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Abstract Broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope glycoprotein (Env) have the capacity to delay viral rebound when administered to people with HIV-1 (PWH) during anti-retroviral therapy (ART) interruption. To further enhance the performance of bNAbs through their Fc effector functions, in particular NK cell-mediated killing of HIV-1 infected cells, we have produced a panel of glyco-engineered (afucosylated) bNAbs with enhanced affinity for Fc gamma receptor IIIa. These afucosylated anti-HIV-1 bNAbs enhance NK cell activation and degranulation compared to fucosylated counterparts even at low antigen density. NK cells from PWH expressing exhaustion markers PD-1 and TIGIT are activated in a similar fashion by afucosylated bNAbs as NK cell from HIV-1 negative individuals. Killing of HIV-1 infected cells is most effective with afucosylated bNAbs 2G12, N6, PGT151 and PGDM1400, whereas afucosylated PGT121 and non-neutralizing antibody A32 only induce minor NK cell-mediated killing. These data indicate that the approach angle and affinity of Abs influence the capacity to induce antibody-dependent cellular cytotoxicity. Thus, afucosylated bNAbs have the capacity to induce NK cell-mediated killing of infected cells, which warrants further investigation of afucosylated bNAb administration in vivo, aiming for reduction of the viral reservoir and ART free durable control.

Neutralization of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by human sera is a strong correlate of protection against symptomatic and severe Coronavirus Disease 2019 (COVID-19). The emergence of antigenically distinct SARS-CoV-2 variants of concern (VOCs) and the relatively rapid waning of serum antibody titers, however, raises questions about the sustainability of serum protection. In addition to serum neutralization, other antibody functionalities and the memory B cell (MBC) response are suggested to help maintaining this protection. In this study, we investigate the breadth of spike (S) protein-specific serum antibodies that mediate effector functions by interacting with Fc-gamma receptor IIa (FcγRIIa) and FcγRIIIa, and of the receptor binding domain (RBD)-specific MBCs, following a primary SARS-CoV-2 infection with the D614G, Alpha, Beta, Gamma, Delta, Omicron BA.1 or BA.2 variant. Irrespectively of the variant causing the infection, the breadth of S protein-specific serum antibodies that interact with FcγRIIa and FcγRIIIa and the RBD-specific MBC responses exceeded the breadth of serum neutralization, although the Alpha-induced B cell response seemed more strain-specific. Between VOC groups, both quantitative and qualitative differences in the immune responses were observed, suggesting differences in immunogenicity. Overall, this study contributes to the understanding of protective humoral and B cell responses in the light of emerging antigenically distinct VOCs, and highlights the need to study the immune system beyond serum neutralization to gain a better understanding of the protection against emerging variants.

Summary: Zoonotic events by sarbecoviruses have sparked an epidemic (severe acute respiratory syndrome coronavirus [SARS-CoV]) and a pandemic (SARS-CoV-2) in the past two decades. The continued risk of spillovers from animals to humans is an ongoing threat to global health and a pan-sarbecovirus vaccine would be an important contribution to pandemic preparedness. Here, we describe multivalent virosome-based vaccines that present stabilized spike proteins from four sarbecovirus strains, one from each clade. A cocktail of four monovalent virosomes or a mosaic virosome preparation induced broad sarbecovirus binding and neutralizing antibody responses in mice. Pre-existing immunity against SARS-CoV-2 and extending the intervals between immunizations enhanced antibody responses. These results should inform the development of a pan-sarbecovirus vaccine, as part of our efforts to prepare for and/or avoid a next pandemic.

