Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients.

The SARS-CoV-2 pandemic has unprecedented implications for public health, social life, and the world economy. Because approved drugs and vaccines are limited or not available, new options for COVID-19 treatment and prevention are in high demand. To identify SARS-CoV-2-neutralizing antibodies, we analyzed the antibody response of 12 COVID-19 patients from 8 to 69 days after diagnosis. By screening 4,313 SARS-CoV-2-reactive B cells, we isolated 255 antibodies from different time points as early as 8 days after diagnosis. Of these, 28 potently neutralized authentic SARS-CoV-2 with IC100 as low as 0.04 µg/mL, showing a broad spectrum of variable (V) genes and low levels of somatic mutations. Interestingly, potential precursor sequences were identified in naive B cell repertoires from 48 healthy individuals who were sampled before the COVID-19 pandemic. Our results demonstrate that SARS-CoV-2-neutralizing antibodies are readily generated from a diverse pool of precursors, fostering hope for rapid induction of a protective immune response upon vaccination.

Single-cell analysis of memory B cells from top neutralizers reveals multiple sites of vulnerability within HCMV Trimer and Pentamer.

Human cytomegalovirus (HCMV) can cause severe diseases in fetuses, newborns, and immunocompromised individuals. Currently, no vaccines are approved, and treatment options are limited. Here, we analyzed the human B cell response of four HCMV top neutralizers from a cohort of 9,000 individuals. By single-cell analyses of memory B cells targeting the pentameric and trimeric HCMV surface complexes, we identified vulnerable sites on the shared gH/gL subunits as well as complex-specific subunits UL128/130/131A and gO. Using high-resolution cryogenic electron microscopy, we revealed the structural basis of the neutralization mechanisms of antibodies targeting various binding sites. Moreover, we identified highly potent antibodies that neutralized a broad spectrum of HCMV strains, including primary clinical isolates, that outperform known antibodies used in clinical trials. Our study provides a deep understanding of the mechanisms of HCMV neutralization and identifies promising antibody candidates to prevent and treat HCMV infection.

Analysis of antibodies from HCV elite neutralizers identifies genetic determinants of broad neutralization.

The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates.

Correction: Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV.

[This corrects the article DOI: 10.1371/journal.ppat.1008560.].

Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV.

Human cytomegalovirus (HCMV) causes serious complications to immune compromised hosts. Dendritic cells (iDCgB) expressing granulocyte-macrophage colony-stimulating factor, interferon-alpha and HCMV-gB were developed to promote de novo antiviral adaptive responses. Mice reconstituted with a human immune system (HIS) were immunized with iDCgB and challenged with HCMV, resulting into 93% protection. Immunization stimulated the expansion of functional effector memory CD8+ and CD4+ T cells recognizing gB. Machine learning analyses confirmed bone marrow T/CD4+, liver B/IgA+ and spleen B/IgG+ cells as predictive biomarkers of immunization (≈87% accuracy). CD8+ and CD4+ T cell responses against gB were validated. Splenic gB-binding IgM-/IgG+ B cells were sorted and analyzed at a single cell level. iDCgB immunizations elicited human-like IgG responses with a broad usage of various IgG heavy chain V gene segments harboring variable levels of somatic hypermutation. From this search, two gB-binding human monoclonal IgGs were generated that neutralized HCMV infection in vitro. Passive immunization with these antibodies provided proof-of-concept evidence of protection against HCMV infection. This HIS/HCMV in vivo model system supported the validation of novel active and passive immune therapies for future clinical translation.

Evaluation of a Rapid Antigen Test To Detect SARS-CoV-2 Infection and Identify Potentially Infectious Individuals.

