Physicochemical properties of CDRH3s and VH gene segment mutation frequencies

Germinal center markers are expressed in lymphoid follicles.

Schematic overview laser microdissection

The limited mechanistic understanding of Human Cytomegalovirus (HCMV) infection and neutralization has hindered the effective application of antibodies for HCMV prevention and therapy. To address this, we conducted high-resolution cryo-EM analysis of 17 human monoclonal antibodies targeting HCMV glycoproteins gH and gL. This analysis revealed a landscape of vulnerable epitopes, including detailed binding modes, spatial orientations, and structural features associated with distinct neutralization phenotypes. By comprehensively characterizing the neutralizing properties of these antibodies and their combinations, we identified synergistic antibody cocktails that enhance the neutralization capacity of individual antibodies. Notably, while single antibodies only partially neutralized HCMV, their synergistic combinations achieved substantially more complete inhibition. Together, these findings delineate structural correlates of HCMV neutralization and establish functional principles for antibody-based combinatorial targeting of HCMV.

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

Clinical data on Bundibugyo virus (BDBV) infection are limited, and no approved virus-specific treatment exists. We describe the case of a previously healthy 39-year-old healthcare worker who acquired BDBV infection while working in Ituri Province, Democratic Republic of the Congo (DRC), and was medically evacuated to Germany. He received MBP134, an investigational combination of two monoclonal antibodies, under an FDA Emergency Investigational New Drug (eIND) authorization, together with remdesivir and supportive care. Viral RNA concentrations were highest in oropharyngeal swabs and plasma, and became undetectable in blood, throat swabs, urine, and stool by day 13 after symptom onset and in semen by day 25. The patient’s serum demonstrated neutralizing activity against the current BDBV outbreak strain and other orthoebolaviruses. We detected BDBV-induced antibody responses not attributable to MBP134 or prior vaccination, providing evidence of endogenous humoral immunity following combined antiviral treatment. The patient recovered and was discharged on day 22 after symptom onset. This case report provides detailed insights into the clinical course, virologic dynamics and immune responses in BDBV infection during combined antiviral treatment, while larger clinical studies are needed to assess the efficacy and clinical utility of these therapeutic approaches. In a case report of an individual with Bundibugyo virus infection, treatment with emergency use of the monoclonal antibody cocktail MBP134 with remdesivir and supportive care treatment resulted in recovery and discharge 22 days after symptom onset, supporting further evaluation of MBP134.

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.

The highly pathogenic avian influenza viruses of subtype H5N1 represent a major threat to animal and public health. The current panzootic with H5 clade 2.3.4.4b has caused numerous, widespread outbreaks in various domestic and wild avian species with high mortalities, massive losses, and a high frequency of spillover events to unexpected novel mammalian hosts, such as dairy cows. The global H5N1 situation raises serious concerns about zoonotic risks due to effective mammal-to-mammal transmission. Therefore, it is critical to increase surveillance intensity of a broadened species range, particularly at the human-animal interface. For this purpose, reliable and cost-effective serological tools that are easy to perform and suitable for high-throughput screening are critically needed. The newly developed double-antigen enzyme-linked immunosorbent assay format employing a luminescence-based detection technology has demonstrated compliance with such prerequisites. The assay allowed sensitive and specific detection of antibodies directed against H5 hemagglutinin of clade 2.3.4.4b in a wide range of birds and mammals, including humans. Furthermore, it allowed differentiating H5 anti-head-specific from cross-reacting anti-stalk antibodies, which represents a valuable feature with regard to the monitoring of future vaccination programs with H5-specific vaccines. Thus, the assay is a significant contribution to existing serological diagnostic tests for a clade-optimized and species-independent detection of influenza A virus antibodies. IMPORTANCE:The ongoing highly pathogenic avian influenza virus H5N1 panzootic has caused numerous outbreaks in domestic and wild animals, with frequent spillover events to unexpected host species, which underscores the importance of intensified surveillance. However, sensitive and specific multi-species serological assays represent a major gap. For this purpose, we developed a novel double-antigen enzyme-linked immunosorbent assay that employs an innovative luminescence-based readout strategy. The test allowed a highly sensitive and specific detection of H5-specific antibodies in a wide range of avian and mammalian species, including humans. It therefore represents a valuable contribution to improving species-independent serological diagnostic tools for the detection of influenza A virus antibodies.

