The Impact of First-Time SARS-CoV-2 Infection on Human Anelloviruses.

Members of the Anelloviridae family dominate the blood virome, emerging early in life. The anellome, representing the variety of anelloviruses within an individual, stabilizes by adulthood. Despite their supposedly commensal nature, elevated anellovirus concentrations under immunosuppressive treatment indicate an equilibrium controlled by immunity. Here, we investigated whether anelloviruses are sensitive to the immune activation that accompanies a secondary infection. As a model, we investigated 19 health care workers (HCWs) with initial SARS-CoV-2 infection, with blood sampling performed pre and post infection every 4 weeks in a 3-month-follow-up during the early 2020 COVID-19 pandemic. A concurrently followed control group (n = 27) remained SARS-CoV-2-negative. Serum anellovirus loads were measured using qPCR. A significant decrease in anellovirus load was found in the first weeks after SARS-CoV-2 infection, whereas anellovirus concentrations remained stable in the uninfected control group. A restored anellovirus load was seen approximately 10 weeks after SARS-CoV-2 infection. For five subjects, an in-time anellome analysis via Illumina sequencing could be performed. In three of the five HCWs, the anellome visibly changed during SARS-CoV-2 infection and returned to baseline in two of these cases. In conclusion, anellovirus loads in blood can temporarily decrease upon an acute secondary infection.

Broad SARS-CoV-2 neutralization by monoclonal and bispecific antibodies derived from a Gamma-infected individual.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has remained a medical threat due to the evolution of multiple variants that acquire resistance to vaccines and prior infection. Therefore, it is imperative to discover monoclonal antibodies (mAbs) that neutralize a broad range of SARS-CoV-2 variants. A stabilized spike glycoprotein was used to enrich antigen-specific B cells from an individual with a primary Gamma variant infection. Five mAbs selected from those B cells showed considerable neutralizing potency against multiple variants, with COVA309-35 being the most potent against the autologous virus, as well as Omicron BA.1 and BA.2, and COVA309-22 having binding and neutralization activity against Omicron BA.4/5, BQ.1.1, and XBB.1. When combining the COVA309 mAbs as cocktails or bispecific antibodies, the breadth and potency were improved. In addition, the mechanism of cross-neutralization of the COVA309 mAbs was elucidated by structural analysis. Altogether these data indicate that a Gamma-infected individual can develop broadly neutralizing antibodies.

Virological and immunological correlates of HIV posttreatment control after temporal antiretroviral therapy during acute HIV infection.

OBJECTIVE: People with HIV rarely control viral replication after cessation of antiretroviral therapy (ART). We present a person with HIV with extraordinary posttreatment control (PTC) for over 23 years after temporary ART during acute HIV infection (AHI) leading to a new insight in factors contributing to PTC. DESIGN/METHODS: Viral reservoir was determined by HIV qPCR, Intact Proviral DNA Assay, and quantitative viral outgrowth assay. Viral replication kinetics were determined in autologous and donor PBMC. IgG levels directed against HIV envelope and neutralizing antibodies were measured. Immune phenotyping of T cells and HIV-specific T-cell responses were analyzed by flow cytometry. RESULTS: The case presented with AHI and a plasma viral load of 2.7 million copies/ml. ART was initiated 2 weeks after diagnosis and interrupted after 26 months. Replicating virus was isolated shortly after start ART. At 18 years after treatment interruption, HIV-DNA in CD4 + T cells and low levels of HIV-RNA in plasma (<5 copies/ml) were detectable. Stable HIV envelope glycoprotein-directed IgG was present during follow-up, but lacked neutralizing activity. Strong antiviral CD8 + T-cell responses, in particular targeting HIV-gag, were detected during 25 years follow-up. Moreover, we found a P255A mutation in an HLA-B∗44 : 02 restricted gag-epitope, which was associated with decreased replication. CONCLUSION: We describe an exceptional case of PTC, which is likely associated with sustained potent gag-specific CD8 + T-cell responses in combination with a replication attenuating escape mutation in gag. Understanding the initiation and preservation of the HIV-specific T-cell responses could guide the development of strategies to induce HIV control.

