Physicochemical properties of CDRH3s and VH gene segment mutation frequencies

Germinal center markers are expressed in lymphoid follicles.

Schematic overview laser microdissection

PDAC patients show activated or non-activated tumor draining lymph nodes.

Broadly neutralizing antibodies (bNAbs) against HIV-1 can suppress viremia in vivo and inform vaccine development. Here we characterized 007, a V3 glycan site bNAb exhibiting high levels of antiviral activity against multiclade pseudovirus panels. 007 targets an N332 gp120 glycan-independent V3 epitope, a site of the HIV-1 envelope protein (Env) vulnerability to which only weakly neutralizing antibodies had previously been identified. Functional analyses demonstrated distinct binding and neutralization profiles compared to classical V3 glycan site bNAbs. A 007 Fab-Env cryogenic electron microscopy structure revealed contacts with the V3 324 GD/NIR 327 motif and interactions with N156 gp120 and N301 gp120 glycans. In contrast to classical V3 bNAbs, 007 binding to Env does not depend on the N332 gp120 glycan, rendering it resistant to common escape mutations. Structures of 007 IgG-Env trimer complexes showed two Env trimers crosslinked by three bivalent IgGs. Bivalent 007 IgG was more potent than monovalent 007 IgG heterodimer, suggesting a role for avidity in potent neutralization. Finally, in HIV-1 ADA -infected humanized mice, 007 caused transient decline of viremia and overcame classical V3 escape mutations, highlighting 007’s potential for HIV-1 prevention, therapy, functional cure and vaccine design.

BACKGROUND:Infections with respiratory viruses such as SARS-CoV-2 and influenza are significant international public health concerns. While patients with cancer remain the most vulnerable group, they show poor vaccine response in general. Immunological data in this population are limited and mainly focus on serological parameters. However, in these patients, cellular, and especially T-cell, responses often seem to be induced more reliably than humoral responses. OBJECTIVE:To gain further insights into vaccine-induced immunity, the RESPONSE study will analyze the effect of early and late booster vaccination on humoral and cellular responses, with special focus on T cell-induced immune responses. In addition, we aim to investigate factors influencing humoral and cellular vaccine-induced immunity in patients with hematological and oncological malignancies, including state of disease, treatment, and demographic factors. METHODS:Humoral immune responses will be assessed by measuring binding and neutralizing antibodies using standardized assays. Cellular immunity will be evaluated using functional assays such as flow cytometry and FluoroSpot, as well as in-depth analyses using additional exploratory assays as appropriate. Immune responses will be correlated with clinical parameters, including disease status, treatment, and demographic factors. RESULTS:This study was initiated following ethics approval and is currently recruiting participants. Enrollment commenced on March 25, 2025, and is ongoing, whereas biosample collection and follow-up visits are nearing completion for most participants. Final data cleaning, dataset integration, and statistical analyses of adaptive immune responses are planned from the third quarter of 2026 onward. CONCLUSIONS:This study intends to lay a foundation for a structured translational research platform on vaccination to aim for best protection from infection by different respiratory pathogens. Long-term objectives are reaching best possible protection from vaccine-preventable disease with a first focus on influenza infection. In addition, we plan to investigate vaccine-induced immune responses to the recently approved respiratory syncytial virus vaccine using this platform and possibly extend this to further vaccines in the future. Urgent questions, such as the influence of different targeted therapies on vaccine immune response, will be part of these projects. TRIAL REGISTRATION:ClinicalTrials.gov NCT06612515; https://clinicaltrials.gov/study/NCT06612515. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID):DERR1-10.2196/88520.

Abstract Orthoebolaviruses such as Ebola virus (EBOV), Sudan virus (SUDV) and Bundibugyo virus (BDBV) can cause severe disease with high case-fatality rates. While licensed EBOV vaccines and therapeutic antibodies protect against EBOV infection, no single monoclonal antibody currently provides broad protection across multiple orthoebolaviruses. Here, we analyzed the humoral immune response of an rVSV-EBOV vaccinee to identify pan- orthoebolavirus -neutralizing antibodies. Using BDBV- and SUDV-glycoproteins for single B cell-sorting, we identified B10, which neutralized authentic EBOV and SUDV, with potent activity against SUDV compared with established cross-reactive antibodies. Structural analysis mapped antibody B10 binding to the pan- orthoebolavirus conserved GP2-stalk/HR2 region, associated with asymmetric trimer destabilization and spike opening. In vivo, B10 showed significant prophylactic efficacy in an EBOV mouse model and partial protection with antiviral activity in a SUDV mouse model. Together, these findings demonstrate that rVSV-EBOV vaccination induced the development of a broadly orthoebolavirus-neutralizing antibody that holds exeptional therapeutic potential.