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.

Five individuals from a single-family cluster, one rVSV-ZEBOV-vaccinated adult and four unvaccinated children aged 1–7 years, received investigational MBP134 as post-exposure prophylaxis (PEP) 5–6 days after high- to intermediate-risk occupational or household exposure to Bundibugyo ebolavirus (BDBV). MBP134 is a broadly neutralizing monoclonal antibody cocktail that has conferred protection against BDBV in non-human primates but to our knowledge has not previously been evaluated as PEP in humans. Given the absence of approved preventive interventions against BDBV and the substantial case fatality rate of Bundibugyo virus disease (BVD), MBP134 was administered as an individual treatment attempt under emergency Investigational New Drug (eIND) authorizations. Administration was well tolerated, with no treatment-related adverse events. Throughout the 21-day monitoring period, all family members remained free of clinical or laboratory evidence of BVD, as assessed by daily medical evaluation and serial PCR testing. Plasma Orthoebolavirus-reactive antibodies were detected after infusion, persisted throughout follow-up and mediated broad neutralizing activity against multiple Orthoebolavirus species, including authentic BDBV. Notably, endogenous BDBV-reactive IgM and IgA were detected in the adult following high-risk exposure despite persistently negative PCR results. Together, these findings support prospective evaluation of MBP134 as PEP for high-risk adult and pediatric contacts during BDBV outbreaks. In a case series of an adult and four children with high- to intermediate-risk exposure to Bundibugyo ebolavirus, emergency use post-exposure prophylaxis monoclonal antibody cocktail (MBP134) treatment was well tolerated with no evidence of Bundibugyo virus disease, supporting future evaluation of MBP134 as a post-exposure prophylaxis for outbreaks.

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.

Broadly neutralizing antibody (bNAb) 1-18 is a promising tool for future clinical strategies against HIV-1 infection. To enhance 1-18's clinical potential, we introduced half-life-extending LS mutations and evaluated the resulting investigational bNAb candidate, BNT351. LS mutations increased the affinity of BNT351 to human neonatal Fc receptor by 20-fold, resulting in a long half-life of 10-14 days in Tg32 mice and 18 days in non-human primates, with a predicted human half-life of ∼50 days. BNT351 retained 1-18's exceptional neutralization potency and breadth against a 119 multiclade panel, and neutralized a pseudovirus panel of circulating HIV-1 clade C strains with high potency. BNT351 fully suppressed viremia in HIV-1-infected humanized CD34+ NSG mice without eliciting resistant viral variants. Additionally, we observed no off-target binding of BNT351 to a panel of ∼6,500 human proteins, and no developability concerns. This favorable preclinical evaluation supported initiation of a phase 1 clinical trial with BNT351 (NCT07392372).

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

C3 expression in TLS-high patients

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.

Abstract Major histocompatibility complexes (MHC) govern antigen presentation and T-cell receptor (TCR) selection. Accurate in vivo modeling of human immunity therefore requires physiological human MHC–TCR interactions. Humanized NOD-scid-IL2Rγc null (NSG) mice engrafted with human CD34⁺ hematopoietic stem cells are widely used to provide preclinical platforms for the development of advanced therapies; however, interactions between murine MHC and human TCR can promote xenoreactivity and alter T-cell development. Here, we investigated how elimination of murine MHC together with different conditioning regimens shapes human T-cell maturation in vivo . CD34⁺ cells from ten cord blood donors were transplanted into conventional NSG mice or murine MHC-deficient NSG derivatives (DKO) following either sublethal irradiation or myeloablative busulfan conditioning. Integrated analyses combining flow cytometry, plasma cytokine profiling, and bulk and single-cell TCR sequencing revealed marked differences in T-cell differentiation across models. Busulfan-conditioned DKO mice developed highly proliferative, activated, and cytotoxic T cells together with clonally expanded TCR repertoires. In contrast, irradiated NSG mice preferentially accumulated naïve, NKT, and regulatory T-cell populations. Busulfan-conditioned DKO mice showed no evidence of xenogeneic graft-versus-host disease and represent a refined enabling platform for human T-cell development and provide a foundation for future preclinical evaluation of advanced gene and cell therapies.