IgG antibodies contain a conserved N-linked glycosylation site at Asn297 in the Fc region, and variations in Fc glycosylation critically influence antibody effector functions. While inflammatory signals during immunization are known to reduce IgG Fc galactosylation and sialylation, the counter-regulatory pathways that enhance these modifications remain poorly understood. Here, we investigated the association of the anti-inflammatory cytokine IL-10 with germinal center (GC) responses and IgG Fc glycosylation following protein immunization in mice. Blockade of IL-10 receptor signaling after immunization with a model protein and the adjuvant Alum reduced Fc galactosylation and sialylation of antigen-specific IgG1 and was associated with decreased expression of the sialyltransferase St6gal1 in antigen-specific GC B cells and plasma cells. Although IFNγ is known to suppress IgG Fc galactosylation and sialylation, Alum immunization paradoxically induced both high levels of Fc galactosylation and sialylation as well as a high frequency of IFNγ-producing CD4+ T follicular cells. Using IL-10 reporter mice, we resolved this apparent discrepancy by identifying a substantial population of IL-10⁺ IFNγ⁺ double-producing T follicular cells following Alum immunization. Ex vivo murine and human B cell culture experiments further showed that IL-10 counteracts IFNγ-mediated downregulation of St6gal1. Together, these findings identify an association between IL-10 signaling and increased St6gal1 expression in GC B cells and PCs, as well as increased IgG Fc galactosylation and sialylation. Furthermore, the data identify a population of IL-10⁺ IFNγ⁺ T follicular cells that may contribute to the IL-10-associated glycosylation changes. Our study supports a link between cytokine environments and IgG Fc glycosylation and provides a framework for future studies investigating how IL-10-associated pathways shape IgG glycosylation in vaccination and inflammatory disease settings.

Correlates of protection against symptomatic and severe breakthrough SARS-CoV-2 infections are well characterized. However, correlates of protection against virus transmission are poorly defined due to a lack of evidence in well-designed prospective clinical trials. We studied a Croatian cohort of individuals with documented household exposure to SARS-CoV-2 in late 2022/early 2023. Sera were acquired at the time of the COVID-19 diagnosis of the index case and before symptom onset of the test case. Samples were comprehensively analyzed for predictors of protection against virus transmission. PCR-negative participants at day 0 were recalled 14 days later and re-tested by PCR and IgM ELISA to identify any virus transmission, including asymptomatic ones. Out of nearly 200 tested serological parameters, several serological features differed between the participants that became PCR-positive during the study period and those that remained PCR-negative, although none remained significant after correction for multiple testing in the full cohort. Titers of variant-specific neutralizing antibody showed the biggest difference and were higher in the uninfected subgroup. Since recent antigenic exposure may confound results, we identified the recipients exhibiting IgM seroconversion and censored them from the study. This refinement clearly separated infected and uninfected individuals by variant-specific neutralization titers and IgA1 responses to BA4/5 RBD, which remained significant after correction. Therefore, our data indicate that high IgA1 and neutralizing titers may be predictive serum correlates of protection against SARS-CoV-2 transmission in intense contacts among household members, but only if variant-specific antigen is used in serological assays.

Vaccination with tumor-(neo) antigen plus adjuvant is emerging as a promising cancer-therapy. However, as different adjuvants induce distinct immune cell and antibody (Ab) responses, selecting the right adjuvants remains challenging. Here, we evaluated the following vaccine adjuvants to promote protection against tumor-growth in mice and correlated IgG subclass and Fc N-glycosylation responses: Alum; the toll-like receptor activators Poly(I:C) and MPLA; Alum-Poly(I:C); and the more inflammatory water-in-oil adjuvants Montanide, IFA, CFA, and M.tb.-enriched (e)CFA. While Alum and Montanide failed to protect, MPLA and IFA tended to protect, and Poly(I:C), Alum-Poly(I:C), CFA, and eCFA significantly protected against tumor-growth. Across all adjuvants, tumor-protection correlated with the induction of highly activating IgG2(c/b) Abs and afucosylated (F0) IgG1 Abs, the latter showing up to 5

