Systematic evaluation of B-cell clonal family inference approaches.

The reconstruction of clonal families (CFs) in B-cell receptor (BCR) repertoire analysis is a crucial step to understand the adaptive immune system and how it responds to antigens. The BCR repertoire of an individual is formed throughout life and is diverse due to several factors such as gene recombination and somatic hypermutation. The use of Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using next generation sequencing enabled the generation of full BCR repertoires that also include rare CFs. The reconstruction of CFs from AIRR-seq data is challenging and several approaches have been developed to solve this problem. Currently, most methods use the heavy chain (HC) only, as it is more variable than the light chain (LC). CF reconstruction options include the definition of appropriate sequence similarity measures, the use of shared mutations among sequences, and the possibility of reconstruction without preliminary clustering based on V- and J-gene annotation. In this study, we aimed to systematically evaluate different approaches for CF reconstruction and to determine their impact on various outcome measures such as the number of CFs derived, the size of the CFs, and the accuracy of the reconstruction. The methods were compared to each other and to a method that groups sequences based on identical junction sequences and another method that only determines subclones. We found that after accounting for data set variability, in particular sequencing depth and mutation load, the reconstruction approach has an impact on part of the outcome measures, including the number of CFs. Simulations indicate that unique junctions and subclones should not be used as substitutes for CF and that more complex methods do not outperform simpler methods. Also, we conclude that different approaches differ in their ability to correctly reconstruct CFs when not considering the LC and to identify shared CFs. The results showed the effect of different approaches on the reconstruction of CFs and highlighted the importance of choosing an appropriate method.

Control of complement-induced inflammatory responses to SARS-CoV-2 infection by anti-SARS-CoV-2 antibodies.

Dysregulated immune responses contribute to the excessive and uncontrolled inflammation observed in severe COVID-19. However, how immunity to SARS-CoV-2 is induced and regulated remains unclear. Here, we uncover the role of the complement system in the induction of innate and adaptive immunity to SARS-CoV-2. Complement rapidly opsonizes SARS-CoV-2 particles via the lectin pathway. Complement-opsonized SARS-CoV-2 efficiently induces type-I interferon and pro-inflammatory cytokine responses via activation of dendritic cells, which are inhibited by antibodies against the complement receptors (CR) 3 and 4. Serum from COVID-19 patients, or monoclonal antibodies against SARS-CoV-2, attenuate innate and adaptive immunity induced by complement-opsonized SARS-CoV-2. Blocking of CD32, the FcγRII antibody receptor of dendritic cells, restores complement-induced immunity. These results suggest that opsonization of SARS-CoV-2 by complement is involved in the induction of innate and adaptive immunity to SARS-CoV-2 in the acute phase of infection. Subsequent antibody responses limit inflammation and restore immune homeostasis. These findings suggest that dysregulation of the complement system and FcγRII signaling may contribute to severe COVID-19.

Long trimer-immunization interval and appropriate adjuvant reduce immune responses to the soluble HIV-1-envelope trimer base.

Soluble 'SOSIP'-stabilized HIV-1 envelope glycoprotein (Env) trimers elicit dominant antibody responses targeting their glycan-free base regions, potentially diminishing neutralizing responses. Previously, using a nonhuman primate model, we demonstrated that priming with fusion peptide (FP)-carrier conjugate immunogens followed by boosting with Env trimers reduced the anti-base response. Further, we demonstrated that longer immunization intervals further reduced anti-base responses and increased neutralization breadth. Here, we demonstrate that long trimer-boosting intervals, but not long FP immunization intervals, reduce the anti-base response. Additionally, we identify that FP priming before trimer immunization enhances antibody avidity to the Env trimer. We also establish that adjuvants Matrix M and Adjuplex further reduce anti-base responses and increase neutralizing titers. FP priming, long trimer-immunization interval, and an appropriate adjuvant can thus reduce anti-base antibody responses and improve Env-directed vaccine outcomes.

A panel of hepatitis C virus glycoproteins for the characterization of antibody responses using antibodies with diverse recognition and neutralization patterns.

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.

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.

Into the Dark Serum Proteome: Personalized Features of IgG1 and IgA1 Repertoires in Severe COVID-19 Patients.