PurposePrevious studies have demonstrated that the majority of patients with an inborn error of immunity (IEI) develop a spike (S)-specific IgG antibody and T-cell response after two doses of the mRNA-1273 COVID-19 vaccine, but little is known about the response to a booster vaccination. We studied the immune responses 8 weeks after booster vaccination with mRNA-based COVID-19 vaccines in 171 IEI patients. Moreover, we evaluated the clinical outcomes in these patients one year after the start of the Dutch COVID-19 vaccination campaign.MethodsThis study was embedded in a large prospective multicenter study investigating the immunogenicity of COVID-19 mRNA-based vaccines in IEI (VACOPID study). Blood samples were taken from 244 participants 8 weeks after booster vaccination. These participants included 171 IEI patients (X-linked agammaglobulinemia (XLA;N=11), combined immunodeficiency (CID;N=4), common variable immunodeficiency (CVID;N=45), isolated or undefined antibody deficiencies (N=108) and phagocyte defects (N=3)) and 73 controls. SARS-CoV-2-specific IgG titers, neutralizing antibodies, and T-cell responses were evaluated. One year after the start of the COVID-19 vaccination program, 334 study participants (239 IEI patients and 95 controls) completed a questionnaire to supplement their clinical data focusing on SARS-CoV-2 infections.ResultsAfter booster vaccination, S-specific IgG titers increased in all COVID-19 naive IEI cohorts and controls, when compared to titers at 6 months after the priming regimen. The fold-increases did not differ between controls and IEI cohorts. SARS-CoV-2-specific T-cell responses also increased equally in all cohorts after booster vaccination compared to 6 months after the priming regimen. Most SARS-CoV-2 infections during the study period occurred in the period when the Omicron variant had become dominant. The clinical course of these infections was mild, although IEI patients experienced more frequent fever and dyspnea compared to controls and their symptoms persisted longer.ConclusionOur study demonstrates that mRNA-based booster vaccination induces robust recall of memory B-cell and T-cell responses in most IEI patients. One-year clinical follow-up demonstrated that SARS-CoV-2 infections in IEI patients were mild. Given our results, we support booster campaigns with newer variant-specific COVID-19 booster vaccines to IEI patients with milder phenotypes.

A vaccine against Hepatitis C virus (HCV) is urgently needed to limit the spread of HCV. The large antigenic diversity of the HCV glycoprotein E1E2 makes it difficult to design a vaccine but also to fully understand the antibody response after infection or vaccination.Here we designed a panel of HCV pseudoparticles (HCVpps) that cover a wide range of genetically and antigenically diverse E1E2s. We validate our panel using neutralization and a binding antibody multiplex assay (BAMA). The panel of HCVpps includes E1E2 glycoproteins from acute and chronically infected cases in the Netherlands, as well as E1E2 glycoproteins from previously reported HCVs.Using eight monoclonal antibodies targeting multiple antigenic regions on E1E2, we could categorize four groups of neutralization sensitive viruses with viruses showing neutralization titers over a 100-fold range. One HCVpp (AMS0230) was extremely neutralization resistant and only neutralized by AR4-targeting antibodies. In addition, using binding antibody multiplex competition assay, we delineated mAb epitopes and their interactions. The binding and neutralization sensitivity of the HCVpps were confirmed using patient sera. At the end, eleven HCVpps with unique antibody binding and neutralization profiles were selected as the final panel for standardized HCV antibody assessments.In conclusion, this HCVpp panel can be used to evaluate antibody binding and neutralization breadth and potency as well as delineate the epitopes targeted in sera from patients or candidate vaccine trials. The HCVpp panel in combination with the established antibody competition assay present highly valuable tools for HCV vaccine development and evaluation.

Objectives: Preventive measures against COVID-19 are essential for pregnant women. Pregnant women are particularly vulnerable to emerging infectious pathogens due to alterations in their physiology. We aimed to determine the optimum timing of vaccination to protect pregnant women and their neonates from COVID-19. Methods: A prospective observational longitudinal cohort study in pregnant women who received COVID-19 vaccination. We collected blood samples to evaluate levels of antispike, receptor binding domain and nucleocapsid antibodies against SARS-CoV-2 before vaccination and 15 days after the first and second vaccination. We determined the neutralizing antibodies from mother-infant dyads in maternal and umbilical cord blood at birth. If available, immunoglobulin A was measured in human milk. Results: We included 178 pregnant women. Median antispike immunoglobulin G levels increased significantly from 1.8 to 5431 binding antibody units/ml and receptor binding domain from 6 to 4466 binding antibody units/ml. Virus neutralization showed similar results between different weeks of gestation at vaccination (P >0.3). Conclusion: We advise vaccination in the early second trimester of pregnancy for the optimum balance between the maternal antibody response and placental antibody transfer to the neonate.