Therapeutic monoclonal antibodies (mAbs) can be functionally enhanced via Fc engineering. To determine whether pairs of mAbs with different Fc modifications can be combined for functional complementarity, we investigated the in vitro activity of two HIV-1 mAb libraries, each equipped with 60 engineered Fc variants. Our findings demonstrate that the impact of Fc engineering on Fc functionality is dependent on the specific Fab clone. Notably, combinations of Fc variants of the same Fab specificity exhibited limited enhancement in functional breadth compared to combinations involving two distinct Fabs. This suggests that the strategic selection of complementary Fc modifications can enhance both functional activity and breadth. Furthermore, while some combinations of Fc variants displayed additive functional effects, others were detrimental, suggesting that the functional outcome of Fc mutations is not easily predicted. Collectively, these results provide preliminary evidence supporting the potential of complementary Fc modifications in mAb combinations. Future studies will be essential to identify the optimal Fc modifications that maximize in vivo efficacy.

Mosaic HIV-1 vaccines have been shown to elicit robust humoral and cellular immune responses in people living with HIV-1 (PLWH), that had started antiretroviral therapy (ART) during acute infection. We evaluated the safety and immunogenicity of 2 mosaic vaccine regimens in virologically suppressed individuals that had initiated ART during the chronic phase of infection, exemplifying the majority of PLWH. In this double-blind, placebo-controlled phase 1 trial (IPCAVD013/HTX1002) 25 ART-suppressed PLWH were randomized to receive Ad26.Mos4.HIV/MVA-Mosaic (Ad26/MVA) (n = 10) or Ad26.Mos4.HIV/Ad26.Mos4.HIV plus adjuvanted gp140 protein (Ad26/Ad26+gp140) (n = 9) or placebo (n = 6). Primary endpoints included safety and tolerability and secondary endpoints included HIV-specific binding and neutralizing antibody titers and HIV-specific T cell responses. Both vaccine regimens were well tolerated with pain/tenderness at the injection site and fatigue, myalgia/chills and headache as the most commonly reported solicited local and grade 3 systemic adverse events, respectively. In the Ad26/Ad26+gp140 group, Env-specific IFN-γ T cell responses showed a median 12-fold increase while responses to Gag and Pol increased 1.8 and 2.4-fold, respectively. The breadth of T cell responses to individual peptide subpools increased from 11.0 pre-vaccination to 26.0 in the Ad26/Ad26+gp140 group and from 10.0 to 14.5 in the Ad26/MVA group. Ad26/Ad26+gp140 vaccination increased binding antibody titers against vaccine-matched clade C Env 5.5-fold as well as augmented neutralizing antibody titers against Clade C pseudovirus by 7.2-fold. Both vaccine regimens were immunogenic, while the addition of the protein boost resulted in additional T cell and augmented binding and neutralizing antibody titers. These data suggest that the Ad26/Ad26+gp140 regimen should be tested further.