T cells surround B-cell cluster in PDAC TLS.

TLS in PDAC patients are active germinal centers.

Gene expression TLS-high vs. TLS-low tumor tissue

Next-generation sequencing (NGS) read characteristics

Measuring antibody binding titers to viral proteins is essential to assess population immunity and monitor viral escape. We present a multiplexable flow cytometry protocol to quantify antibody binding to native-like, cell-surface-expressed antigens and demonstrate the method using influenza A virus hemagglutinin. We describe steps for the transfection of human suspension cells, cell staining and fixation, data acquisition, and analysis. In a single run, up to 500 samples can be measured simultaneously against three antigens per well within three days. For complete details on the use and execution of this protocol, please refer to Daniel et al.1.

The transmission of influenza A virus H5N1 clade 2.3.4.4b from cattle to humans highlights the risk of an H5N1 pandemic. Pre-existing immunity strongly impacts the course and severity of viral infections, making detailed knowledge of antibodies against the spilled-over strain crucial. Here, we assessed humoral immunity against H5N1 A/Texas/37/2024 in H5N1-naive individuals. We performed complementary binding and neutralization assays on 66 individuals and ranked activities among a panel of 76 influenza A virus isolates. We detected low but distinct cross-neutralizing titers against A/Texas/37/2024, with a 3.9- to 15.6-fold reduction compared with selected H1N1 or H3N2 strains. By cloning and characterizing 136 memory B cell-derived monoclonal antibodies, we identified potent A/Texas/37/2024-neutralizing antibodies in five out of six individuals we investigated. These antibodies cross-neutralized H1, competed with antibodies targeting the hemagglutinin (HA) stem, and protected mice from lethal H5N1 challenge. Our findings demonstrate partial pre-existing humoral immunity to A/Texas/37/2024 in H5N1-naive individuals.

CD38 and FoxP3 cells surround B-cell clusters in TLS.

Administration of HIV-1 neutralizing antibodies can suppress viremia and prevent infection in vivo. However, clinical use is challenged by broad envelope sequence diversity and rapid emergence of viral escape1-9. Here, we performed single B cell profiling of 32 top HIV-1 elite neutralizers to identify broadly neutralizing antibodies (bNAbs) with highest potency and breadth for clinical application. From 831 expressed monoclonal antibodies, we identified 04_A06, a new VH1-2-encoded CD4 binding site bNAb with remarkable breadth and potency against extended multiclade pseudovirus panels (GeoMean IC50 = 0.059 μg/ml, breadth = 98.5%, 332 virus strains). Moreover, 04_A06 was not susceptible to classic viral CD4bs escape variants and maintained full viral suppression in HIV-1-infected humanized mice. Structural analyses revealed that antiviral activity is mediated by an unusually long 11-amino acid heavy chain insertion. This insertion facilitates inter-protomer contacts and interactions with highly conserved residues on the adjacent gp120 protomer. Finally, 04_A06 demonstrated high activity against contemporaneously circulating viruses from the Antibody Mediated Prevention (AMP) trials (GeoMean IC50 = 0.082 μg/ml, breadth = 98.4%, 191 virus strains) and in silico modeling for 04_A06LS predicted HIV-1 prevention efficacy of >93%. Thus, 04_A06 will provide unique opportunities for effective treatment and prevention strategies of HIV-1 infection.

