Emanuele Marra

Building on seminal studies which utilized RAG2-deficient mice engrafted with human tumors and reconstituted with human PBMCs to investigate antitumor immunity, the field still lacks robust preclinical systems that faithfully recapitulate human HLA-restricted T cell responses. Here, we describe a humanized murine platform—the HLA-matched Immuno-avatar model—engineered to evaluate DNA-based cancer vaccines delivered by electroporation. The model is based on the adoptive transfer of splenocytes from immunocompetent, vaccinated HLA-A0201 transgenic (HHK) donors into immunodeficient RAG2−/− IL-2Rγ−/− recipients, which are subsequently xenografted with a human colon carcinoma cell line and treated with a DNA vaccine encoding HLA-restricted epitopes of carcinoembryonic antigen (CEA). Transfer of splenocytes from CEA-vaccinated donors induced a robust and statistically significant antitumor effect in recipient mice. This humanized, HLA-matched, and functionally immunocompetent platform provides a flexible and translationally relevant system for the preclinical evaluation of both off-the-shelf and personalized cancer vaccines targeting evolving tumor antigen repertoires.

Background/Objectives: The ErbB protein family plays a critical role in the progression of various solid tumors, and HER3 has been implicated in resistance mechanisms to multiple cancer therapies due to its ability to form heterodimers with other ErbB receptors, thereby activating pathways that promote tumor growth and survival. This study aimed to generate and characterize humanized monoclonal antibodies against HER3 to inhibit its function and evaluate their potential as therapeutic agents. Methods: Murine monoclonal antibodies TK-A3 and TK-A4 were humanized and tested for binding to ErbB3 and competition with neuregulin-1β (NRG). Specificity was assessed by ELISA, and epitope identified by X-ray crystallography. Downstream signaling was analyzed by western blot for phosphorylated ErbB3, Akt, and MAPK. Antitumor activity was evaluated in vitro and in a pancreatic cancer xenograft model. A toxicology study was also conducted. Results: TK-hu A3 and TK-hu A4 bound specifically to ErbB3 without cross-reactivity to other ErbB receptors. The ErbB3-TK-hu A3 Fab structure revealed the binding epitope. Both antibodies competed with NRG, inhibiting ErbB3, Akt, and MAPK phosphorylation in a dose-dependent manner. They suppressed cancer cell survival in vitro, and TK-hu A3 significantly delayed tumor growth in vivo. The toxicology study indicated good tolerability. Conclusions: TK-hu A3 emerged as the lead candidate, showing specific HER3 targeting, strong pathway inhibition, and antitumor efficacy in vivo. Beyond standalone use, it could support novel strategies such as T-cell engagers, ADCs, CAR-T, and bispecific antibodies. These findings highlight TK-hu A3 as a promising therapy for HER3-positive, treatment-resistant cancers, meriting further development.

Crohn’s and ulcerative colitis are common conditions associated with inflammatory bowel disease as well as intestinal flora and epithelial barrier dysfunction. A novel fermented Lactobacillus brevis (AL0035) herein assayed in a trinitro benzene sulfonic acid (TNBS)-induced colitis mice model after oral administration significantly counteracted the body weight loss and improves the disease activity index and histological injury scores. AL0035 significantly decreased the mRNA and protein expression of different pro-inflammatory cytokines (TNFalpha, IL-1beta, IL-6, IL-12, IFN-gamma) and enhanced the expression of IL-10. In addition, the probiotic promoted the expression of tight junction proteins, such as ZO-1, keeping the intestinal mucosal barrier function to attenuate colitis symptoms in mice. Markers of inflammation cascade such as myeloperoxidase (MPO) and PPAR-gamma measured in the colon were also modified by AL0035 treatment. AL0035 was also able to reduce different lymphocyte markers’ infiltration in the colon (GATA-3, T-Bet, NK1.1) and monocyte chemoattractant protein-1 (MCP-1/CCL2), a key chemokine involved in the migration and infiltration of monocytes/macrophages in the immunological surveillance of tissues and inflammation. In colonic microbiota profile analysis through 16S rRNA sequencing, AL0035 increased the microbial diversity depleted by TNBS administration and the relative abundance of the Lactobacillaceae and Lachnospiraceae families, whereas it decreased the abundance of Proteobacteria. Altogether, these data indicated that AL0035 could lower the severity of colitis induced by TNBS by regulating inflammatory cytokines, increasing the expression of tight junction proteins and modulating intestinal microbiota, thus preventing tissue damage induced by colitis.

The COVID-19 pandemic, once a global crisis, is now largely under control, a testament to the extraordinary global efforts involving vaccination and public health measures. However, the relentless evolution of SARS-CoV-2, leading to the emergence of new variants, continues to underscore the importance of remaining vigilant and adaptable. Monoclonal antibodies (mAbs) have stood out as a powerful and immediate therapeutic response to COVID-19. Despite the success of mAbs, the evolution of SARS-CoV-2 continues to pose challenges and the available antibodies are no longer effective. New variants require the ongoing development of effective antibodies. In the present study, we describe the generation and characterization of neutralizing mAbs against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein by combining plasmid DNA and recombinant protein vaccination. By integrating genetic immunization for rapid antibody production and the potent immune stimulation enabled by protein vaccination, we produced a rich pool of antibodies, each with unique binding and neutralizing specificities, tested with the ELISA, BLI and FACS assays and the pseudovirus assay, respectively. Here, we present a panel of mAbs effective against the SARS-CoV-2 variants up to Omicron BA.1 and BA.5, with the flexibility to target emerging variants. This approach ensures the preparedness principle is in place to address SARS-CoV-2 actual and future infections.