BACKGROUND:Recently, it has been questioned whether vaccination of patients with inflammatory (auto)immune diseases under anti-tumor necrosis factor (TNF) treatment leads to impaired vaccine-induced immune responses and protection against breakthrough infections. However, the effects of TNF blockade on short- and long-term immune responses after repeated vaccination remain unclear. Vaccination studies have shown that initial short-term IgG antibodies (Abs) carry highly galactosylated and sialylated Fc glycans, whilst long-term IgG Abs have low levels of galactosylation and sialylation and are most likely generated by long-lived plasma cells (PCs) derived primarily from the germinal center (GC) response. Thus, IgG Fc glycosylation patterns may be applicable to distinguish short- and long-term vaccine responses after repeated vaccination under the influence of anti-TNF treatment. METHODS:We used COVID-19 vaccination as a model to investigate vaccine-induced IgG subclass levels and Fc glycosylation patterns, B cell subsets, and effector functions of short- and long-term Ab responses after up to three vaccinations in patients on anti-TNF or other immunosuppressive treatments and in healthy individuals. Using TriNetX, a global healthcare database, we determined the risk of SARS-CoV-2 breakthrough infections in vaccinated patients treated with anti-TNF or other immunosuppressive drugs. RESULTS:Anti-TNF treatment reduced the long-term abundance of all anti-S IgG subclasses with low levels of galactosylation and sialylation. Re-activation of potential memory B cells initially generated highly galactosylated and sialylated IgG antibodies, which were progressively reduced after each booster dose in anti-TNF-treated patients, especially in the elderly. The reduced short- and long-term IgG (1) levels in anti-TNF-treated patients correlated with diminished functional activity and an increased risk for the development of COVID-19. CONCLUSIONS:The data suggest that anti-TNF treatment reduces both GC-dependent long-lived PCs and GC-dependent memory B cell-derived short-lived PCs, hence both the long- and short-term IgG subclass responses, respectively, after repeated vaccination. We propose that anti-TNF therapy, especially in the elderly, reduces the benefit of booster vaccination.

DNA, RNA, and proteins are synthesized using template molecules, but glycosylation is not believed to be constrained by a template. However, if cellular environment is the only determinant of glycosylation, all sites should receive the same glycans on average. This template-free assertion is inconsistent with observations of microheterogeneity-wherein each site receives distinct and reproducible glycan structures. Here, we test the assumption of template-free glycan biosynthesis. Through structural analysis of site-specific glycosylation data, we find protein-sequence and structural features that predict specific glycan features. To quantify these relationships, we present a new amino acid substitution matrix that describes glycoimpact-how glycosylation varies with protein structure. High-glycoimpact amino acids co-evolve with glycosites, and glycoimpact is high when estimates of amino acid conservation and variant pathogenicity diverge. We report hundreds of disease variants near glycosites with high-glycoimpact, including several with known links to aberrant glycosylation (e.g., Oculocutaneous Albinism, Jakob-Creutzfeldt disease, Gerstmann-Straussler-Scheinker, and Gaucher's Disease). Finally, we validate glycoimpact quantification by studying oligomannose-complex glycan ratios on HIV ENV, differential sialylation on IgG3 Fc, differential glycosylation on SARS-CoV-2 Spike, and fucose-modulated function of a tuberculosis monoclonal antibody. In all, we show glycan biosynthesis is accurately guided by specific, genetically-encoded rules, and this presents a plausible refutation to the assumption of template-free glycosylation. ### Competing Interest Statement This work is associated with a provisional patent filed by the authors, and Augment Biologics, founded by BK and NEL.

Background Accurate quantitation of immune markers is crucial for ensuring reliable assessment of vaccine efficacy against infectious diseases. This study was designed to confirm standardised performance of SARS-CoV-2 assays used to evaluate COVID-19 vaccine candidates at the initial seven laboratories (in North America, Europe, and Asia) of the Coalition for Epidemic Preparedness Innovations (CEPI) Centralized Laboratory Network (CLN). Methods Three ELISAs (pre -spike protein, receptor binding domain, and nucleocapsid), a microneutralisation assay (MNA), a pseudotyped virus -based neutralisation assay (PNA), and an IFN-gamma T -cell ELISpot assay were developed, validated or qualified, and transferred to participating laboratories. Immune responses were measured in ELISA laboratory units (ELU) for ELISA, 50% neuralisation dilution (ND50) for MNA, 50% neutralisation titre (NT50) for PNA, and spot -forming units for the ELISpot assay. Replicate assay results of well characterised panels and controls of blood samples from individuals with or without SARS-CoV-2 infection were evaluated by geometric mean ratios, standard deviation, linear regression, and Spearman correlation analysis for consistency, accuracy, and linearity of quantitative measurements across all laboratories. Findings High reproducibility of results across all laboratories was demonstrated, with interlaboratory precision of 4.1-7.7% coefficient of variation for all three ELISAs, 3.8-19.5% for PNA, and 17.1-24.1% for MNA, over a linear range of 11-30 760 ELU per mL for the three ELISAs, 14-7876 NT50 per mL for PNA, and 21-25 587 ND50 per mL for MNA. The MNA was also adapted for detection of neutralising antibodies against the major SARS-CoV-2 variants of concern. The results of PNA and MNA (r=0.864) and of ELISA and PNA (r=0.928) were highly correlated. The IFN-gamma ELISpot interlaboratory variability was 15.9-49.9% coefficient of variation. Sensitivity and specificity were close to 100% for all assays. Interpretation The CEPI CLN provides accurate quantitation of anti-SARS-CoV-2 immune response across laboratories to allow direct comparisons of different vaccine formulations in different geographical areas. Lessons learned from this programme will serve as a model for faster responses to future pandemic threats and roll -out of effective vaccines. Funding CEPI. Copyright (c) 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY -NC -ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