The repeated spill-over of Influenza A virus H5N1 clade 2.3.4.4b from cattle to humans highlights the risk of a human H5N1 pandemic. Given the impact of pre-existing immunity on the course and severity of viral infections, we assessed in detail the humoral immunity against the H5N1 A/Texas/37/2024 isolate in H5N1-naive individuals. To this end, we performed complementary binding and neutralization assays on 66 subjects and ranked activities among a panel of 76 influenza A virus isolates. We detected low but distinct cross-neutralizing titers against A/Texas/37/2024 with a 3.9 to 15.6-fold reduction compared to selected H1N1 or H3N2 strains. Moreover, by cloning and evaluating 136 monoclonal antibodies from single memory B cells, we identified potent A/Texas/37/2024-neutralizing monoclonal antibodies in five out of six investigated individuals. These antibodies predominantly utilize VH1-69 gene segments, cross-neutralize H1, and compete with antibodies targeting the HA stem. Our findings demonstrate partial pre-existing humoral immunity to A/Texas/37/2024 in H5N1-naive individuals. ### Competing Interest Statement DR, MM, CK, FK, and ML are members of the non-profit Center for Predictive Analysis of Viral Evolution (Previr). LG, HG, CK and FK are inventors on patent applications on virus neutralizing antibodies filed by the University of Cologne and have received payments from the University of Cologne for licensed patents.

Abstract Tertiary lymphoid structures (TLS) in cancer are considered ectopic hotspots for immune activation that are similar to lymphoid follicles in secondary lymphoid organs (SLO). This study elucidates shared and TLS/SLO-specific features in pancreatic ductal adenocarcinoma (PDAC). TLS abundance was related to superior survival and T-cell abundance in 110 treatment-naïve PDAC samples, underlining their clinical relevance. Immunofluorescence microscopy identified structural homologies between TLSs and SLOs. In RNA expression analyses of laser-microdissected TLSs and paired SLOs, we observed largely overlapping expression patterns of immune-related gene clusters but distinct expression patterns of T-cell and complement-associated genes. Immune cells in TLS expressed essential markers of germinal center formation. Increased activation of tumor-draining lymph nodes in patients with high numbers of TLSs highlights the relevance of these tumor-related structures to systemic immune response. In line with this, we identified an overlap of expanded B-cell receptor clonotypes in TLSs and SLOs, which suggests a vivid cross-talk between the two compartments. We conclude that combined therapeutic approaches exploiting TLS-mediated antitumor immune responses may improve susceptibility of PDAC to immunotherapy.

Administration of HIV-1 neutralizing antibodies can suppress viremia and prevent infection in vivo. However, clinical use is challenged by envelope diversity and rapid viral escape. Here, we performed single B cell profiling of 32 top HIV-1 elite neutralizers to identify broadly neutralizing antibodies with highest antiviral activity. From 831 expressed monoclonal antibodies, we identified 04_A06, a VH1-2-encoded broadly neutralizing antibody to the CD4 binding site with remarkable breadth and potency against multiclade pseudovirus panels (geometric mean half-maximal inhibitory concentration = 0.059 µg ml−1, breadth = 98.5

Broadly neutralizing antibodies (bNAbs) against HIV-1 can suppress viremia in vivo and inform vaccine development. Here, we characterized 007, a V3 glycan site bNAb exhibiting high levels of antiviral activity against multiclade pseudovirus panels1-3 (GeoMean IC50 = 0.012 μg/mL, breadth = 69%, 217 virus strains) by targeting a N332gp120 glycan-independent V3 epitope, a site of Env vulnerability to which only weakly neutralizing antibodies had previously been identified. Functional analyses demonstrated distinct binding and neutralization profiles compared to classical V3 glycan site bNAbs. A 007 Fab-Env cryo-EM structure revealed contacts with the V3 324GD/NIR327 motif and interactions with N156gp120 and N301gp120 glycans. In contrast to classical V3 bNAbs, 007 binding to Env does not depend on the N332gp120 glycan, rendering it resistant to common escape mutations. Structures of 007 IgG-Env trimer complexes showed two Env trimers crosslinked by three bivalent IgGs, and bivalent 007 IgG was up to ~300-fold more potent than monovalent 007 IgG heterodimer, suggesting a role for avidity in potent neutralization. Finally, in HIV-1ADA-infected humanized mice, 007 caused transient decline of viremia and overcame classical V3 escape mutations, highlighting 007's potential for HIV-1 prevention, therapy, functional cure, and vaccine design.