The identification and isolation of highly infectious SARS-CoV-2-infected individuals is an important public health strategy. Rapid antigen detection tests (RADT) are promising tools for large-scale screenings due to timely results and feasibility for on-site testing. Nonetheless, the diagnostic performance of RADT in detecting infectious individuals is not yet fully determined. In this study, RT-qPCR and virus culture of RT-qPCR-positive samples were used to evaluate and compare the performance of the Standard Q COVID-19 Ag test in detecting SARS-CoV-2-infected and possibly infectious individuals. To this end, two combined oro- and nasopharyngeal swabs were collected at a routine SARS-CoV-2 diagnostic center. A total of 2,028 samples were tested, and 118 virus cultures were inoculated. SARS-CoV-2 infection was detected in 210 samples by RT-qPCR, representing a positive rate of 10.36%. The Standard Q COVID-19 Ag test yielded a positive result in 92 (4.54%) samples resulting in an overall sensitivity and specificity of 42.86 and 99.89%, respectively. For adjusted CT values of <20 (n = 14), <25 (n = 57), and <30 (n = 88), the RADT reached sensitivities of 100, 98.25, and 88.64%, respectively. All 29 culture-positive samples were detected by the RADT. Although the overall sensitivity was low, the Standard Q COVID-19 Ag test reliably detected patients with high RNA loads. In addition, negative RADT results fully corresponded with the lack of viral cultivability in Vero E6 cells. These results indicate that RADT can be a valuable tool for the detection of individuals with high RNA loads that are likely to transmit SARS-CoV-2.

LMP1 and EBNA2 constitute a minimal set of EBV genes for transformation of human B cells.

Introduction: Epstein-Barr virus (EBV) infection in humans is associated with a wide range of diseases including malignancies of different origins, most prominently B cells. Several EBV latent genes are thought to act together in B cell immortalization, but a minimal set of EBV genes sufficient for transformation remains to be identified. Methods: Here, we addressed this question by transducing human peripheral B cells from EBV-negative donors with retrovirus expressing the latent EBV genes encoding Latent Membrane Protein (LMP) 1 and 2A and Epstein-Barr Nuclear Antigen (EBNA) 2. Results: LMP1 together with EBNA2, but not LMP1 alone or in combination with LMP2A was able to transform human primary B cells. LMP1/EBNA2-immortalized cell lines shared surface markers with EBV-transformed lymphoblastoid cell lines (LCLs). They showed sustained growth for more than 60 days, albeit at a lower growth rate than EBV-transformed LCLs. LMP1/EBNA2-immortalized cell lines generated tumors when transplanted subcutaneously into severely immunodeficient NOG mice. Conclusion: Our results identify a minimal set of EBV proteins sufficient for B cell transformation.

Mapping the neutralizing specificity of human anti-HIV serum by deep mutational scanning.

Understanding the specificities of human serum antibodies that broadly neutralize HIV can inform prevention and treatment strategies. Here we describe a deep mutational scanning system that can measure the effects of combinations of mutations to HIV envelope (Env) on neutralization by antibodies and polyclonal serum. We first show that this system can accurately map how all functionally tolerated mutations to Env affect neutralization by monoclonal antibodies. We then comprehensively map Env mutations that affect neutralization by a set of human polyclonal sera known to target the CD4-binding site that neutralize diverse strains of HIV. The neutralizing activities of these sera target different epitopes, with most sera having specificities reminiscent of individual characterized monoclonal antibodies, but one sera targeting two epitopes within the CD4 binding site. Mapping the specificity of the neutralizing activity in polyclonal human serum will aid in assessing anti-HIV immune responses to inform prevention strategies.

Mapping the neutralizing specificity of human anti-HIV serum by deep mutational scanning.

Understanding the specificities of human serum antibodies that broadly neutralize HIV can inform prevention and treatment strategies. Here, we describe a deep mutational scanning system that can measure the effects of combinations of mutations to HIV envelope (Env) on neutralization by antibodies and polyclonal serum. We first show that this system can accurately map how all functionally tolerated mutations to Env affect neutralization by monoclonal antibodies. We then comprehensively map Env mutations that affect neutralization by a set of human polyclonal sera that neutralize diverse strains of HIV and target the site engaging the host receptor CD4. The neutralizing activities of these sera target different epitopes, with most sera having specificities reminiscent of individual characterized monoclonal antibodies, but one serum targeting two epitopes within the CD4-binding site. Mapping the specificity of the neutralizing activity in polyclonal human serum will aid in assessing anti-HIV immune responses to inform prevention strategies.

Enhanced SARS-CoV-2 humoral immunity following breakthrough infection builds upon the preexisting memory B cell pool.