AbstractBackgroundWhile correlates of protection against symptomatic and severe breakthrough SARS-CoV-2 infections are well characterized, correlates of protection against virus transmission are incompletely understood.MethodsWe studied a Croatian cohort of individuals with documented household exposure to SARS-CoV-2 in December 2022. Sera were acquired prior to symptom onset, at the time of the COVID-19 diagnosis of the index cases, and comprehensively analyzed for correlates of protection against virus transmission. We monitored participants for 14 days and tested them with PCR at the end of the observation period to identify any virus transmission, including asymptomatic ones.InterpretationOut of nearly 200 tested serological parameters, 22 features were significantly different between the infected and the uninfected participants. Titers of variant-specific neutralizing antibody showed the biggest difference and were significantly higher in the uninfected subgroup. Some infected individuals with strong IgM responses to the spike antigen showed robust neutralization titers as well. Since IgM is likely an indication of recent antigenic exposure, data were reanalyzed by excluding such values. This refined analysis showed a complete segregation of infected and uninfected individuals into groups with low and high variant-specific neutralization titers. Therefore, our data indicate that high neutralizing titers are correlates of protection against SARS-CoV-2 transmission in intense contacts among household members.FundingThis research was funded by the Impulse and Networking fund of the Helmholtz Association through the grant PIE-0008 to LCS and VH-NG-19-28 to YCB and by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy - EXC 2155 - project number 390874280 to LCS. BL and MH received funding within the RESPINOW project from the Federal Ministry of Education and Research under the grant number 031L0298A.Research in ContextEvidence before this studyPre-existing immunity to SARS-CoV-2, whether from prior infections or vaccinations, has been shown to primarily protect against severe disease rather than preventing infection altogether. Many current studies examining this phenomenon focus on cohorts with breakthrough infections occurring a certain time after their last vaccination. However, these studies often lack precise information about when the individuals were infected and their serological status immediately before the infection.Added value of this studyUnlike other studies, we focused on a cohort of individuals with a confirmed SARS-CoV-2-positive household member. Serum samples were collected before symptom onset, coinciding with the COVID-19 diagnosis of the index cases. We analyzed various serum features to comprehensively assess their ability to protect not only against severe disease but also against virus transmission. Our findings revealed that individuals who remained uninfected had significantly higher concentrations of neutralizing antibodies compared to those who became infected.Implications of the available evidenceThis finding suggests that neutralizing antibodies serve as a correlate of protection against virus transmission and could inform booster strategies based not on a fixed timeline but on antibody levels dropping below a specific threshold. However, due to the limited sample size of our study, larger studies are needed to confirm these results and establish an exact threshold.

