Merkel cell carcinomas (MCC) typically arise from the clonal integration of the Merkel cell polyomavirus. Immunogenic viral oncoproteins then lead to tumorigenesis. Oncoprotein-specific T cells are essential for anti-MCC immunity, but it is unclear whether B cells promote tumor control. In this study, we analyzed the frequency and phenotype of viral oncoprotein-specific and total B cells in blood samples from 47 patients with MCC and tumor samples from another 19 patients with MCC. The phenotype of blood B cells did not correlate with the outcomes of patients with MCC. In contrast, all 11 patients with robust oncoprotein-specific antibody-secreting and/or germinal center B cells in tumors experienced long-term MCC control. In vitro, B cells engineered to be specific for viral oncoproteins increased the sensitivity of oncoprotein-specific CD4+ T cells by more than 50-fold. Together, our findings suggest that cancer-specific B cells promote antitumor immunity via increased responses by T cells and that cancer-specific augmentation of B cells could be therapeutically relevant. See related Spotlight, p. 716.

Additional analyses of CD4+ T cells in MCC tumors.

Overall Frequency of T cells, B cells, and MCC cells in tumor samples does not associate with MCC progression after treatment.

Multiplex immunohistochemistry staining of MCC tumor microarrays.

Additional analyses of antibody-secreting B cells in MCC tumor samples.

PBMC cohort patient history summaries.

Gene editing strategies that do not rely on viral vectors are being explored for their potential to support durable biologics production. While clinical trials have shown that adeno-associated virus encoding broadly neutralizing antibodies can protect against HIV, these interventions often yield limited, short-lived responses. The development of non-viral gene editing approaches in hematopoietic stem and progenitor cells holds promise for long-term antibody production. In this study, we evaluated CRISPR-Cas9 and CRISPR-Cas12a for gene knockin at the immunoglobulin heavy chain locus in non-human primate (NHP) hematopoietic stem and progenitor cells (HSPCs). Delivering the nuclease as a protein alongside a custom DNA template, we optimized editing with Cas12a and demonstrated higher knockin efficiency and fewer non-specific edits than with Cas9. Transplantation of edited NHP HSPCs into MISTRG mice led to engraftment, B cell differentiation, and transgene expression of a reporter transgene or anti-HIV antibody after gp120 antigen immunization with detectable titers in circulation. These findings demonstrate the feasibility of using non-viral knockin in HSPCs as a potential strategy for sustained biologics production in the treatment of chronic diseases. Future work will assess the efficacy of this approach in an NHP model of HIV infection.

Higher frequency of antigen-experienced B cells in blood of female patients near diagnosis associates with MCC progression.

The frequency of T-Ag-specific B cells in the blood does not associate with levels of T-Ag-specific antibodies in the blood or outcome.

Tumor mIHC cohort patient history summaries.

Tumor sample cohort patient history summaries.

The continued and increasing prevalence of syphilis worldwide highlights the need for an effective syphilis vaccine to complement public health measures. Previous work demonstrated that immunization of the rabbit animal model with vaccine candidates derived from the T. pallidum endothelial cell adhesin Tp0751 could reduce dissemination of T. pallidum to lymph nodes. In those studies, a proportion of animals exhibited complete inhibition of treponemal dissemination and others exhibited partial or no inhibition of treponemal dissemination, consistent with results expected from an outbred animal model. In the current study we further characterized the Tp0751-specific antibody response in immunized animals that showed inhibition of T. pallidum dissemination. To do this, we generated Tp0751 tetramers to identify Tp0751-specific B cells before and after immunization. Using this approach, we found a robust expansion of Tp0751-specific B cells in the blood and spleens of immunized animals compared to unimmunized control animals. Ten antibodies from Tp0751-immunized rabbits were cloned and binding to specific structural regions of the Tp0751 protein was assessed using epitope mapping assays and structural modeling. Importantly, nine out of the ten antibodies cloned from Tp0751 tetramer-binding B cells were able to significantly inhibit T. pallidum attachment to human endothelial cells in vitro, including antibodies exhibiting weaker binding to Tp0751. Combined, our results provide a proof-of-principle that Tp0751-based subunit vaccines can stimulate strong B cell responses resulting in the production of antibodies able to inhibit T. pallidum attachment to endothelial cells.