Serum proteomics has matured and is now able to monitor hundreds of proteins quantitatively in large cohorts of patients. However, the fine characteristics of some of the most dominant proteins in serum, the immunoglobulins, are in these studies often ignored, due to their vast, and highly personalized, diversity in sequences. Here, we focus exclusively on these personalized features in the serum proteome and distinctively chose to study individual samples from a low diversity population: elderly donors infected by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2). By using mass spectrometry-based methods, immunoglobulin IgG1 and IgA1 clonal repertoires were monitored quantitatively and longitudinally in more than 50 individual serum samples obtained from 17 Corona virus disease 2019 patients admitted to intensive care units. These clonal profiles were used to examine how each patient reacted to a severe SARS-CoV-2 infection. All 17 donors revealed unique polyclonal repertoires and substantial changes over time, with several new clones appearing following the infection, in a few cases leading to a few, very high, abundant clones dominating their repertoire. Several of these clones were de novo sequenced through combinations of top-down, middle-down, and bottom-up proteomics approaches. This revealed sequence features in line with sequences deposited in the SARS-CoV-specific antibody database. In other patients, the serological Ig profiles revealed the treatment with tocilizumab, that subsequently dominated their serological IgG1 repertoire. Tocilizumab clearance could be monitored, and a half-life of approximately 6 days was established. Overall, our longitudinal monitoring of IgG1 and IgA1 repertoires of individual donors reveals that antibody responses are highly personalized traits of each patient, affected by the disease and the chosen clinical treatment. The impact of these observations argues for a more personalized and longitudinal approach in patients' diagnostics, both in serum proteomics as well as in monitoring immune responses.

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.

Assessing immunogenicity barriers of the HIV-1 envelope trimer.

Understanding the balance between epitope shielding and accessibility on HIV-1 envelope (Env) trimers is essential to guide immunogen selection for broadly neutralizing antibody (bnAb) based vaccines. To investigate the antigenic space of Env immunogens, we created a strategy based on synthetic, high diversity, Designed Ankyrin Repeat Protein (DARPin) libraries. We show that DARPin Antigenicity Analysis (DANA), a purely in vitro screening tool, has the capability to extrapolate relevant information of antigenic properties of Env immunogens. DANA screens of stabilized, soluble Env trimers revealed that stronger trimer stabilization led to the selection of highly mutated DARPins with length variations and framework mutations mirroring observations made for bnAbs. By mimicking heterotypic prime-boost immunization regimens, DANA may be used to select immunogen combinations that favor the selection of trimer-reactive binders. This positions DANA as a versatile strategy for distilling fundamental antigenic features of immunogens, complementary to preclinical immunogenicity testing.

Physical activity in lactating women influences SARS-CoV-2-specific antibodies in human milk.

Background: Moderate exercise results in a significant increase in serum and salivary immunoglobulins. Maternal physical activity might therefore also be a factor influencing antibody levels in human milk. This study aims to determine the influence of physical activity on SARS-CoV-2-specific Immunoglobulin A (IgA) in human milk and Immunoglobulin G (IgG) in serum. Methods: In this prospective cross-sectional cohort study, all lactating women in the Netherlands were eligible to participate. SARS-CoV-2-specific IgA in human milk and IgG in serum were determined using an enzyme-linked immunosorbent assay (ELISA). Data on performed physical activity was collected using the Short Questionnaire to Assess Health enhancing physical activity (SQUASH), which includes intensity and duration of the performed activity. Findings: In total, 356 out of 2312 lactating women tested positive for SARS-CoV-2-specific antibodies in serum. Of them, 323 filled in the questionnaire and were included in the analysis. An association between the activity score and SARS-CoV-2-specific antibodies in human milk (B = 1·035, 95·0% CI = 1·019 to 1·052, p = 0·042) and serum (B = 1·019, 95·0% CI = 1·009 to 1·029, p = 0·048) was demonstrated. No association was found between the duration of physical activity and SARS-CoV-2-specific antibodies in human milk or serum. Interpretation: Our findings suggest that physical activity is beneficial for the levels of SARS-CoV-2-specific antibodies in human milk and serum, with the intensity of the physical activity being the most important contributor to this relationship. A higher level of antibodies in human milk might provide better immunological protection for infants against COVID-19.