There is currently no prophylactic vaccine available for human immunodeficiency virus (HIV). Research efforts have resulted in improved immunogens that mimic the native envelope (Env) glycoprotein structure. Recently, a novel triple tandem trimer (TTT) platform has been used to generate a plasmid encoding Env immunogen (pBG505-TTT) that expresses only as trimers, making it more suitable for nucleic acid vaccines. We have previously demonstrated that adenosine deaminase-1 (ADA-1) is critical to the T follicular helper (TFH) function and improves vaccine immune responses in vivo . In this study, we demonstrate that co-delivery of plasmid-encoded adenosine deaminase 1 (pADA) with pBG505-TTT enhances the magnitude, durability, isotype switching and functionality of HIV-specific antibodies in a dose-sparing manner. Co-delivery of the molecular immune modulator ADA-1 also enhances HIV-specific T cell polyfunctionality, activation, and degranulation as well as memory B cell responses. These data demonstrate that pADA enhances HIV-specific cellular and humoral immunity, making ADA-1 a promising immune modulator for HIV-targeting vaccines.

Schistosomiasis is a disease caused by parasitic flatworms of the Schistosoma spp., and is increasingly recognized to alter the immune system, and the potential to respond to vaccines. The impact of endemic infections on protective immunity is critical to inform vaccination strategies globally. We assessed the influence of Schistosoma mansoni worm burden on multiple host vaccine-related immune parameters in a Ugandan fishing cohort (n = 75) given three doses of a Hepatitis B (HepB) vaccine at baseline and multiple timepoints post-vaccination. We observed distinct differences in immune responses in instances of higher worm burden, compared to low worm burden or non-infected. Concentrations of pre-vaccination serum schistosome-specific circulating anodic antigen (CAA), linked to worm burden, showed a significant bimodal distribution associated with HepB titers, which was lower in individuals with higher CAA values at month 7 post-vaccination (M7). Comparative chemokine/cytokine responses revealed significant upregulation of CCL19, CXCL9 and CCL17 known to be involved in T cell activation and recruitment, in higher CAA individuals, and CCL17 correlated negatively with HepB titers at month 12 post-vaccination. We show that HepB-specific CD4+ T cell memory responses correlated positively with HepB titers at M7. We further established that those participants with high CAA had significantly lower frequencies of circulating T follicular helper (cTfh) subpopulations pre- and post-vaccination, but higher regulatory T cells (Tregs) post-vaccination, suggesting changes in the immune microenvironment in high CAA could favor Treg recruitment and activation. Additionally, we found that changes in the levels of innate-related cytokines/chemokines CXCL10, IL-1β, and CCL26, involved in driving T helper responses, were associated with increasing CAA concentration. This study provides further insight on pre-vaccination host responses to Schistosoma worm burden which will support our understanding of vaccine responses altered by pathogenic host immune mechanisms and memory function and explain abrogated vaccine responses in communities with endemic infections.

Background: Cases of adolescents and young adults developing myocarditis after vaccination with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–targeted mRNA vaccines have been reported globally, but the underlying immunoprofiles of these individuals have not been described in detail. Methods: From January 2021 through February 2022, we prospectively collected blood from 16 patients who were hospitalized at Massachusetts General for Children or Boston Children’s Hospital for myocarditis, presenting with chest pain with elevated cardiac troponin T after SARS-CoV-2 vaccination. We performed extensive antibody profiling, including tests for SARS-CoV-2–specific humoral responses and assessment for autoantibodies or antibodies against the human-relevant virome, SARS-CoV-2–specific T-cell analysis, and cytokine and SARS-CoV-2 antigen profiling. Results were compared with those from 45 healthy, asymptomatic, age-matched vaccinated control subjects. Results: Extensive antibody profiling and T-cell responses in the individuals who developed postvaccine myocarditis were essentially indistinguishable from those of vaccinated control subjects, despite a modest increase in cytokine production. A notable finding was that markedly elevated levels of full-length spike protein (33.9±22.4 pg/mL), unbound by antibodies, were detected in the plasma of individuals with postvaccine myocarditis, whereas no free spike was detected in asymptomatic vaccinated control subjects (unpaired t test; P <0.0001). Conclusions: Immunoprofiling of vaccinated adolescents and young adults revealed that the mRNA vaccine–induced immune responses did not differ between individuals who developed myocarditis and individuals who did not. However, free spike antigen was detected in the blood of adolescents and young adults who developed post-mRNA vaccine myocarditis, advancing insight into its potential underlying cause.