Discriminating cross-reactivity in polyclonal IgG1 responses against SARS-CoV-2 variants of concern.

Existing assays to measure antibody cross-reactivity against different SARS-CoV-2 spike (S) protein variants lack the discriminatory power to provide insights at the level of individual clones. Using a mass spectrometry-based approach we are able to monitor individual donors' IgG1 clonal responses following a SARS-CoV-2 infection. We monitor the plasma clonal IgG1 profiles of 8 donors who had experienced an infection by either the wild type Wuhan Hu-1 virus or one of 3 VOCs (Alpha, Beta and Gamma). In these donors we chart the full plasma IgG1 repertoires as well as the IgG1 repertoires targeting the SARS-CoV-2 spike protein trimer VOC antigens. The plasma of each donor contains numerous anti-spike IgG1 antibodies, accounting for <0.1% up to almost 10% of all IgG1s. Some of these antibodies are VOC-specific whereas others do recognize multiple or even all VOCs. We show that in these polyclonal responses, each clone exhibits a distinct cross-reactivity and also distinct virus neutralization capacity. These observations support the need for a more personalized look at the antibody clonal responses to infectious diseases.

Broad SARS-CoV-2 Neutralization by Monoclonal and Bispecific Antibodies Derived from a Gamma-infected Individual.

The worldwide pandemic caused by SARS-CoV-2 has remained a human medical threat due to the continued evolution of multiple variants that acquire resistance to vaccines and prior infection. Therefore, it is imperative to discover monoclonal antibodies (mAbs) that neutralize a broad range of SARS-CoV-2 variants for therapeutic and prophylactic use. A stabilized autologous SARS-CoV-2 spike glycoprotein was used to enrich antigen-specific B cells from an individual with a primary Gamma variant infection. Five mAbs selected from those B cells showed considerable neutralizing potency against multiple variants of concern, with COVA309-35 being the most potent against the autologous virus, as well as against Omicron BA.1 and BA.2. When combining the COVA309 mAbs as cocktails or bispecific antibody formats, the breadth and potency was significantly improved against all tested variants. In addition, the mechanism of cross-neutralization of the COVA309 mAbs was elucidated by structural analysis. Altogether these data indicate that a Gamma-infected individual can develop broadly neutralizing antibodies.

Recent infection with HCoV-OC43 may be associated with protection against SARS-CoV-2 infection.

Antibodies against seasonal human coronaviruses (HCoVs) are known to cross-react with SARS-CoV-2, but data on cross-protective effects of prior HCoV infections are conflicting. In a prospective cohort of healthcare workers (HCWs), we studied the association between seasonal HCoV (OC43, HKU1, 229E and NL63) nucleocapsid protein IgG and SARS-CoV-2 infection during the first pandemic wave in the Netherlands (March 2020 - June 2020), by 4-weekly serum sampling. HCW with HCoV-OC43 antibody levels in the highest quartile, were less likely to become SARS-CoV-2 seropositive when compared with those with lower levels (6/32, 18.8%, versus 42/97, 43.3%, respectively: p = 0.019; HR 0.37, 95% CI 0.16-0.88). We found no significant association with HCoV-OC43 spike protein IgG, or with antibodies against other HCoVs. Our results indicate that the high levels of HCoV-OC43-nucleocapsid antibodies, as an indicator of a recent infection, are associated with protection against SARS-CoV-2 infection; this supports and informs efforts to develop pancoronavirus vaccines.

Antigenic cartography using sera from sequence-confirmed SARS-CoV-2 variants of concern infections reveals antigenic divergence of Omicron.

Large-scale vaccination campaigns have prevented countless hospitalizations and deaths due to COVID-19. However, the emergence of SARS-CoV-2 variants that escape from immunity challenges the effectiveness of current vaccines. Given this continuing evolution, an important question is when and how to update SARS-CoV-2 vaccines to antigenically match circulating variants, similarly to seasonal influenza viruses where antigenic drift necessitates periodic vaccine updates. Here, we studied SARS-CoV-2 antigenic drift by assessing neutralizing activity against variants of concern (VOCs) in a set of sera from patients infected with viral sequence-confirmed VOCs. Infections with D614G or Alpha strains induced the broadest immunity, whereas individuals infected with other VOCs had more strain-specific responses. Omicron BA.1 and BA.2 were substantially resistant to neutralization by sera elicited by all other variants. Antigenic cartography revealed that Omicron BA.1 and BA.2 were antigenically most distinct from D614G, associated with immune escape, and possibly will require vaccine updates to ensure vaccine effectiveness.

