CARs derived from broadly neutralizing, human monoclonal antibodies identified by single B cell sorting target hepatitis B virus-positive cells.

To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs.

Blocking the angiopoietin-2-dependent integrin ß-1 signaling axis abrogates small cell lung cancer invasion and metastasis.

Small cell lung cancer (SCLC) is the most aggressive lung cancer entity with an extremely limited therapeutic outcome. Most patients are diagnosed at an extensive stage. However, the molecular mechanisms driving SCLC invasion and metastasis remain largely elusive. We used an autochthonous SCLC mouse model and matched samples from patients with primary and metastatic SCLC to investigate the molecular characteristics of tumor metastasis. We demonstrate that tumor cell invasion and liver metastasis in SCLC are triggered by an Angiopoietin-2 (ANG-2)/Integrin ß-1-dependent pathway in tumor cells, mediated by focal adhesion kinase/Src kinase signaling. Strikingly, CRISPR-Cas9 KO of Integrin ß-1 or blocking Integrin ß-1 signaling by an anti-ANG-2 treatment abrogates liver metastasis formation in vivo. Interestingly, analysis of a unique collection of matched samples from patients with primary and metastatic SCLC confirmed a strong increase of Integrin ß-1 in liver metastasis in comparison with the primary tumor. We further show that ANG-2 blockade combined with PD-1-targeted by anti-PD-1 treatment displays synergistic treatment effects in SCLC. Together, our data demonstrate a fundamental role of ANG-2/Integrin ß-1 signaling in SCLC cells for tumor cell invasion and liver metastasis and provide a potentially new effective treatment strategy for patients with SCLC.

SARS-CoV-2 Test-to-Stay in Daycare.

BACKGROUND AND OBJECTIVES: Test-to-stay concepts apply serial testing of children in daycare after exposure to SARS-CoV-2 without use of quarantine. This study aims to assess the safety of a test-to-stay screening in daycare facilities. METHODS: 714 daycare facilities and approximately 50 000 children ≤6 years in Cologne, Germany participated in a SARS-CoV-2 Pool-polymerase chain reaction (PCR) screening from March 2021 to April 2022. The screening initially comprised post-exposure quarantine and was adapted to a test-to-stay approach during its course. To assess safety of the test-to-stay approach, we explored potential changes in frequencies of infections among children after the adaptation to the test-to-stay approach by applying regression discontinuity in time (RDiT) analyses. To this end, PCR-test data were linked with routinely collected data on reported infections in children and analyzed using ordinary least squares regressions. RESULTS: 219 885 Pool-PCRs and 352 305 Single-PCRs were performed. 6440 (2.93%) Pool-PCRs tested positive, and 17 208 infections in children were reported. We estimated that during a period of 30 weeks, the test-to-stay concept avoided between 7 and 20 days of quarantine per eligible daycare child. RDiT revealed a 26% reduction (Exp. Coef: 0.74, confidence interval 0.52-1.06) in infection frequency among children and indicated no significant increase attributable to the test-to-stay approach. This result was not sensitive to adjustments for 7-day incidence, season, SARS-CoV-2 variant, and socioeconomic status. CONCLUSIONS: Our analyses provide evidence that suggest safety of the test-to-stay approach compared with quarantine measures. This approach offers a promising option to avoid use of quarantine after exposure to respiratory pathogens in daycare settings.

Semen enhances transmitted/founder HIV-1 infection and only marginally reduces antiviral activity of broadly neutralizing antibodies.