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.

Vaccine-induced neutralizing antibody responses to seasonal influenza virus H1N1 strains are not enhanced during subsequent pandemic H1N1 infection.

The first exposure to influenza is presumed to shape the B-cell antibody repertoire, leading to preferential enhancement of the initially formed responses during subsequent exposure to viral variants. Here, we investigated whether this principle remains applicable when there are large genetic and antigenic differences between primary and secondary influenza virus antigens. Because humans usually have a complex history of influenza virus exposure, we conducted this investigation in influenza-naive cynomolgus macaques. Two groups of six macaques were immunized four times with influenza virus-like particles (VLPs) displaying either one (monovalent) or five (pentavalent) different hemagglutinin (HA) antigens derived from seasonal H1N1 (H1N1) strains. Four weeks after the final immunization, animals were challenged with pandemic H1N1 (H1N1pdm09). Although immunization resulted in robust virus-neutralizing responses to all VLP-based vaccine strains, there were no cross-neutralization responses to H1N1pdm09, and all animals became infected. No reductions in viral load in the nose or throat were detected in either vaccine group. After infection, strong virus-neutralizing responses to H1N1pdm09 were induced. However, there were no increases in virus-neutralizing titers against four of the five H1N1 vaccine strains; and only a mild increase was observed in virus-neutralizing titer against the influenza A/Texas/36/91 vaccine strain. After H1N1pdm09 infection, both vaccine groups showed higher virus-neutralizing titers against two H1N1 strains of intermediate antigenic distance between the H1N1 vaccine strains and H1N1pdm09, compared with the naive control group. Furthermore, both vaccine groups had higher HA-stem antibodies early after infection than the control group. In conclusion, immunization with VLPs displaying HA from antigenically distinct H1N1 variants increased the breadth of the immune response during subsequent H1N1pdm09 challenge, although this phenomenon was limited to intermediate antigenic variants.

Identification of new drugs to counteract anti-spike IgG-induced hyperinflammation in severe COVID-19.

Previously, we and others have shown that SARS-CoV-2 spike-specific IgG antibodies play a major role in disease severity in COVID-19 by triggering macrophage hyperactivation, disrupting endothelial barrier integrity, and inducing thrombus formation. This hyperinflammation is dependent on high levels of anti-spike IgG with aberrant Fc tail glycosylation, leading to Fcγ receptor hyperactivation. For development of immune-regulatory therapeutics, drug specificity is crucial to counteract excessive inflammation whereas simultaneously minimizing the inhibition of antiviral immunity. We here developed an in vitro activation assay to screen for small molecule drugs that specifically counteract antibody-induced pathology. We identified that anti-spike-induced inflammation is specifically blocked by small molecule inhibitors against SYK and PI3K. We identified SYK inhibitor entospletinib as the most promising candidate drug, which also counteracted anti-spike-induced endothelial dysfunction and thrombus formation. Moreover, entospletinib blocked inflammation by different SARS-CoV-2 variants of concern. Combined, these data identify entospletinib as a promising treatment for severe COVID-19.

Signatures of VH1-69-derived hepatitis C virus neutralizing antibody precursors defined by binding to envelope glycoproteins.

An effective preventive vaccine for hepatitis C virus (HCV) remains a major unmet need. Antigenic region 3 (AR3) on the E1E2 envelope glycoprotein complex overlaps with the CD81 receptor binding site and represents an important epitope for broadly neutralizing antibodies (bNAbs) and is therefore important for HCV vaccine design. Most AR3 bNAbs utilize the VH1-69 gene and share structural features that define the AR3C-class of HCV bNAbs. In this work, we identify recombinant HCV glycoproteins based on a permuted E2E1 trimer design that bind to the inferred VH1-69 germline precursors of AR3C-class bNAbs. When presented on nanoparticles, these recombinant E2E1 glycoproteins efficiently activate B cells expressing inferred germline AR3C-class bNAb precursors as B cell receptors. Furthermore, we identify critical signatures in three AR3C-class bNAbs that represent two subclasses of AR3C-class bNAbs that will allow refined protein design. These results provide a framework for germline-targeting vaccine design strategies against HCV.

