmission (preprint)

BackgroundIt is not yet clear to what extent SARS-CoV-2 infection rates in children reflect community transmission, nor whether infection rates differ between primary schoolchildren and young teenagers.MethodsA cross-sectional serosurvey compared the SARS-CoV2 attack-rate in a sample of 362 children recruited from September 21 to October 6, 2020 in primary (ages 6-12) or lower secondary school (ages 12-15) in a municipality with low community transmission (Pelt) to a municipality with high community transmission (Alken) in Belgium. Children were equally distributed over grades and regions. Blood samples were tested for the presence of antibodies to SARS-CoV-2 with an enzyme-linked immunosorbent assay. ResultsWe found anti-SARS-CoV-2 antibodies in 4.4% of children in the low transmission region and in 14.4% of children in the high transmission region. None of the primary schoolchildren were seropositive in the low transmission region, whereas the seroprevalence among primary and secondary schoolchildren did not differ significantly in the high transmission region. None of the seropositive children suffered from severe disease. Children who were in contact with a confirmed case (RR: 3.8; 95%CI: 1.7 – 8.3), who participated in extracurricular activities (RR: 5.6; 95%CI: 1.2 – 25.3) or whose caregiver is a healthcare worker who had contact with COVID-19 patients (RR: 2.2; 95%CI: 1.0 – 4.6), were at higher risk of seropositivity.ConclusionIf SARS-CoV2 circulation in the community is high, this will be reflected in the pediatric population with similar infection rates in children aged 6-12 years and 12-15 years. 

SARS-CoV-2 Prevalence and Seroprevalence among Healthcare Workers in Belgian Hospitals: Baseline Results of a Prospective Cohort Study (preprint)

Background: Given the current SARS-CoV-2 pandemic and the occurrence of a second wave, assessing the burden of disease among health care workers (HCWs) is crucial. We aim to document the prevalence of SARS-CoV-2 and the seroprevalence of anti-SARS-CoV-2 IgG among HCWs in Belgian hospitals, and to study potential risk factors for the infection in order to guide infection prevention and control (IPC) measures in healthcare institutions. Methods: We performed a cross-sectional analysis of the baseline results (April 22 - April 26) of an ongoing cohort study. All staff who were present in the hospital during the sampling period and whose profession involved contact with patients were eligible. Fourteen hospitals across Belgium and 50 HCW per hospital were randomly selected. RT-qPCR was performed to detect SARS-CoV-2 RNA on nasopharyngeal swabs, and a semi-quantitative IgG ELISA was used to detect anti-SARS-CoV-2 antibodies in sera. Individual characteristics likely to be associated with seropositivity were collected using an online questionnaire. Findings: 698 participants completed the questionnaire; 80.8% were women, median age was 39.5, and 58.5% were nurses. Samples were collected on all 699 participants. The weighted anti-SARS-CoV-2 IgG seroprevalence was 7.7% (95%CI, 4.7%-12.2%), while 1.1% (95%CI, 0.4%-3.0%) of PCR results were positive. Unprotected contact with a confirmed case was the only factor associated with seropositivity (PR 2.16, 95% CI, 1.4-3.2). Interpretation: Most Belgian HCW did not show evidence of SARS-CoV-2 infection by late April 2020, and unprotected contact was the most important risk factor. This confirms the importance of widespread availability of protective equipment and use of adequate IPC measures in hospital settings.

Evaluating SARS-CoV-2 spike and nucleocapsid proteins as targets for IgG antibody detection in severe and mild COVID-19 cases using a Luminex bead-based assay (preprint)

Large-scale serosurveillance of severe acute respiratory syndrome coronavirus type 2 (SARS- CoV-2) will only be possible if serological tests are sufficiently reliable, rapid and inexpensive. Current assays are either labour-intensive and require specialised facilities (e.g. virus neutralization assays), or expensive with suboptimal specificity (e.g. commercial ELISAs). Bead-based assays offer a cost-effective alternative and allow for multiplexing to test for antibodies of other pathogens. Here, we compare the performance of four antigens for the detection of SARS-CoV-2 specific IgG antibodies in a panel of sera that includes both severe (n=40) and mild (n=52) cases, using a neutralization and a Luminex bead-based assay. While we show that neutralising antibody levels are significantly lower in mild than in severe cases, we demonstrate that a combination of recombinant nucleocapsid protein (NP), receptor- binding domain (RBD) and the whole spike protein (S1S2) results in a highly sensitive (96%) and specific (99%) bead-based assay that can detect IgG antibodies in both groups. Although S1-specific IgG levels correlate most strongly with neutralizing antibody levels, they fall below the detection threshold in 10% of the cases in our Luminex assay. In conclusion, our data supports the use of RBD, NP and S1S2 for the development of SARS-CoV-2 serological bead- based assays. Finally, we argue that low antibody levels in mild/asymptomatic cases might complicate the epidemiological assessment of large-scale surveillance studies.