Topically applied microbicides may play a critical role in preventing sexual transmission of human immunodeficiency virus type 1 (HIV-1); however, their efficacy can be compromised by amyloid fibrils present in semen, which significantly increase HIV-1 infectivity. This phenomenon may have contributed to the failure of most microbicide candidates in clinical settings. Understanding the impact of semen on microbicide effectiveness is thus crucial. In our study, we evaluated the influence of semen on the neutralizing activity of broadly neutralizing antibodies (bNAbs), including PG16, PGT121, 10-1074, 3BNC117, and VRC01, which are potential microbicide candidates. We found that semen enhances infection of HIV-1 transmitted/founder viruses but only marginally affects the neutralizing activity of tested antibodies, suggesting their potential for microbicide application. Our findings underscore the need to consider semen-mediated enhancement when evaluating and developing microbicides and highlight the potential of incorporating HIV-1 bNAbs in formulations to enhance efficacy and mitigate HIV-1 transmission during sexual encounters.IMPORTANCEThis study examined the impact of semen on the development of microbicides, substances used to prevent the transmission of HIV-1 during sexual activity. Semen contains certain components that can render the virus more infectious, posing a challenge to microbicide effectiveness. Researchers specifically investigated the effect of semen on a group of powerful antibodies called broadly neutralizing antibodies, which can neutralize a large spectrum of different HIV-1 variants. The results revealed that semen only had a minimal effect on the antibodies' ability to neutralize the virus. This is promising because it suggests that these antibodies could still be effective in microbicides, even in the presence of semen. Understanding this interaction is crucial for developing better strategies to prevent HIV-1 transmission. By incorporating the knowledge gained from this study, scientists can now focus on creating microbicides that consider the impact of semen, bringing us closer to more effective prevention methods.

Hybrid immunity to SARS-CoV-2 in patients with chronic lymphocytic leukemia.

OBJECTIVE: Preventing severe COVID-19 remains a priority globally, particularly in the immunocompromised population. As shown in healthy individuals, immunity against SARS-CoV-2 can be yielded by previous infection, vaccination, or both (hybrid immunity). The objective of this observation study was to investigate hybrid immunity in patients with chronic lymphocytic leukemia (CLL). METHODS/RESULTS: Blood samples of six patients with CLL were collected 55 days after fourth COVID-19 vaccination. All patients had a SARS-CoV-2 infection within 12 months before the second booster (fourth vaccination). SARS-CoV-2 spike receptor binding domain (RBD)-specific IgG antibodies were detectable in 6/6 (100.0%) CLL patients after four compared to 4/6 (66.7%) after three vaccinations. The median number of SARS-CoV-2 spike-specific T cells after repeated booster vaccination plus infection was 166 spot-forming cells (SFC) per million peripheral blood mononuclear cells. Overall, 5/5 (100%) studied patients showed a detectable increase in T cell activity. CONCLUSION: Our data reveal an increase of cellular and humoral immune response in CLL patients after fourth COVID-19 vaccination combined with SARS-CoV-2 infection, even in those undergoing B cell-depleting treatment. Patients with prior vaccination failure now show a specific IgG response. Future research should explore the duration and effectiveness of hybrid immunity considering various factors like past infection and vaccination rates, types and numbers of doses, and emerging variants.

Torque Teno Virus load is associated with CDC stage and CD4+ cell count in people living with HIV but unrelated to AIDS-defining events and Human Pegivirus load.

BACKGROUND: Torque Teno Virus (TTV) is a non-enveloped, circular single-strand DNA virus and part of the human virome. The replication of TTV was related to the immune status in patients treated with immunosuppressive drugs after organ transplantation. We hypothesize that TTV load could be an additional marker for immune function in people living with HIV (PLWH). METHODS: In this analysis serum samples of PLWH from the RESINA multicenter cohort were reanalysed for TTV. Investigated clinical and epidemiological parameters included Pegivirus (HPgV) load, age, sex, HIV load, CD4+ cell count (CDC 1, 2, 3) and CDC clinical stages (1993 CDC classification system, A, B, C) before initiation of antiretroviral treatment. Regression analysis was used to detect possible associations among parameters. RESULTS: Our analysis confirmed TTV as a strong predictor of CD4+ cell count and CDC class 3. This relationship was used to propose a first classification of TTV load in regard to clinical stage. We found no association with clinical CDC stages A, B and C. HPgV load was inversely correlated with HIV load but not TTV load. CONCLUSIONS: TTV load was associated with immunodeficiency in PLWH. Neither TTV- nor HIV load were predictive for the clinical categories of HIV infection.

Five-year single-center analysis of cytomegalovirus viremia in kidney transplant recipients and possible implication for novel prophylactic therapy approaches.