To fully understand the potential shortcomings of SARS-CoV-2 vaccination, it is necessary to delineate the properties of the antibodies elicited, during immunization, and also infection. Through investigation of the SARS-CoV-2 spike-reactive B cell repertoire, authors identify following infection, a subset of B cells enriched and almost exclusively target a non-neutralizing S2 epitope present in aberrant forms.

ABSTRACT Few studies have comprehensively compared severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine-induced and hybrid B- and T-cell responses in people with HIV (PWH) to those in comparable controls without HIV. We included 195 PWH and 246 comparable controls from the AGEhIV COVID-19 substudy. A positive nucleocapsid antibody (INgezim IgA/IgM/IgG) or self-reported PCR test defined prior SARS-CoV-2 infection. SARS-CoV-2 anti-spike (anti-S) IgG titers and anti-S IgG production by memory B cells were assessed. Neutralizing antibody titers were determined in a subset of participants. T-cell responses were assessed by gamma interferon (IFN-γ) release and activation-induced marker assay. We estimated mean differences in postvaccination immune responses (β) between levels of determinants. Anti-S IgG titers and anti-S IgG production by memory B cells were not different between PWH and controls. Prior SARS-CoV-2 infection (β = 0.77), receiving mRNA vaccine (β = 0.56), female sex (β = 0.24), fewer days between last vaccination and sampling (β = 0.07), and a CD4/CD8 ratio of

Abstract Current SARS-CoV-2 vaccines are effective, but long-term protection is threatened by the emergence of virus variants. We generated a virosome vaccine containing the Beta spike protein and compared its immunogenicity in mice to a virosome vaccine containing the original Wuhan spike. Two administrations of the virosomes induced potent SARS-CoV-2 neutralizing antibodies in both vaccine groups. The level of autologous neutralization in Beta-vaccinated mice was similar to the level of autologous neutralization in Wuhan-vaccinated mice. However, heterologous neutralization to the Wuhan strain in Beta-vaccinated mice was 4.7-fold lower than autologous neutralization, whereas heterologous neutralization to the Beta strain in Wuhan-vaccinated mice was reduced by only 1.9-fold compared to autologous neutralization levels. In addition, neutralizing activity against the D614G, Alpha and Delta variants was also significantly lower after Beta spike vaccination than after Wuhan spike vaccination. Our results show that Beta spike vaccination induces inferior neutralization breadth. These results are informative for programs aimed to develop broadly active SARS-CoV-2 vaccines.

Abstract The emergence of SARS-CoV-2 variants that are more resistant to antibody-mediated neutralization pose a new hurdle in combating the COVID-19 pandemic. Although vaccines based on the original Wuhan sequence have been shown to be effective at preventing COVID-19, their efficacy is likely to be decreased against more neutralization-resistant variants-of-concern (VOC), in particular, the Beta variant originating in South Africa. We assessed, in mice, rabbits, and non-human primates, whether a third vaccination with experimental Wuhan-based Spike vaccines could alleviate this problem. Our data show that a third immunization improves neutralizing antibody titers against the variants-of-concern, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). After three vaccinations, the level of neutralization against Beta was similar to the level of neutralization against the original strain after two vaccinations, suggesting that simply providing a third immunization could nullify the reduced activity of current vaccines against VOC.