A public antibody class recognizes an S2 epitope exposed on open conformations of SARS-CoV-2 spike.

Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigate the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We show that ∼82% of SARS-CoV-2 S-reactive B cells harbor a naive phenotype, which represents an unusually high fraction of total human naive B cells (∼0.1%). Approximately 10% of these naive S-reactive B cells share an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.

The effect of SARS-CoV-2 vaccination on post-acute sequelae of COVID-19 (PASC): A prospective cohort study.

BACKGROUND: Symptoms of post-acute sequelae of COVID-19 (PASC) may improve following SARS-CoV-2 vaccination. However few prospective data that also explore the underlying biological mechanism are available. We assessed the effect of vaccination on symptomatology of participants with PASC, and compared antibody dynamics between those with and without PASC. METHODS: RECoVERED is a prospective cohort study of adult patients with mild to critical COVID-19, enrolled from illness onset. Among participants with PASC, vaccinated participants were exact-matched 1:1 on age, sex, obesity status and time since illness onset to unvaccinated participants. Between matched pairs, we compared the monthly mean numbers of symptoms over a 3-month follow-up period, and, using exact logistic regression, the proportion of participants who fully recovered from PASC. Finally, we assessed the association between PACS status and rate of decay of spike- and RBD-binding IgG titers up to 9 months after illness onset using Bayesian hierarchical linear regression. FINDINGS: Of 349 enrolled participants, 316 (90.5%) had ≥3 months of follow-up, of whom 186 (58.9%) developed PASC. Among 36 matched pairs with PASC, the mean number of symptoms reported each month during 3 months of follow-up were comparable between vaccinated and unvaccinated groups. Odds of full recovery from PASC also did not differ between matched pairs (OR 1.57 [95%CI 0.46-5.84]) within 3 months after the matched time-point. The median half-life of spike- and RBD-binding IgG levels were, in days (95%CrI), 233 (183-324) and 181 (147-230) among participants with PASC, and 170 (125-252) and 144 (113-196) among those without PASC, respectively. INTERPRETATION: Our study found no strong evidence to suggest that vaccination improves symptoms of PASC. This was corroborated by comparable spike- and RBD-binding IgG waning trajectories between those with and without PASC, refuting any immunological basis for a therapeutic effect of vaccination on PASC.

Decreased Passive Immunity to Respiratory Viruses through Human Milk during the COVID-19 Pandemic.

Infants may develop severe viral respiratory tract infections because their immune system is still developing in the first months after birth. Human milk provides passive humoral immunity during the first months of life. During the COVID-19 pandemic, circulation of common respiratory viruses was virtually absent due to the preventative measures resulting in reduced maternal exposure. Therefore, we hypothesized that this might result in lower antibody levels in human milk during the pandemic and, subsequently, decreased protection of infants against viral respiratory tract infections. We assessed antibody levels against respiratory syncytial virus (RSV), Influenza virus, and several seasonal coronaviruses in different periods of the COVID-19 pandemic in serum and human milk using a Luminex assay. IgG levels against RSV, Influenza, HCoV-OC43, HCoV-HKU1, and HCoV-NL63 in human milk were reduced with a factor of 1.7 (P < 0.001), 2.2 (P < 0.01), 2.6 (P < 0.05), 1.4 (P < 0.01), and 2.1 (P < 0.001), respectively, since the introduction of the COVID-19 restrictions. Furthermore, we observed that human milk of mothers that experienced COVID-19 contained increased levels of IgG and IgA binding to other respiratory viruses. Passive immunity via human milk against common respiratory viruses was reduced during the COVID-19 pandemic, which may have consequences for the protection of breastfed infants against respiratory infections. IMPORTANCE Passive immunity derived from antibodies in human milk is important for protecting young infants against invading viruses. During the COVID-19 pandemic, circulation of common respiratory viruses was virtually absent due to preventative measures. In this study, we observed a decrease in human milk antibody levels against common respiratory viruses several months into the COVID-19 pandemic. This waning of antibody levels might partially explain the previously observed surge of hospitalizations of infants, mostly due to RSV, when preventative hygiene measures were lifted. Knowledge of the association between preventative measures, antibody levels in human milk and subsequent passive immunity in infants might help predict infant hospital admissions and thereby enables anticipation to prevent capacity issues. Additionally, it is important in the consideration for strategies for future lockdowns to best prevent possible consequences for vulnerable infants.