The human immune response must continuously adapt to newly emerging SARS-CoV-2 variants. To investigate how B cells respond to repeated SARS-CoV-2 antigen exposure by Wu01 booster vaccination and Omicron breakthrough infection, we performed a molecular longitudinal analysis of the memory B cell pool. We demonstrate that a subsequent breakthrough infection substantially increases the frequency of B cells encoding SARS-CoV-2-neutralizing antibodies. However, this is not primarily attributable to maturation, but to selection of preexisting B cell clones. Moreover, broadly reactive memory B cells arose early and even neutralized highly mutated variants like XBB.1.5 that the individuals had not encountered. Together, our data show that SARS-CoV-2 immunity is largely imprinted on Wu01 over the course of multiple antigen contacts but can respond to new variants through preexisting diversity.

Probabilities of developing HIV-1 bNAb sequence features in uninfected and chronically infected individuals.

HIV-1 broadly neutralizing antibodies (bNAbs) are able to suppress viremia and prevent infection. Their induction by vaccination is therefore a major goal. However, in contrast to antibodies that neutralize other pathogens, HIV-1-specific bNAbs frequently carry uncommon molecular characteristics that might prevent their induction. Here, we perform unbiased sequence analyses of B cell receptor repertoires from 57 uninfected and 46 chronically HIV-1- or HCV-infected individuals and learn probabilistic models to predict the likelihood of bNAb development. We formally show that lower probabilities for bNAbs are predictive of higher HIV-1 neutralization activity. Moreover, ranking bNAbs by their probabilities allows to identify highly potent antibodies with superior generation probabilities as preferential targets for vaccination approaches. Importantly, we find equal bNAb probabilities across infected and uninfected individuals. This implies that chronic infection is not a prerequisite for the generation of bNAbs, fostering the hope that HIV-1 vaccines can induce bNAb development in uninfected people.

The unique ORF8 protein from SARS-CoV-2 binds to human dendritic cells and induces a hyper-inflammatory cytokine storm.

The novel coronavirus pandemic, first reported in December 2019, was caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection leads to a strong immune response and activation of antigen-presenting cells, which can elicit acute respiratory distress syndrome (ARDS) characterized by the rapid onset of widespread inflammation, the so-called cytokine storm. In response to viral infections, monocytes are recruited into the lung and subsequently differentiate into dendritic cells (DCs). DCs are critical players in the development of the acute lung inflammation that causes ARDS. Here we focus on the interaction of a specific SARS-CoV-2 open reading frame protein, ORF8, with DCs. We show that ORF8 binds to DCs, causes a pre-maturation of differentiating DCs, and induces the secretion of multiple proinflammatory cytokines by these cells. In addition, we identified DC-SIGN as a possible interaction partner of ORF8 on DCs. Blockade of ORF8 leads to reduced production of IL-1ß, IL-6, IL-12p70, TNF-α, MCP-1 (also named CCL2), and IL-10 by DCs. Therefore, a neutralizing antibody blocking the ORF8-mediated cytokine and chemokine response could be an improved therapeutical strategy against severe SARS-CoV-2.

Dynamics and durability of HIV-1 neutralization are determined by viral replication.

Human immunodeficiency virus type 1 (HIV-1)-neutralizing antibodies (nAbs) that prevent infection are the main goal of HIV vaccine discovery. But as no nAb-eliciting vaccines are yet available, only data from HIV-1 neutralizers-persons with HIV-1 who naturally develop broad and potent nAbs-can inform about the dynamics and durability of nAb responses in humans, knowledge which is crucial for the design of future HIV-1 vaccine regimens. To address this, we assessed HIV-1-neutralizing immunoglobulin G (IgG) from 2,354 persons with HIV-1 on or off antiretroviral therapy (ART). Infection with non-clade B viruses, CD4+ T cell counts <200 µl-1, being off ART and a longer time off ART were independent predictors of a more potent and broad neutralization. In longitudinal analyses, we found nAb half-lives of 9.3 and 16.9 years in individuals with no- or low-level viremia, respectively, and 4.0 years in persons who newly initiated ART. Finally, in a potent HIV-1 neutralizer, we identified lower fractions of serum nAbs and of nAb-encoding memory B cells after ART initiation, suggesting that a decreasing neutralizing serum activity after antigen withdrawal is due to lower levels of nAbs. These results collectively show that HIV-1-neutralizing responses can persist for several years, even at low antigen levels, suggesting that an HIV-1 vaccine may elicit a durable nAb response.