To the Editor, Immunoglobulin E (IgE) plays a crucial role in allergic reactions, including systemic anaphylaxis, by binding to the highaffinity IgE receptor FcεRI (FcεRIα + FcεRIβ + FcεRIγ) on mast cells and basophils and, upon allergenmediated aggregation, inducing the release of inflammatory mediators.1– 3 The allergic potential of IgE antibodies (Abs) is further affected by IgE glycosylationdependent interactions with membranebound and soluble carbohydratebinding proteins (e.g., galectins, siglecs, and Ctype lectin receptors) as well as different structural features (E14).1,3 Many healthy individuals harbor allergenspecific IgE Abs in the absence of an allergic disease (E5, E6), suggesting qualitative differences in IgEtriggered responses. Similarly, not all allergic individuals develop severe disease forms (e.g., asthma), and it is still unclear why allergen immunotherapy (AIT) is effective in some individuals but not in all (E6). Different natural or AITinduced inflammatory states of the specific Tand Bcell response may lead, as described for IgG (E7),4 to differently glycosylated IgE Abs that contribute distinctly to the induction of allergic responses. IgE Abs have several conserved Nglycosylation sites in the constant region of their heavy chains. Most of them are of the complex type and potentially galactosylated and terminal sialylated2,5 (Figure 1A,B). By comparing the effects of sialylated versus enzymatically desialylated IgE monoclonal (m)Abs, a recent study suggested that highly sialylated IgE Abs have a stronger potential to induce allergic reactions than lower sialylated IgE Abs.6 Notably, this observation is opposite to the findings that sialylation dampens the inflammatory potential of IgG, IgA, and IgM Abs (E8E10).4,7 Here, we further investigated the role of IgE sialylation on FcεRIα interaction, mast cell and basophil loading and activation, and IgEmediated passive systemic anaphylaxis (PSA) in mice. Therefore, we generated an antiTNP (2,4,6trinitrophenol) murine IgE mAb with three different sialylation levels: (i) originally sialylated (originally sial; glycosylation form generated by the hybridoma cell line), (ii) enzymatically desialylated (desial), and (iii) enzymatically additional sialylated (high sial) (Figure 1B– D). Both enzymatic IgE glycan modifications did not influence antigen binding (Figure 1E). Desialylated antiTNP IgE mAbs showed a stronger interaction with human and murine FcεRIα than originally sialylated and additionally sialylated IgE mAbs, whereas the latter two sialylated IgE glycoforms showed a comparable low or no interaction with FcεRIα in these assays, respectively (Figure 1F). This observation was unexpected due to the mentioned findings about the lower allergic potential of desialylated compared to sialylated IgE Abs6 but is in line with a stronger interaction of desialylated IgG Abs with Fcγ receptors than sialylated IgG Abs (E8). A comparable trend was observed when human mast cells differentiated from CD34+ blood cells were activated with the three antiTNP IgE glycoforms and TNPOVA as antigen (Figure S1). Moreover, less sialylated antiTNP IgE mAbs showed an enhanced loading on the human mast cell line LAD2 as well as on human blood basophils, which correlated positively with their activation (Figure 1G– J and Figure S1). Low sensitivity of the FcεRIα interaction assay and of the differentiated human mast cell assay and/or additional factors to FcεRIα binding might explain the higher loading and activation potentials of originally sialylated (orig. sial) compared to additional sialylated (high sial) IgE Abs in the LAD2 mast cell and blood basophil assays. Together, these data suggest that the higher interaction of de−/less sialylated IgE with mast cells and basophils correlate with their stronger activation potential. Next, we compared the potential of the three antiTNP IgE mAb glycoforms in an IgEmediated passive systemic anaphylaxis (PSA) mouse model (Figure 2A and Figure S1). In accordance with the data described by Shade et al., 20206 but in contrast to the expectation based on our in vitro data, intravenous (i.v.) injection of desialylated antiTNP IgE mAbs, followed by i.v. injection of TNPcoupled ovalbumin (TNPOVA) as “allergen” 6 or 24 h later, hardly induced any anaphylaxis, as measured by the drop in body core/rectal temperature, compared to the sialylated IgE mAbs (Figure 2B and Figure S1). However, we realized that desialylated antiTNP IgE mAbs could neither be detected in serum nor on blood basophils already 6 h after injection (Figure 2C,D and Figure S1). These data suggested a highly reduced serum halflife of desialylated IgE mAbs, which has also been described for desialylated IgA Abs in an asialoglycoprotein receptordependent manner (E11). To counteract the potential quick clearance of desialylated IgE mAbs, we blocked asialoglycoprotein receptors with an excess of desialylated (asialo)α1acid glycoprotein (AGP; orosomucoid) (E11) before sensitizing mice with the antiTNP IgE glycoforms (Figure 2E