Antibody responses against SARS-CoV-2 variants induced by four different SARS-CoV-2 vaccines in health care workers in the Netherlands: A prospective cohort study.

BACKGROUND: Emerging and future SARS-CoV-2 variants may jeopardize the effectiveness of vaccination campaigns. Therefore, it is important to know how the different vaccines perform against diverse SARS-CoV-2 variants. METHODS AND FINDINGS: In a prospective cohort of 165 SARS-CoV-2 naive health care workers in the Netherlands, vaccinated with either one of four vaccines (BNT162b2, mRNA-1273, AZD1222 or Ad26.COV2.S), we performed a head-to-head comparison of the ability of sera to recognize and neutralize SARS-CoV-2 variants of concern (VOCs; Alpha, Beta, Gamma, Delta and Omicron). Repeated serum sampling was performed 5 times during a year (from January 2021 till January 2022), including before and after booster vaccination with BNT162b2. Four weeks after completing the initial vaccination series, SARS-CoV-2 wild-type neutralizing antibody titers were highest in recipients of mRNA-1273, followed by recipients of BNT162b2 (geometric mean titers (GMT) of 358 [95% CI 231-556] and 214 [95% CI 153-299], respectively; p<0.05), and substantially lower in those vaccinated with the adenovirus vector-based vaccines AZD1222 and Ad26.COV2.S (GMT of 18 [95% CI 11-30] and 14 [95% CI 8-25] IU/ml, respectively; p<0.001). VOCs neutralization was reduced in all vaccine groups, with the greatest reduction in neutralization GMT observed against the Omicron variant (fold change 0.03 [95% CI 0.02-0.04], p<0.001). The booster BNT162b2 vaccination increased neutralizing antibody titers for all groups with substantial improvement against the VOCs including the Omicron variant. We used linear regression and linear mixed model analysis. All results were adjusted for possible confounding of age and sex. Study limitations include the lack of cellular immunity data. CONCLUSIONS: Overall, this study shows that the mRNA vaccines appear superior to adenovirus vector-based vaccines in inducing neutralizing antibodies against VOCs four weeks after initial vaccination and after booster vaccination, which implies the use of mRNA vaccines for both initial and booster vaccination.

Optimization of Anti-SARS-CoV-2 Neutralizing Antibody Therapies: Roadmap to Improve Clinical Effectiveness and Implementation.

One of the major breakthroughs to combat the current Coronavirus Disease 2019 (COVID-19) pandemic has been the development of highly effective vaccines against the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Still, alternatives are needed for individuals who are at high risk of developing severe COVID-19 and are not protected by vaccination. Monoclonal antibodies against the spike protein of SARS-CoV-2 have been shown to be effective as prophylaxis and treatment against COVID-19. However, the emergence of variants of concern (VOCs) challenges the efficacy of antibody therapies. This review describes the neutralization resistance of the clinically-approved monoclonal antibody therapies against the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P1), Delta (B.1.617.2), and the Omicron (B.1.1.529) variants. To guide the development of monoclonal antibody therapies and to anticipate on the continuous evolution of SARS-CoV-2, we highlight different strategies to broaden the antibody activity by targeting more conserved epitopes and/or simultaneously targeting multiple sites of vulnerability of the virus. This review further describes the contribution of antibody Fc effector functions to optimize the antibody efficacy. In addition, the main route of SARS-CoV-2 antibody administration is currently intravenously and dictates a monthly injection when used as prophylactic. Therefore, we discusses the concept of long-acting antibodies (LAABs) and non-intravenously routes of antibody administration in order to broaden the clinical applicability of antibody therapies.