Background Coronavirus disease 2019 (COVID-19) emerged in late December 2019 and was declared pandemic in March 2020 by the World Health Organization, causing clinically acute respiratory manifestations and corresponding symptoms, pathological inflammation and multi-organ dysfunctions. The total commitment of the scientific community to develop therapeutics to deal with this global emergency in the shortest possible period was unprecedented. In a very short time, several vaccines were approved by the EMA (European Medicines Agency) and the FDA (Food and Drug Administration). Despite this, it is conceivable that COVID-19 will continue to spread globally through evolving variants in more or less cyclic waves. With these perspectives, it is essential to quickly develop additional therapeutic tools to deal with the next wave of infection. Methods In the present study we describe the development and characterization of neutralizing mouse monoclonal antibodies (mAbs) against the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) protein. Results The mAbs identified are able to specifically detect the RBD of SARS-CoV-2 Spike protein in all tested applications, including enzyme-linked immunosorbent assay (ELISA), flow cytometry (FACS) and bio-layer interferometry. In addition, we show that these mAbs efficiently block entry of both SARS-CoV-2 pseudoparticles carrying the spike protein of the original SARS-CoV-2 strain and a broad set of variants of concern (VOC). Conclusions Here we report a panel of monoclonal antibodies that target RBD and inhibit SARS-CoV-2 variants infection and enable the isolation of novel therapeutic tools to neutralize SARS-CoV-2 virus

Objective. Periodontitis is a common disorder that leads to the loss of both tooth and personal well‐being, contributing to worsen the risk for metabolic and cardiovascular diseases. Recently, probiotics, characterized by rapid oral dispersion, have been topically used. Here, we present data of a mucoadhesive gel containing probiotics, capable of ensuring a slow release of bacteria to prevent and treat periodontitis. Methods. An original mucoadhesive gel (AL0005) that is anhydrous and of food grade, loaded with the blend of lactobacilli and plants’ dry extracts, has been assayed. Results. The release kinetics of the bacterial mixture in different experimental models in vitro, including simulated saliva or physiological solutions, showed a significant and stable release for 5–8 hours. In one in vivo study of a mouse model of periodontitis, a locally applied mucoadhesive gel enriched with probiotic strains improved significantly the tissue pathology when compared with vehicle‐exposed mice. Conclusions. Together, the results suggest that this mucoadhesive gel can be useful in the normalization of the gum bacterial flora and improvement of the tissue pathology of gum disorders.

The COVID-19 pandemic has highlighted genetic vaccination as a powerful and cost-effective tool to counteract infectious diseases. Invasive fungal infections (IFI) remain a major challenge among immune compromised patients, particularly those undergoing allogeneic hematopoietic bone marrow transplantation (HSCT) or solid organ transplant (SOT) both presenting high morbidity and mortality rates. Candidiasis and Aspergillosis are the major fungal infections among these patients and the failure of current antifungal therapies call for new therapeutic aids. Vaccination represents a valid alternative, and proof of concept of the efficacy of this approach has been provided at clinical level. This review will analyze current understanding of antifungal immunology, with a particular focus on genetic vaccination as a suitable strategy to counteract these diseases.

COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight the current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful “tool” to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.

COVID-19 has rapidly spread all over the world, progressing into a pandemic. This situation has urgently impelled many companies and public research institutes to concentrate their efforts on research for effective therapeutics. Here, we outline the strategies and targets currently adopted in developing a vaccine against SARS-CoV-2. Based on previous evidence and experience with SARS and MERS, the primary focus has been the Spike protein, considered as the ideal target for COVID-19 immunotherapies.

Fungal infections have aroused much interest over the last years because of their involvement in several human diseases. Immunocompromission due to transplant-related therapies and malignant cancer treatments are risk factors for invasive fungal infections, but also aggressive surgery, broad-spectrum antibiotics and prosthetic devices are frequently associated with infectious diseases. Current therapy is based on the administration of antifungal drugs, but the occurrence of resistant strains to the most common molecules has become a serious health-care problem. New antifungal agents are urgently needed and it is essential to identify fungal molecular targets that could offer alternatives for development of treatments. The fungal cell wall and plasma membrane are the most important structures that offer putative new targets which can be modulated in order to fight microbial infections. The development of monoclonal antibodies against new targets is a valid therapeutic strategy, both to solve resistance problems and to support the immune response, especially in immunocompromised hosts. In this review, we summarize currently used antifungal agents and propose novel therapeutic approaches, including new fungal molecular targets to be considered for drug development.