BACKGROUND: Cytomegalovirus (CMV) infections are a common complication after kidney transplantation (KTx) and negatively affecting patient outcome. Valganciclovir (VGC) prophylaxis is often limited by drug-induced side effects and dose reduction due to decline in kidney function. METHOD: In the present study, episodes of CMV viremia in the first year after KTx in a cohort of 316 recipients were analyzed retrospectively to identify risk factors linked to persistent infections. RESULTS: In the studied cohort, 18.7% of patients showed a high-risk (HR) constellation (D+/R-) for CMV infections. CMV viremia affected 22% of our cohort, with HR patients being the most affected cohort (44.1%). Within this group, most viremic events (65.3%) occurred while patients were still on prophylactic therapy, showing significantly higher viral loads and a longer duration compared to seropositive recipients. CONCLUSION: The analysis at hand revealed that detection of viremia under ongoing antiviral prophylaxis bears an increased risk for sustained viral replication and antiviral drug resistance in HR patients. We identified low estimated glomerular filtration rate (eGFR) and lower dose VGC prophylaxis post-KTx as a risk factor for breakthrough infections in HR patients in our single center cohort. These patients might benefit from a closer CMV monitoring or novel prophylactic agents as letermovir.

[Use of AI-based applications by hospital staff: task profiles and qualification requirements]. / Einsatz von KI-basierten Anwendungen durch Krankenhauspersonal: Aufgabenprofile und Qualifizierungsbedarfe.

BACKGROUND: Artificial intelligence (AI) is becoming increasingly important for the future development of hospitals. To unlock the large potential of AI, job profiles of hospital staff members need to be further developed in the direction of AI and digitization skills through targeted qualification measures. This affects both medical and non-medical processes along the entire value chain in hospitals. The aim of this paper is to provide an overview of the skills required to deal with smart technologies in a clinical context and to present measures for training employees. METHODS: As part of the "SmartHospital.NRW" project in 2022, we conducted a literature review as well as interviews and workshops with experts. AI technologies and fields of application were identified. RESULTS: Key findings include adapted and new task profiles, synergies and dependencies between individual task profiles, and the need for a comprehensive interdisciplinary and interprofessional exchange when using AI-based applications in hospitals. DISCUSSION: Our article shows that hospitals need to promote digital health literacy skills for hospital staff members at an early stage and at the same time recruit technology- and AI-savvy staff. Interprofessional exchange formats and accompanying change management are essential for the use of AI in hospitals.

Bridging the gap - estimation of 2022/2023 SARS-CoV-2 healthcare burden in Germany based on multidimensional data from a rapid epidemic panel.

OBJECTIVES: Throughout the SARS-CoV-2 pandemic, Germany like other countries lacked adaptive population-based panels to monitor the spread of epidemic diseases. METHODS: To fill a gap in population-based estimates needed for winter 2022/23 we resampled in the German SARS-CoV-2 cohort study MuSPAD in mid-2022, including characterization of systemic cellular and humoral immune responses by interferon-γ-release assay (IGRA) and CLIA/IVN assay. We were able to confirm categorization of our study population into four groups with differing protection levels against severe COVID-19 courses based on literature synthesis. Using these estimates, we assessed potential healthcare burden for winter 2022/23 in different scenarios with varying assumptions on transmissibility, pathogenicity, new variants, and vaccine booster campaigns in ordinary differential equation models. RESULTS: We included 9921 participants from eight German regions. While 85% of individuals were located in one of the two highest protection categories, hospitalization estimates from scenario modeling were highly dependent on viral variant characteristics ranging from 30-300% compared to the 02/2021 peak. Our results were openly communicated and published to an epidemic panel network and a newly established modeling network. CONCLUSIONS: We demonstrate feasibility of a rapid epidemic panel to provide complex immune protection levels for inclusion in dynamic disease burden modeling scenarios.

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.

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.

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.

Somatic hypermutation introduces bystander mutations that prepare SARS-CoV-2 antibodies for emerging variants.

Somatic hypermutation (SHM) drives affinity maturation and continues over months in SARS-CoV-2-neutralizing antibodies (nAbs). However, several potent SARS-CoV-2 antibodies carry no or only a few mutations, leaving the question of how ongoing SHM affects neutralization unclear. Here, we reverted variable region mutations of 92 antibodies and tested their impact on SARS-CoV-2 binding and neutralization. Reverting higher numbers of mutations correlated with decreasing antibody functionality. However, for some antibodies, including antibodies of the public clonotype VH1-58, neutralization of Wu01 remained unaffected. Although mutations were dispensable for Wu01-induced VH1-58 antibodies to neutralize Alpha, Beta, and Delta variants, they were critical for Omicron BA.1/BA.2 neutralization. We exploited this knowledge to convert the clinical antibody tixagevimab into a BA.1/BA.2 neutralizer. These findings broaden our understanding of SHM as a mechanism that not only improves antibody responses during affinity maturation but also contributes to antibody diversification, thus increasing the chances of neutralizing viral escape variants.