Immunogenicity of the mRNA-1273 COVID-19 vaccine in adult patients with inborn errors of immunity.

BACKGROUND: Patients with inborn errors of immunity (IEI) are at increased risk of severe coronavirus disease-2019 (COVID-19). Effective vaccination against COVID-19 is therefore of great importance in this group, but little is known about the immunogenicity of COVID-19 vaccines in these patients. OBJECTIVES: We sought to study humoral and cellular immune responses after mRNA-1273 COVID-19 vaccination in adult patients with IEI. METHODS: In a prospective, controlled, multicenter study, 505 patients with IEI (common variable immunodeficiency [CVID], isolated or undefined antibody deficiencies, X-linked agammaglobulinemia, combined B- and T-cell immunodeficiency, phagocyte defects) and 192 controls were included. All participants received 2 doses of the mRNA-1273 COVID-19 vaccine. Levels of severe acute respiratory syndrome coronavirus-2-specific binding antibodies, neutralizing antibodies, and T-cell responses were assessed at baseline, 28 days after first vaccination, and 28 days after second vaccination. RESULTS: Seroconversion rates in patients with clinically mild antibody deficiencies and phagocyte defects were similar to those in healthy controls, but seroconversion rates in patients with more severe IEI, such as CVID and combined B- and T-cell immunodeficiency, were lower. Binding antibody titers correlated well to the presence of neutralizing antibodies. T-cell responses were comparable to those in controls in all IEI cohorts, with the exception of patients with CVID. The presence of noninfectious complications and the use of immunosuppressive drugs in patients with CVID were negatively correlated with the antibody response. CONCLUSIONS: COVID-19 vaccination with mRNA-1273 was immunogenic in mild antibody deficiencies and phagocyte defects and in most patients with combined B- and T-cell immunodeficiency and CVID. Lowest response was detected in patients with X-linked agammaglobulinemia and in patients with CVID with noninfectious complications. The assessment of longevity of immune responses in these vulnerable patient groups will guide decision making for additional vaccinations.

A single mRNA vaccine dose in COVID-19 patients boosts neutralizing antibodies against SARS-CoV-2 and variants of concern.

The urgent need for, but limited availability of, SARS-CoV-2 vaccines worldwide has led to widespread consideration of dose-sparing strategies. Here, we evaluate the SARS-CoV-2-specific antibody responses following BNT162b2 vaccination in 150 previously SARS-CoV-2-infected individuals from a population-based cohort. One week after first vaccine dose, spike protein antibody levels are 27-fold higher and neutralizing antibody titers 12-fold higher, exceeding titers of fully vaccinated SARS-CoV-2-naive controls, with minimal additional boosting after the second dose. Neutralizing antibody titers against four variants of concern increase after vaccination; however, overall neutralization breadth does not improve. Pre-vaccination neutralizing antibody titers and time since infection have the largest positive effect on titers following vaccination. COVID-19 severity and the presence of comorbidities have no discernible impact on vaccine response. In conclusion, a single dose of BNT162b2 vaccine up to 15 months after SARS-CoV-2 infection offers higher neutralizing antibody titers than 2 vaccine doses in SARS-CoV-2-naive individuals.

Cross-reactive antibodies after SARS-CoV-2 infection and vaccination.

Current SARS-CoV-2 vaccines are losing efficacy against emerging variants and may not protect against future novel coronavirus outbreaks, emphasizing the need for more broadly protective vaccines. To inform the development of a pan-coronavirus vaccine, we investigated the presence and specificity of cross-reactive antibodies against the spike (S) proteins of human coronaviruses (hCoV) after SARS-CoV-2 infection and vaccination. We found an 11- to 123-fold increase in antibodies binding to SARS-CoV and MERS-CoV as well as a 2- to 4-fold difference in antibodies binding to seasonal hCoVs in COVID-19 convalescent sera compared to pre-pandemic healthy donors, with the S2 subdomain of the S protein being the main target for cross-reactivity. In addition, we detected cross-reactive antibodies to all hCoV S proteins after SARS-CoV-2 vaccination in macaques and humans, with higher responses for hCoV more closely related to SARS-CoV-2. These findings support the feasibility of and provide guidance for development of a pan-coronavirus vaccine.