Antibody-based immunotherapies are promising; however, their application remains limited to acute diseases due to rapid clearance of antibodies in vivo. Some chronic conditions could benefit from sustained therapeutic antibody expression. One such instance is human immunodeficiency virus type 1 (HIV-1), where the efficiency of broadly neutralizing antibodies by passive immunization has been limited in clinical trials. B cell editing to enable sustained production of an antibody of interest in vivo could address this issue. However, the long-term potential of this approach and feasibility to perform editing in B cells from people living with HIV remain to be determined. We investigated editing of rhesus macaque B cells from healthy or simian/human immunodeficiency virus (SHIV)-infected animals to model this approach. An antibody-encoding cassette was inserted in the immunoglobulin locus by CRIPSR-Cas9-mediated ex vivo B cell editing. Similar indel efficiencies were achieved in B cells from both uninfected and infected animals, and expression of the antibody of interest was detected in up to 10% of uninfected B cells. This study paves the way for future in vivo work to assess the long-term potential of this approach and its impact on B cell development and function in an immunocompetent in vivo nonhuman primate model of HIV persistence and cure.

Non-viral gene editing offers a practical alternative to viral delivery for durable biologics production. Clinical trials have shown that adeno-associated virus encoding broadly neutralizing antibodies can protect against HIV, but result in limited, short-lived responses. The development of non-viral gene editing approaches in hematopoietic stem and progenitor cells holds promise for long-term antibody production. In this study, we evaluated CRISPR/Cas9 and CRISPR/Cas12a for gene knock-in at the immunoglobulin heavy chain locus in non-human primate hematopoietic stem and progenitor cells. Delivering the nuclease as a protein alongside a custom DNA template, we optimized editing with Cas12a and demonstrated higher knock-in efficiency and fewer non-specific edits than Cas9. Transplantation of edited non-human primate hematopoietic stem and progenitor cells into MISTRG mice led to engraftment, B cell differentiation, and transgene expression of a reporter transgene or anti-HIV antibody after HIV immunization with detectable anti-HIV antibody titers in peripheral blood circulation. These findings demonstrate the feasibility of using non-viral gene editing in HSPC as a potential strategy for sustained biologics production in the treatment of chronic diseases such as HIV. Future work will assess the efficacy of this model in a non-human primate model of HIV infection.

The study of Ag-specific lymphocytes has been a key advancement in immunology over the past few decades. The development of multimerized probes containing Ags, peptide:MHC complexes, or other ligands was one innovation allowing the direct study of Ag-specific lymphocytes by flow cytometry. Although these types of study are now common and performed by thousands of laboratories, quality control and assessment of probe quality are often minimal. In fact, many of these types of probe are made in-house, and protocols vary between laboratories. Although peptide:MHC multimers can often be obtained from commercial sources or core facilities, few such services exist for Ag multimers. To ensure high quality and consistency with ligand probes, we have developed an easy and robust multiplexed approach using commercially available beads able to bind Abs specific for the ligand of interest. Using this assay, we have sensitively assessed the performance of peptide:MHC and Ag tetramers and have found considerable batch-to-batch variability in performance and stability over time more easily than using murine or human cell-based assays. This bead-based assay can also reveal common production errors such as miscalculation of Ag concentration. This work could set the stage for the development of standardized assays for all commonly used ligand probes to limit laboratory-to-laboratory technical variation and experimental failure caused by probe underperformance.

Respiratory syncytial virus (RSV), human metapneumovirus (HMPV), and human parainfluenza virus types one (HPIV1) and three (HPIV3) can cause severe disease and death in immunocompromised patients, the elderly, and those with underlying lung disease. A protective monoclonal antibody exists for RSV, but clinical use is limited to high-risk infant populations. Hence, therapeutic options for these viruses in vulnerable patient populations are currently limited. Here, we present the discovery, in vitro characterization, and in vivo efficacy testing of two cross-neutralizing monoclonal antibodies, one targeting both HPIV3 and HPIV1 and the other targeting both RSV and HMPV. The 3 × 1 antibody is capable of targeting multiple parainfluenza viruses; the MxR antibody shares features with other previously reported monoclonal antibodies that are capable of neutralizing both RSV and HMPV. We obtained structures using cryo-electron microscopy of these antibodies in complex with their antigens at 3.62 Å resolution for 3 × 1 bound to HPIV3 and at 2.24 Å for MxR bound to RSV, providing a structural basis for in vitro binding and neutralization. Together, a cocktail of 3 × 1 and MxR could have clinical utility in providing broad protection against four of the respiratory viruses that cause significant morbidity and mortality in at-risk individuals.