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.

Discovery of highly neutralizing human antibodies targeting Pseudomonas aeruginosa.

Drug-resistant Pseudomonas aeruginosa (PA) poses an emerging threat to human health with urgent need for alternative therapeutic approaches. Here, we deciphered the B cell and antibody response to the virulence-associated type III secretion system (T3SS) in a cohort of patients chronically infected with PA. Single-cell analytics revealed a diverse B cell receptor repertoire directed against the T3SS needle-tip protein PcrV, enabling the production of monoclonal antibodies (mAbs) abrogating T3SS-mediated cytotoxicity. Mechanistic studies involving cryoelectron microscopy identified a surface-exposed C-terminal PcrV epitope as the target of highly neutralizing mAbs with broad activity against drug-resistant PA isolates. These anti-PcrV mAbs were as effective as treatment with conventional antibiotics in vivo. Our study reveals that chronically infected patients represent a source of neutralizing antibodies, which can be exploited as therapeutics against PA.

15-month post-COVID syndrome in outpatients: Attributes, risk factors, outcomes, and vaccination status - longitudinal, observational, case-control study.

Background: While the short-term symptoms of post-COVID syndromes (PCS) are well-known, the long-term clinical characteristics, risk factors and outcomes of PCS remain unclear. Moreover, there is ongoing discussion about the effectiveness of post-infection vaccination against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) to aid in PCS recovery. Methods: In this longitudinal and observational case-control study we aimed at identifying long-term PCS courses and evaluating the effects of post-infection vaccinations on PCS recovery. Individuals with initial mild COVID-19 were followed for a period of 15 months after primary infection. We assessed PCS outcomes, distinct symptom clusters (SC), and SARS-CoV-2 immunoglobulin G (IgG) levels in patients who received SARS-CoV-2 vaccination, as well as those who did not. To identify potential associating factors with PCS, we used binomial regression models and reported the results as odds ratios (OR) with 95% confidence intervals (95%CI). Results: Out of 958 patients, follow-up data at 15 month after infection was obtained for 222 (23.2%) outpatients. Of those individuals, 36.5% (81/222) and 31.1% (69/222) were identified to have PCS at month 10 and 15, respectively. Fatigue and dyspnea (SC2) rather than anosmia and ageusia (SC1) constituted PCS at month 15. SARS-CoV-2 IgG levels were equally distributed over time among age groups, sex, and absence/presence of PCS. Of the 222 patients, 77.0% (171/222) were vaccinated between 10- and 15-months post-infection, but vaccination did not affect PCS recovery at month 15. 26.3% of unvaccinated and 25.8% of vaccinated outpatients improved from PCS (p= .9646). Baseline headache (SC4) and diarrhoea (SC5) were risk factors for PCS at months 10 and 15 (SC4: OR 1.85 (95%CI 1.04-3.26), p=.0390; SC5: OR 3.27(95%CI 1.54-6.64), p=.0009). Conclusion: Based on the specific symptoms of PCS our findings show a shift in the pattern of recovery. We found no effect of SARS-CoV-2 vaccination on PCS recovery and recommend further studies to identify predicting biomarkers and targeted PCS therapeutics.

HIV-1 neutralizing antibodies provide sterilizing immunity by blocking infection of the first cells.

Neutralizing antibodies targeting HIV-1 Env have been shown to protect from systemic infection. To explore whether these antibodies can inhibit infection of the first cells, challenge viruses based on simian immunodeficiency virus (SIV) were developed that use HIV-1 Env for entry into target cells during the first replication cycle, but then switch to SIV Env usage. Antibodies binding to Env of HIV-1, but not SIV, block HIV-1 Env-mediated infection events after rectal exposure of non-human primates to the switching challenge virus. After natural exposure to HIV-1, such a reduction of the number of first infection events should be sufficient to provide sterilizing immunity in the strictest sense in most of the exposed individuals. Since blocking infection of the first cells avoids the formation of latently infected cells and reduces the risk of emergence of antibody-resistant mutants, it may be the best mode of protection.

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.