Saliva SARS-CoV-2 Antibody Prevalence in Children.

COVID-19 patients produce circulating and mucosal antibodies. In adults, specific saliva antibodies have been detected. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We therefore assessed SARS-CoV-2-specific antibody prevalence in serum and saliva in children in the Netherlands. We assessed SARS-CoV-2 antibody prevalence in serum and saliva of 517 children attending medical services in the Netherlands (irrespective of COVID-19 exposure) from April to October 2020. The prevalence of SARS-CoV-2 spike (S), receptor binding domain (RBD), and nucleocapsid (N)-specific IgG and IgA were evaluated with an exploratory Luminex assay in serum and saliva and with the Wantai SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay in serum. Using the Wantai assay, the RBD-specific antibody prevalence in serum was 3.3% (95% confidence interval [CI]. 1.9 to 5.3%). With the Luminex assay, we detected heterogeneity between antibodies for S, RBD, and N antigens, as IgG and IgA prevalence ranged between 3.6 and 4.6% in serum and between 0 and 4.4% in saliva. The Luminex assay also revealed differences between serum and saliva, with SARS-CoV-2-specific IgG present in saliva but not in serum for 1.5 to 2.7% of all children. Using multiple antigen assays, the IgG prevalence for at least two out of three antigens (S, RBD, or N) in serum or saliva can be calculated as 3.8% (95% CI, 2.3 to 5.6%). Our study displays the heterogeneity of the SARS-CoV-2 antibody response in children and emphasizes the additional value of saliva antibody detection and the combined use of different antigens. IMPORTANCE Comprehending humoral immunity to SARS-CoV-2, including in children, is crucial for future public health and vaccine strategies. Others have suggested that mucosal antibody measurement could be an important and more convenient tool to evaluate humoral immunity compared to circulating antibodies. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We show the heterogeneity of SARS-CoV-2 antibodies, in terms of both antigen specificity and differences between circulating and mucosal antibodies, emphasizing the additional value of saliva antibody detection next to detection of antibodies in serum.

Emerging SARS-CoV-2 variants of concern evade humoral immune responses from infection and vaccination.

Emerging SARS-CoV-2 variants of concern (VOCs) pose a threat to human immunity induced by natural infection and vaccination. We assessed the recognition of three VOCs (B.1.1.7, B.1.351, and P.1) in cohorts of COVID-19 convalescent patients (n = 69) and Pfizer-BioNTech vaccine recipients (n = 50). Spike binding and neutralization against all three VOCs were substantially reduced in most individuals, with the largest four- to sevenfold reduction in neutralization being observed against B.1.351. While hospitalized patients with COVID-19 and vaccinees maintained sufficient neutralizing titers against all three VOCs, 39% of nonhospitalized patients exhibited no detectable neutralization against B.1.351. Moreover, monoclonal neutralizing antibodies show sharp reductions in their binding kinetics and neutralizing potential to B.1.351 and P.1 but not to B.1.1.7. These data have implications for the degree to which pre-existing immunity can protect against subsequent infection with VOCs and informs policy makers of susceptibility to globally circulating SARS-CoV-2 VOCs.

Time since SARS-CoV-2 infection and humoral immune response following BNT162b2 mRNA vaccination.