Fourth mRNA COVID-19 vaccination in immunocompromised patients with haematological malignancies (COBRA KAI): a cohort study.

Background: Patients with haematological malignancies have impaired antibody responses to SARS-CoV-2 vaccination. We aimed to investigate whether a fourth mRNA COVID-19 vaccination improved antibody quantity and quality. Methods: In this cohort study, conducted at 5 sites in the Netherlands, we compared antibody concentrations 28 days after 4 mRNA vaccinations (3-dose primary series plus 1 booster vaccination) in SARS-CoV-2 naive, immunocompromised patients with haematological malignancies to those obtained by age-matched, healthy individuals who had received the standard primary 2-dose mRNA vaccination schedule followed by a first booster mRNA vaccination. Prior to and 4 weeks after each vaccination, peripheral blood samples and data on demographic parameters and medical history were collected. Concentrations of antibodies that bind spike 1 (S1) and nucleocapsid (N) protein of SARS-CoV-2 were quantified in binding antibody units (BAU) per mL according to the WHO International Standard for COVID-19 serological tests. Seroconversion was defined as an S1 IgG concentration >10 BAU/mL and a previous SARS-CoV-2 infection as N IgG >14.3 BAU/mL. Antibody neutralising activity was tested using lentiviral-based pseudoviruses expressing spike protein of SARS-CoV-2 wild-type (D614G), Omicron BA.1, and Omicron BA.4/5 variants. This study is registered with EudraCT, number 2021-001072-41. Findings: Between March 24, 2021 and May 4, 2021, 723 patients with haematological diseases were enrolled, of which 414 fulfilled the inclusion criteria for the current analysis. Although S1 IgG concentrations in patients significantly improved after the fourth dose, they remained significantly lower compared to those obtained by 58 age-matched healthy individuals after their first booster (third) vaccination. The rise in neutralising antibody concentration was most prominent in patients with a recovering B cell compartment, although potent responses were also observed in patients with persistent immunodeficiencies. 19% of patients never seroconverted, despite 4 vaccinations. Patients who received their first 2 vaccinations when they were B cell depleted and the third and fourth vaccination during B cell recovery demonstrated similar antibody induction dynamics as patients with normal B cell numbers during the first 2 vaccinations. However, the neutralising capacity of these antibodies was significantly better than that of patients with normal B cell numbers after two vaccinations. Interpretation: A fourth mRNA COVID-19 vaccination improved S1 IgG concentrations in the majority of patients with a haematological malignancy. Vaccination during B cell depletion may pave the way for better quality of antibody responses after B cell reconstitution. Funding: The Netherlands Organisation for Health Research and Development and Amsterdam UMC.

Robust Vaccine-Induced as Well as Hybrid B- and T-Cell Immunity across SARS-CoV-2 Vaccine Platforms in People with HIV.

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 (ß) 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 (ß = 0.77), receiving mRNA vaccine (ß = 0.56), female sex (ß = 0.24), fewer days between last vaccination and sampling (ß = 0.07), and a CD4/CD8 ratio of <1.0 (ß = -0.39) were independently associated with anti-S IgG titers, but HIV status was not. Neutralization titers against the ancestral and Delta and Omicron SARS-CoV-2 variants were not different between PWH and controls. IFN-γ release was higher in PWH. Prior SARS-CoV-2 infection (ß = 2.39), HIV-positive status (ß = 1.61), and fewer days between last vaccination and sampling (ß = 0.23) were independently associated with higher IFN-γ release. The percentages of SARS-CoV-2-reactive CD4+ and CD8+ T cells, however, were not different between PWH and controls. Individuals with well-controlled HIV generally mount robust vaccine-induced as well as hybrid B- and T-cell immunity across SARS-CoV-2 vaccine platforms similar to controls. Determinants of a reduced vaccine response were likewise largely similar in both groups and included a lower CD4/CD8 ratio. IMPORTANCE Some studies have suggested that people with HIV may respond less well to vaccines against SARS-CoV-2. We comprehensively compared B- and T-cell responses to different COVID-19 vaccines in middle-aged persons with well-treated HIV and individuals of the same age without HIV, who were also highly comparable in terms of demographics and lifestyle, including those with prior SARS-CoV-2 infection. Individuals with HIV generally mounted equally robust immunity to the different vaccines. Even stronger immunity was observed in both groups after prior SARS-CoV-2 infection. These findings are reassuring with respect to the efficacy of SARS-Cov-2 vaccines for the sizable and increasing global population of people with HIV with access and a good response to HIV treatment.