The number of mutations in the omicron (B.1.1.529) BA.1 variant of concern led to an unprecedented evasion of vaccine induced immunity. However, despite rise in global infections, severe disease did not increase proportionally and is likely linked to persistent recognition of BA.1 by T cells and non-neutralizing opsonophagocytic antibodies. Yet, the emergence of new sublineage BA.2, which is more transmissible than BA.1 despite relatively preserved neutralizing antibody responses, has raised the possibility that BA.2 may evade other vaccine-induced responses. Here, we comprehensively profiled the BNT162b2 vaccine-induced response to several VOCs, including omicron BA.1 and BA.2. While vaccine-induced immune responses were compromised against both omicron sublineages, vaccine-induced antibody isotype titers, and non-neutralizing Fc effector functions were attenuated to the omicron BA.2 spike compared to BA.1. Conversely, FcγR2a and FcγR2b binding was elevated to BA.2, albeit lower than BA.1 responses, potentially contributing to persistent protection against severity of disease.

ABSTRACT An Ad26.RSV.preF/RSV preF protein combination vaccine demonstrated 80.0% vaccine efficacy for the prevention of respiratory syncytial virus (RSV)-mediated lower respiratory tract disease in a phase 2b study. In addition to neutralizing antibodies, Fc-effector functions are associated with protective immunity against RSV. Here, vaccine-induced Fc-effector functions were evaluated for the Ad26.RSV.preF/RSV preF protein vaccine in a subset of participants enrolled in a phase 1/2a study. RSV preF-specific antibody subclasses, isotypes, Fcγ receptor binding, and Fc-effector functions were evaluated on days 1 (pre-vaccination), 15, 29, and 183. Compared with Ad26.RSV.preF or RSV preF protein alone, the combination vaccine induced greater Fc-effector functions, including antibody-dependent neutrophil phagocytosis and antibody-dependent natural killer cell activation. Despite RSV pre-exposure, antibody-dependent neutrophil phagocytosis and antibody-dependent natural killer cell activation were not observed at baseline but were induced de novo following vaccination. Compared with the individual vaccine components, the combination vaccine induced a more polyfunctional antibody response. IMPORTANCE Respiratory syncytial virus (RSV) can cause serious illness in older adults (i.e., those aged ≥60 years). Because options for RSV prophylaxis and treatment are limited, the prevention of RSV-mediated illness in older adults remains an important unmet medical need. Data from prior studies suggest that Fc-effector functions are important for protection against RSV infection. In this work, we show that the investigational Ad26.RSV.preF/RSV preF protein vaccine induced Fc-effector functional immune responses in adults aged ≥60 years who were enrolled in a phase 1/2a regimen selection study of Ad26.RSV.preF/RSV preF protein. These results demonstrate the breadth of the immune responses induced by the Ad26.RSV.preF/RSV preF protein vaccine.