BACKGROUND: To optimise the use of available SARS-CoV-2 vaccines, some advocate delaying second vaccination for individuals infected within six months. We studied whether post-vaccination immune response is equally potent in individuals infected over six months prior to vaccination. METHODS: We tested serum IgG binding to SARS-CoV-2 spike protein and neutralising capacity in 110 healthcare workers, before and after both BNT162b2 messenger RNA (mRNA) vaccinations. We compared outcomes between participants with more recent infection (n = 18, median two months, IQR 2-3), with infection-vaccination interval over six months (n = 19, median nine months, IQR 9-10), and to those not previously infected (n = 73). FINDINGS: Both recently and earlier infected participants showed comparable humoral immune responses after a single mRNA vaccination, while exceeding those of previously uninfected persons after two vaccinations with 2.5 fold (p = 0.003) and 3.4 fold (p < 0.001) for binding antibody levels, and 6.4 and 7.2 fold for neutralisation titres, respectively (both p < 0.001). The second vaccine dose yielded no further substantial improvement of the humoral response in the previously infected participants (0.97 fold, p = 0.92), while it was associated with a 4 fold increase in antibody binding levels and 18 fold increase in neutralisation titres in previously uninfected participants (both p < 0.001). Adjustment for potential confounding of sex and age did not affect these findings. INTERPRETATION: Delaying the second vaccination in individuals infected up to ten months prior may constitute a more efficient use of limited vaccine supplies. FUNDING: Netherlands Organization for Health Research and Development ZonMw; Corona Research Fund Amsterdam UMC; Bill & Melinda Gates Foundation.

Human Milk from Previously COVID-19-Infected Mothers: The Effect of Pasteurization on Specific Antibodies and Neutralization Capacity.

BACKGROUND: Since the outbreak of coronavirus disease 2019 (COVID-19), many put their hopes in the rapid availability of effective immunizations. Human milk, containing antibodies against syndrome coronavirus 2 (SARS-CoV-2), may serve as means of protection through passive immunization. We aimed to determine the presence and pseudovirus neutralization capacity of SARS-CoV-2 specific IgA in human milk of mothers who recovered from COVID-19, and the effect of pasteurization on these antibodies. METHODS: This prospective case control study included lactating mothers, recovered from (suspected) COVID-19 and healthy controls. Human milk and serum samples were collected. To assess the presence of SARS-CoV-2 antibodies we used multiple complementary assays, namely ELISA with the SARS-CoV-2 spike protein (specific for IgA and IgG), receptor binding domain (RBD) and nucleocapsid (N) protein for IgG in serum, and bridging ELISA with the SARS-CoV-2 RBD and N protein for specific Ig (IgG, IgM and IgA in human milk and serum). To assess the effect of pasteurization, human milk was exposed to Holder (HoP) and High Pressure Pasteurization (HPP). RESULTS: Human milk contained abundant SARS-CoV-2 antibodies in 83% of the proven cases and in 67% of the suspected cases. Unpasteurized milk with and without these antibodies was found to be capable of neutralizing a pseudovirus of SARS-CoV-2 in (97% and 85% of the samples respectively). After pasteurization, total IgA antibody levels were affected by HoP, while SARS-CoV-2 specific antibody levels were affected by HPP. Pseudovirus neutralizing capacity of the human milk samples was only retained with the HPP approach. No correlation was observed between milk antibody levels and neutralization capacity. CONCLUSIONS: Human milk from recovered COVID-19-infected mothers contains SARS-CoV-2 specific antibodies which maintained neutralization capacity after HPP. All together this may represent a safe and effective immunization strategy after HPP.

Neutralising Antibodies against Enterovirus and Parechovirus in IVIG Reflect General Circulation: A Tool for Sero-Surveillance.

Non-polio enteroviruses (NPEV) and parechoviruses (PeV) are widespread pathogens that cause significant morbidity. Surveillance is based on culturing or genotyping of virus strains found in clinical samples. Sero-surveillance, by measuring neutralising antibodies (nAb) through virus neutralisation assays (VNA), could provide additional information as it offers a more comprehensive overview of exposure to circulating types in the general population. In our study we evaluated Intravenous immunoglobulins (IVIG) to generate sero-surveillance data. We performed VNA of nineteen NPEV and PeV with Dutch IVIG batches from two different time points (2010 and 2017) and an IVIG batch from Vietnam (2011). We compared our findings with geno- and sero-surveillance data and evaluated changes over time and between the two countries. Our findings show a good correlation with what is known from geno-surveillance data. The highest nAb titres were found against strains from Enterovirus B, while we did not observe nAb titres against strains belonging to Enterovirus C. In conclusion, we demonstrated that sero-surveillance by means of IVIG can be used to obtain insight into circulation of EV and PeV genotypes. This is of particular interest for public health, to evaluate changes over time and population susceptibility to emerging genotypes.