No substantial preexisting B cell immunity against SARS-CoV-2 in healthy adults.

Preexisting immunity against SARS-CoV-2 may have critical implications for our understanding of COVID-19 susceptibility and severity. The presence and clinical relevance of a preexisting B cell immunity remain to be fully elucidated. Here, we provide a detailed analysis of the B cell immunity to SARS-CoV-2 in unexposed individuals. To this end, we extensively investigated SARS-CoV-2 humoral immunity in 150 adults sampled pre-pandemically. Comprehensive screening of donor plasma and purified IgG samples for binding and neutralization in various functional assays revealed no substantial activity against SARS-CoV-2 but broad reactivity to endemic betacoronaviruses. Moreover, we analyzed antibody sequences of 8,174 putatively SARS-CoV-2-reactive B cells at a single cell level and generated and tested 158 monoclonal antibodies. None of these antibodies displayed relevant binding or neutralizing activity against SARS-CoV-2. Taken together, our results show no evidence of competent preexisting antibody and B cell immunity against SARS-CoV-2 in unexposed adults.

Pausing methotrexate prevents impairment of Omicron BA.1 and BA.2 neutralisation after COVID-19 booster vaccination.

OBJECTIVE: The level of neutralising capacity against Omicron BA.1 and BA.2 after third COVID-19 vaccination in patients on paused or continuous methotrexate (MTX) therapy is unclear. METHODS: In this observational cohort study, neutralising serum activity against SARS-CoV-2 wild-type (Wu01) and variant of concern Omicron BA.1 and BA.2 were assessed by pseudovirus neutralisation assay before, 4 and 12 weeks after mRNA booster immunisation in 50 rheumatic patients on MTX, 26 of whom paused the medication. 44 non-immunosuppressed persons (NIP) served as control group. RESULTS: While the neutralising serum activity against SARS-CoV-2 Wu01 and Omicron variants increased 67-73 fold in the NIP after booster vaccination, the serum activity in patients receiving MTX increased only 20-23 fold. Patients who continued MTX treatment during vaccination had significantly lower neutralisation against all variants at weeks 4 and 12 compared with patients who paused MTX and the control group, except for BA.2 at week 12. Patients who paused MTX reached comparably high neutralising capacities as NIP, except for Wu01 at week 12. The duration of the MTX pause after-not before-was associated with a significantly higher neutralisation capacity against all three variants, with an optimal duration at 10 days after vaccination. CONCLUSION: Patients pausing MTX after COVID-19 booster showed a similar vaccine response to NIP. Patients who continued MTX demonstrated an impaired response indicating a potentially beneficial second booster vaccination. Our data also suggest that a 1 week MTX break is sufficient if the last administration of MTX occurs 1-3 days before vaccination.

Discovery of ultrapotent broadly neutralizing antibodies from SARS-CoV-2 elite neutralizers.

A fraction of COVID-19 convalescent individuals mount a potent antibody response to SARS-CoV-2 with cross-reactivity to SARS-CoV-1. To uncover their humoral response in detail, we performed single B cell analysis from 10 SARS-CoV-2 elite neutralizers. We isolated and analyzed 126 monoclonal antibodies, many of which were sarbecovirus cross-reactive, with some displaying merbecovirus- and embecovirus-reactivity. Several isolated broadly neutralizing antibodies were effective against B.1.1.7, B.1.351, B.1.429, B.1.617, and B.1.617.2 variants and 19 prominent potential escape sites. Furthermore, assembly of 716,806 SARS-CoV-2 sequences predicted emerging escape variants, which were also effectively neutralized. One of these broadly neutralizing potent antibodies, R40-1G8, is a IGHV3-53 RBD-class-1 antibody. Remarkably, cryo-EM analysis revealed that R40-1G8 has a flexible binding mode, targeting both "up" and "down" conformations of the RBD. Given the threat of emerging SARS-CoV-2 variants, we demonstrate that elite neutralizers are a valuable source for isolating ultrapotent antibody candidates to prevent and treat SARS-CoV-2 infection.