Although most children experience mild symptoms during acute SARS-CoV-2 infection, some develop the severe post-COVID-19 complication, Multisystem Inflammatory Syndrome in Children (MIS-C). While acute presentations of COVID-19 and MIS-C have been well immunophenotyped, little is known about the lasting immune profile in children after acute illness. Children 2 months–20 years of age presenting with either acute COVID-19 (n = 9) or MIS-C (n = 12) were enrolled in a Pediatric COVID-19 Biorepository at a single medical center. We deeply profiled humoral immune responses and circulating cytokines following pediatric COVID-19 and MIS-C. Twenty-one children and young adults provided blood samples at both acute presentation and 6-month follow-up (mean: 6.5 months; standard deviation: 1.77 months). Pro-inflammatory cytokine elevations resolved after both acute COVID-19 and MIS-C. Humoral profiles continue to mature after acute COVID-19, displaying decreasing IgM and increasing IgG over time, as well as stronger effector functions, including antibody-dependent monocyte activation. In contrast, MIS-C immune signatures, especially anti-Spike IgG1, diminished over time. Here, we show the mature immune signature after pediatric COVID-19 and MIS-C, displaying resolving inflammation with recalibration of the humoral responses. These humoral profiles highlight immune activation and vulnerabilities over time in these pediatric post-infectious cohorts.

SARS-CoV-2 mRNA vaccines confer robust protection against COVID-19, but the emergence of variants has generated concerns regarding the protective efficacy of the currently approved vaccines, which lose neutralizing potency against some variants. Emerging data suggest that antibody functions beyond neutralization may contribute to protection from the disease, but little is known about SARS-CoV-2 antibody effector functions. Here, we profiled the binding and functional capacity of convalescent antibodies and Moderna mRNA-1273 COVID-19 vaccine-induced antibodies across SARS-CoV-2 variants of concern (VOCs). Although the neutralizing responses to VOCs decreased in both groups, the Fc-mediated responses were distinct. In convalescent individuals, although antibodies exhibited robust binding to VOCs, they showed compromised interactions with Fc-receptors. Conversely, vaccine-induced antibodies also bound robustly to VOCs but continued to interact with Fc-receptors and mediate antibody effector functions. These data point to a resilience in the mRNA-vaccine-induced humoral immune response that may continue to offer protection from SARS-CoV-2 VOCs independent of neutralization.

While children have been largely spared from COVID-19 disease, the emergence of viral variants of concern (VOC) with increased transmissibility, combined with fluctuating mask mandates and school re-openings have led to increased infections and disease among children. Thus, there is an urgent need to roll out COVID-19 vaccines to children of all ages. However, whether children respond equivalently to adults to mRNA vaccines and whether dosing will elicit optimal immunity remains unclear. Given the recent announcement of incomplete immunity induced by the pediatric dose of the BNT162b2 vaccine in young children, here we aimed to deeply profile and compare the vaccine-induced humoral immune response in 6-11 year old children receiving the pediatric (50μg) or adult (100μg) dose of the mRNA-1273 vaccine compared to adults and naturally infected children or children that experienced multi inflammatory syndrome in children (MIS-C) for the first time. Children elicited an IgG dominant vaccine induced immune response, surpassing adults at a matched 100μg dose, but more variable immunity at a 50μg dose. Irrespective of titer, children generated antibodies with enhanced Fc-receptor binding capacity. Moreover, like adults, children generated cross-VOC humoral immunity, marked by a decline of omicron receptor binding domain-binding, but robustly preserved omicron Spike-receptor binding, with robustly preserved Fc-receptor binding capabilities, in a dose dependent manner. These data indicate that while both 50μg and 100μg of mRNA vaccination in children elicits robust cross-VOC antibody responses, 100ug of mRNA in children results in highly preserved omicron-specific functional humoral immunity. One-Sentence Summary mRNA vaccination elicits robust humoral immune responses to SARS-CoV-2 in children 6-11 years of age. ### Competing Interest Statement G.A. is a founder and equity holder of Seromyx Systems, a company developing a platform technology that describes the antibody immune response. G.A. is an employee and equity holder of Leyden Labs, a company developing pandemic prevention therapeutics. G.A.s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. All other authors have declared that no conflicts of interest exist.