Immune Responses 6 Months After mRNA-1273 COVID-19 Vaccination and the Effect of a Third Vaccination in Patients with Inborn Errors of Immunity.

PURPOSE: Patients with inborn errors of immunity (IEI) are at increased risk of severe coronavirus disease-2019 (COVID-19). Effective long-term protection against COVID-19 is therefore of great importance in these patients, but little is known about the decay of the immune response after primary vaccination. We studied the immune responses 6 months after two mRNA-1273 COVID-19 vaccines in 473 IEI patients and subsequently the response to a third mRNA COVID-19 vaccine in 50 patients with common variable immunodeficiency (CVID). METHODS: In a prospective multicenter study, 473 IEI patients (including X-linked agammaglobulinemia (XLA) (N = 18), combined immunodeficiency (CID) (N = 22), CVID (N = 203), isolated or undefined antibody deficiencies (N = 204), and phagocyte defects (N = 16)), and 179 controls were included and followed up to 6 months after two doses of the mRNA-1273 COVID-19 vaccine. Additionally, samples were collected from 50 CVID patients who received a third vaccine 6 months after primary vaccination through the national vaccination program. SARS-CoV-2-specific IgG titers, neutralizing antibodies, and T cell responses were assessed. RESULTS: At 6 months after vaccination, the geometric mean antibody titers (GMT) declined in both IEI patients and healthy controls, when compared to GMT 28 days after vaccination. The trajectory of this decline did not differ between controls and most IEI cohorts; however, antibody titers in CID, CVID, and isolated antibody deficiency patients more often dropped to below the responder cut-off compared to controls. Specific T cell responses were still detectable in 77% of controls and 68% of IEI patients at 6 months post vaccination. A third mRNA vaccine resulted in an antibody response in only two out of 30 CVID patients that did not seroconvert after two mRNA vaccines. CONCLUSION: A similar decline in IgG titers and T cell responses was observed in patients with IEI when compared to healthy controls 6 months after mRNA-1273 COVID-19 vaccination. The limited beneficial benefit of a third mRNA COVID-19 vaccine in previous non-responder CVID patients implicates that other protective strategies are needed for these vulnerable patients.

Different configurations of SARS-CoV-2 spike protein delivered by integrase-defective lentiviral vectors induce persistent functional immune responses, characterized by distinct immunogenicity profiles.

Several COVID-19 vaccine strategies utilizing new formulations for the induction of neutralizing antibodies (nAbs) and T cell immunity are still under evaluation in preclinical and clinical studies. Here we used Simian Immunodeficiency Virus (SIV)-based integrase defective lentiviral vector (IDLV) delivering different conformations of membrane-tethered Spike protein in the mouse immunogenicity model, with the aim of inducing persistent nAbs against multiple SARS-CoV-2 variants of concern (VoC). Spike modifications included prefusion-stabilizing double proline (2P) substitutions, mutations at the furin cleavage site (FCS), D614G mutation and truncation of the cytoplasmic tail (delta21) of ancestral and Beta (B.1.351) Spike, the latter mutation to markedly improve IDLV membrane-tethering. BALB/c mice were injected once with IDLV delivering the different forms of Spike or the recombinant trimeric Spike protein with 2P substitutions and FCS mutations in association with a squalene-based adjuvant. Anti-receptor binding domain (RBD) binding Abs, nAbs and T cell responses were detected up to six months from a single immunization with escalating doses of vaccines in all mice, but with different levels and kinetics. Results indicated that IDLV delivering the Spike protein with all the combined modifications, outperformed the other candidates in terms of T cell immunity and level of both binding Abs and nAbs soon after the single immunization and persistence over time, showing the best capacity to neutralize all formerly circulating VoC Alpha, Beta, Gamma and Delta. Although present, the lowest response was detected against Omicron variants (BA.1, BA.2 and BA.4/5), suggesting that the magnitude of immune evasion may be related to the higher genetic distance of Omicron as indicated by increased number of amino acid substitutions in Spike acquired during virus evolution.