Effective high-throughput isolation of fully human antibodies targeting infectious pathogens.

As exemplified by the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there is a strong demand for rapid high-throughput isolation pipelines to identify potent neutralizing antibodies for prevention and therapy of infectious diseases. However, despite substantial progress and extensive efforts, the identification and production of antigen-specific antibodies remains labor- and cost-intensive. We have advanced existing concepts to develop a highly efficient high-throughput protocol with proven application for the isolation of potent antigen-specific antibodies against human immunodeficiency virus 1, hepatitis C virus, human cytomegalovirus, Middle East respiratory syndrome coronavirus, SARS-CoV-2 and Ebola virus. It is based on computationally optimized multiplex primer sets (openPrimeR), which guarantee high coverage of even highly mutated immunoglobulin gene segments as well as on optimized antibody cloning and production strategies. Here, we provide the detailed protocol, which covers all critical steps from sample collection to antibody production within 12-14 d.

Development and characterization of an indirect ELISA to detect SARS-CoV-2 spike protein-specific antibodies.

The current Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) pandemic is a public health emergency of international concern. Sensitive and precise diagnostic tools are urgently needed. In this study, we developed a SARS-CoV-2 spike (S1) protein enzyme-linked immunosorbent assay (ELISA) to detect SARS-CoV-2-specific antibodies. The SARS-CoV-2 S1 ELISA was found to be specific [97.8% (95% CI, 96.7% - 98.5%)], reproducible and precise (intra-assay coefficient of variability (CV) 5.3%, inter-assay CV 7.9%). A standard curve and the interpolation of arbitrary ELISA units per milliliter served to reduce the variability between different tests and operators. Cross-reactivity to other human coronaviruses was addressed by using sera positive for MERS-CoV- and hCoV HKU1-specific antibodies. Monitoring antibody development in various samples of twenty-three and single samples of twenty-nine coronavirus disease 2019 (COVID-19) patients revealed seroconversion and neutralizing antibodies against authentic SARS-CoV-2 in all cases. The comparison of the SARS-CoV-2 (S1) ELISA with a commercially available assay showed a better sensitivity for the in-house ELISA. The results demonstrate a high reproducibility, specificity and sensitivity of the newly developed ELISA, which is suitable for the detection of SARS-CoV-2 S1 protein-specific antibody responses.

Evaluation of a New Spike (S)-Protein-Based Commercial Immunoassay for the Detection of Anti-SARS-CoV-2 IgG.

Background: The investigation of the antibody response to SARS-CoV-2 represents a key aspect in facing the COVID-19 pandemic. In the present study, we compared the new Immundiagnostik IDK® anti-SARS-CoV-2 S1 IgG assay with four widely-used commercial serological assays for the detection of antibodies targeting S (spike) and NC (nucleocapsid) proteins. Methods: Serum samples were taken from an unbiased group of convalescent patients and from a negative control group. Sample were simultaneously analyzed by the new Immundiagnostik IDK® anti-SARS-CoV-2 S1 IgG assay, by the DiaSorin LIAISON® SARS-CoV-2 S1/S2 IgG assay, and by the Euroimmun anti-SARS-CoV-2 S1 IgG ELISA. Antibodies binding NC were detected by the Abbott SARS-CoV-2 IgG assay and by the pan-immunoglobulin immunoassay Roche Elecsys® anti-SARS-CoV-2. Moreover, we investigated samples of a group of COVID-19 convalescent subjects that were primarily tested S1 IgG non-reactive. Samples were also tested by live virus and pseudovirus neutralization tests. Results: Overall, the IDK® anti-SARS-CoV-2 S1 IgG assay showed the highest sensitivity among the evaluated spike (S) protein-based assays. Additionally, the Immundiagnostik assay correlated well with serum-neutralizing activity. Conclusions: The novel IDK® anti-SARS-CoV-2 S1 IgG assay showed high sensitivity and specificity, representing a valid option for use in the routine diagnostic.