BackgroundAs SARS-CoV-2 will likely continue to circulate, low-impact methods become more relevant to monitor antibody-mediated immunity. Saliva sampling could provide a non-invasive method with reduced impact on children. Studies reporting on the differences between systemic and mucosal humoral immunity to SARS-CoV-2 are inconsistent in adults and scarce in children. These differences may be further unraveled by exploring associations to demographic and clinical variables.MethodsTo evaluate the use of saliva antibody assays, we performed a cross-sectional cohort study by collecting serum and saliva of 223 children attending medical services in the Netherlands (irrespective of SARS-CoV-2 exposure, symptoms or vaccination) from May to October 2021. With a Luminex and a Wantai assay, we measured prevalence of SARS-CoV-2 spike (S), receptor binding domain (RBD) and nucleocapsid-specific IgG and IgA in serum and saliva and explored associations with demographic variables.FindingsThe S-specific IgG prevalence was higher in serum 39% (95% CI 32 – 45%) than in saliva 30% (95% CI 24 – 36%) (P ≤ 0.003). Twenty-seven percent (55/205) of children were S-specific IgG positive in serum and saliva, 12% (25/205) were only positive in serum and 3% (6/205) only in saliva. Vaccinated children showed a higher concordance between serum and saliva than infected children. Odds for saliva S-specific IgG positivity were higher in girls compared to boys (aOR 2.63, P = 0.012). Moreover, immunocompromised children showed lower odds for S- and RBD-specific IgG in both serum and saliva compared to healthy children (aOR 0.23 – 0.25, P ≤ 0.050).ConclusionsWe showed that saliva-based antibody assays can be useful for identifying SARS-CoV-2 humoral immunity in a non-invasive manner, and that IgG prevalence may be affected by sex and immunocompromisation. Differences between infection and vaccination, between sexes and between immunocompromised and healthy children should be further investigated and considered when choosing systemic or mucosal antibody measurement.

ABSTRACT 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.

BackgroundEmerging 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 findingsIn 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; pConclusionsOverall, 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.

The most effective treatment for HIV-1, antiretroviral therapy, suppresses viral replication and averts the disease from progression. Nonetheless, there is a need for alternative treatments as it requires daily administration with the possibility of side effects and occurrence of drug resistance. Broadly neutralizing antibodies or nanobodies targeting the HIV-1 envelope glycoprotein are explored as alternative treatment, since they mediate viral suppression and contribute to the elimination of virus-infected cells. Besides neutralization potency and breadth, Fc-mediated effector functions of bNAbs also contribute to the in vivo efficacy. In this study multivalent J3, 2E7 and 1F10 anti-HIV-1 broadly neutralizing nanobodies were generated to improve neutralization potency and IgG1 Fc fusion was utilized to gain Fc-mediated effector functions. Bivalent and trivalent nanobodies, coupled using long glycine-serine linkers, showed increased binding to the HIV-1 Env and enhanced neutralization potency compared to the monovalent variant. Fusion of an IgG1 Fc domain to J3 improved neutralization potency compared to the J3-bihead and restored Fc-mediated effector functions such as antibody-dependent cellular phagocytosis and trogocytosis, and natural killer cell activation. Due to their neutralization breadth and potency and their ability to induce effector functions these nanobody-IgG1 constructs may prove to be valuable towards alternative HIV-1 therapies.

Human coronavirus HKU1 (HCoV-HKU1) is one of the four endemic coronaviruses. It has been suggested that there is a difference in incidence, with PCR-confirmed HCoV-NL63 and HCoV-OC43 infections occurring more commonly, whereas HCoV-HKU1 is the least seen. Lower incidence of HCoV-HKU1 infection has also been observed in serological studies. The current study aimed to investigate antibody dynamics during PCR-confirmed HCoV-HKU1 infections using serum collected during infection and 1 month later. We expressed a new HCoV-HKU1 antigen consisting of both the linker and carboxy-terminal domain of the viral nucleocapsid protein and implemented it in ELISA. We also applied a spike-based Luminex assay on serum samples from PCR-confirmed infections by the four endemic HCoVs. At least half of HCoV-HKU1-infected subjects consistently showed no antibody rise via either assay, and some subjects even exhibited substantial antibody decline. Investigation of self-reported symptoms revealed that HCoV-HKU1-infected subjects rated their illness milder than subjects infected by other HCoVs. In conclusion, HCoV-HKU1 infections reported in this study displayed atypical antibody dynamics and milder symptoms when compared to the other endemic HCoVs.

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.

ABSTRACT 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.