Bispecific antibodies combine breadth, potency, and avidity of parental antibodies to neutralize sarbecoviruses.

SARS-CoV-2 variants evade current monoclonal antibody therapies. Bispecific antibodies (bsAbs) combine the specificities of two distinct antibodies taking advantage of the avidity and synergy provided by targeting different epitopes. Here we used controlled Fab-arm exchange to produce bsAbs that neutralize SARS-CoV and SARS-CoV-2 variants, including Omicron and its subvariants, by combining potent SARS-CoV-2-specific neutralizing antibodies with broader antibodies that also neutralize SARS-CoV. We demonstrated that the parental antibodies rely on avidity for neutralization using bsAbs containing one irrelevant Fab arm. Using mass photometry to measure the formation of antibody:spike complexes, we determined that bsAbs increase binding stoichiometry compared to corresponding cocktails, without a loss of binding affinity. The heterogeneous binding pattern of bsAbs to spike, observed by negative-stain electron microscopy and mass photometry provided evidence for both intra- and inter-spike crosslinking. This study highlights the utility of cross-neutralizing antibodies for designing bivalent agents to combat circulating and future SARS-like coronaviruses.

Understanding repertoire sequencing data through a multiscale computational model of the germinal center.

Sequencing of B-cell and T-cell immune receptor repertoires helps us to understand the adaptive immune response, although it only provides information about the clonotypes (lineages) and their frequencies and not about, for example, their affinity or antigen (Ag) specificity. To further characterize the identified clones, usually with special attention to the particularly abundant ones (dominant), additional time-consuming or expensive experiments are generally required. Here, we present an extension of a multiscale model of the germinal center (GC) that we previously developed to gain more insight in B-cell repertoires. We compare the extent that these simulated repertoires deviate from experimental repertoires established from single GCs, blood, or tissue. Our simulations show that there is a limited correlation between clonal abundance and affinity and that there is large affinity variability among same-ancestor (same-clone) subclones. Our simulations suggest that low-abundance clones and subclones, might also be of interest since they may have high affinity for the Ag. We show that the fraction of plasma cells (PCs) with high B-cell receptor (BcR) mRNA content in the GC does not significantly affect the number of dominant clones derived from single GCs by sequencing BcR mRNAs. Results from these simulations guide data interpretation and the design of follow-up experiments.

Distinct dynamics of antigen-specific induction and differentiation of different CD11c+Tbet+ B-cell subsets.

BACKGROUND: CD11c+Tbet+ B cells are enriched in autoimmunity and chronic infections and also expand on immune challenge in healthy individuals. CD11c+Tbet+ B cells remain an enigmatic B-cell population because of their intrinsic heterogeneity. OBJECTIVES: We investigated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen-specific development and differentiation properties of 3 separate CD11c+ B-cell subsets-age-associated B cells (ABCs), double-negative 2 (DN2) B cells, and activated naive B cells-and compared them to their canonical CD11c- counterparts. METHODS: Dynamics of the response of the 3 CD11c+ B-cell subsets were assessed at SARS-CoV-2 vaccination in healthy donors by spectral flow cytometry. Distinct CD11c+ B-cell subsets were functionally characterized by optimized in vitro cultures. RESULTS: In contrast to a durable expansion of antigen-specific CD11c- memory B cells over time, both ABCs and DN2 cells were strongly expanded shortly after second vaccination and subsequently contracted. Functional characterization of antibody-secreting cell differentiation dynamics revealed that CD11c+Tbet+ B cells were primed for antibody-secreting cell differentiation compared to relevant canonical CD11c- counterparts. CONCLUSION: Overall, CD11c+Tbet+ B cells encompass heterogeneous subpopulations, of which primarily ABCs as well as DN2 B cells respond early to immune challenge and display a pre-antibody-secreting cell phenotype.