Antiviral and Anti-Inflammatory Therapeutic Effect of RAGE-Ig Protein against Multiple SARS-CoV-2 Variants of Concern Demonstrated in K18-hACE2 Mouse and Syrian Golden Hamster Models.

SARS-CoV-2 variants of concern (VOCs) continue to evolve and reemerge with chronic inflammatory long COVID sequelae, necessitating the development of anti-inflammatory therapeutic molecules. Therapeutic effects of the receptor for advanced glycation end products (RAGE) were reported in many inflammatory diseases. However, a therapeutic effect of RAGE in COVID-19 has not been reported. In the present study, we investigated whether and how the RAGE-Ig fusion protein would have an antiviral and anti-inflammatory therapeutic effect in the COVID-19 system. The protective therapeutic effect of RAGE-Ig was determined in vivo in K18-hACE2 transgenic mice and Syrian golden hamsters infected with six VOCs of SARS-CoV-2. The underlying antiviral mechanism of RAGE-Ig was determined in vitro in SARS-CoV-2-infected human lung epithelial cells (BEAS-2B). Following treatment of K18-hACE2 mice and hamsters infected with various SARS-CoV-2 VOCs with RAGE-Ig, we demonstrated (1) significant dose-dependent protection (i.e., greater survival, less weight loss, lower virus replication in the lungs); (2) a reduction of inflammatory macrophages (F4/80+/Ly6C+) and neutrophils (CD11b+/Ly6G+) infiltrating the infected lungs; (3) a RAGE-Ig dose-dependent increase in the expression of type I IFNs (IFN-α and IFN-ß) and type III IFN (IFNλ2) and a decrease in the inflammatory cytokines (IL-6 and IL-8) in SARS-CoV-2-infected human lung epithelial cells; and (4) a dose-dependent decrease in the expression of CD64 (FcgR1) on monocytes and lung epithelial cells from symptomatic COVID-19 patients. Our preclinical findings revealed type I and III IFN-mediated antiviral and anti-inflammatory therapeutic effects of RAGE-Ig protein against COVID-19 caused by multiple SARS-CoV-2 VOCs.

Colorectal Cancer Stem Cell Subtypes Orchestrate Distinct Tumor Microenvironments.

Several classification systems have been developed to define tumor subtypes in colorectal cancer (CRC). One system proposes that tumor heterogeneity derives in part from distinct cancer stem cell populations that co-exist as admixtures of varying proportions. However, the lack of single cell resolution has prohibited a definitive identification of these types of stem cells and therefore any understanding of how each influence tumor phenotypes. Here were report the isolation and characterization of two cancer stem cell subtypes from the SW480 CRC cell line. We find these cancer stem cells are oncogenic versions of the normal Crypt Base Columnar (CBC) and Regenerative Stem Cell (RSC) populations from intestinal crypts and that their gene signatures are consistent with the "Admixture" and other CRC classification systems. Using publicly available single cell RNA sequencing (scRNAseq) data from CRC patients, we determine that RSC and CBC cancer stem cells are commonly co-present in human CRC. To characterize influences on the tumor microenvironment, we develop subtype-specific xenograft models and we define their tumor microenvironments at high resolution via scRNAseq. RSCs create differentiated, inflammatory, slow growing tumors. CBCs create proliferative, undifferentiated, invasive tumors. With this enhanced resolution, we unify current CRC patient classification schema with TME phenotypes and organization.

Cross-protection induced by highly conserved human B, CD4+, and CD8+ T-cell epitopes-based vaccine against severe infection, disease, and death caused by multiple SARS-CoV-2 variants of concern.

Background: The coronavirus disease 2019 (COVID-19) pandemic has created one of the largest global health crises in almost a century. Although the current rate of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections has decreased significantly, the long-term outlook of COVID-19 remains a serious cause of morbidity and mortality worldwide, with the mortality rate still substantially surpassing even that recorded for influenza viruses. The continued emergence of SARS-CoV-2 variants of concern (VOCs), including multiple heavily mutated Omicron sub-variants, has prolonged the COVID-19 pandemic and underscores the urgent need for a next-generation vaccine that will protect from multiple SARS-CoV-2 VOCs. Methods: We designed a multi-epitope-based coronavirus vaccine that incorporated B, CD4+, and CD8+ T- cell epitopes conserved among all known SARS-CoV-2 VOCs and selectively recognized by CD8+ and CD4+ T-cells from asymptomatic COVID-19 patients irrespective of VOC infection. The safety, immunogenicity, and cross-protective immunity of this pan-variant SARS-CoV-2 vaccine were studied against six VOCs using an innovative triple transgenic h-ACE-2-HLA-A2/DR mouse model. Results: The pan-variant SARS-CoV-2 vaccine (i) is safe , (ii) induces high frequencies of lung-resident functional CD8+ and CD4+ TEM and TRM cells , and (iii) provides robust protection against morbidity and virus replication. COVID-19-related lung pathology and death were caused by six SARS-CoV-2 VOCs: Alpha (B.1.1.7), Beta (B.1.351), Gamma or P1 (B.1.1.28.1), Delta (lineage B.1.617.2), and Omicron (B.1.1.529). Conclusion: A multi-epitope pan-variant SARS-CoV-2 vaccine bearing conserved human B- and T- cell epitopes from structural and non-structural SARS-CoV-2 antigens induced cross-protective immunity that facilitated virus clearance, and reduced morbidity, COVID-19-related lung pathology, and death caused by multiple SARS-CoV-2 VOCs.

High frequencies of alpha common cold coronavirus/SARS-CoV-2 cross-reactive functional CD4+ and CD8+ memory T cells are associated with protection from symptomatic and fatal SARS-CoV-2 infections in unvaccinated COVID-19 patients.

Background: Cross-reactive SARS-CoV-2-specific memory CD4+ and CD8+ T cells are present in up to 50% of unexposed, pre-pandemic, healthy individuals (UPPHIs). However, the characteristics of cross-reactive memory CD4+ and CD8+ T cells associated with subsequent protection of asymptomatic coronavirus disease 2019 (COVID-19) patients (i.e., unvaccinated individuals who never develop any COVID-19 symptoms despite being infected with SARS-CoV-2) remains to be fully elucidated. Methods: This study compares the antigen specificity, frequency, phenotype, and function of cross-reactive memory CD4+ and CD8+ T cells between common cold coronaviruses (CCCs) and SARS-CoV-2. T-cell responses against genome-wide conserved epitopes were studied early in the disease course in a cohort of 147 unvaccinated COVID-19 patients who were divided into six groups based on the severity of their symptoms. Results: Compared to severely ill COVID-19 patients and patients with fatal COVID-19 outcomes, the asymptomatic COVID-19 patients displayed significantly: (i) higher rates of co-infection with the 229E alpha species of CCCs (α-CCC-229E); (ii) higher frequencies of cross-reactive functional CD134+CD137+CD4+ and CD134+CD137+CD8+ T cells that cross-recognized conserved epitopes from α-CCCs and SARS-CoV-2 structural, non-structural, and accessory proteins; and (iii) lower frequencies of CCCs/SARS-CoV-2 cross-reactive exhausted PD-1+TIM3+TIGIT+CTLA4+CD4+ and PD-1+TIM3+TIGIT+CTLA4+CD8+ T cells, detected both ex vivo and in vitro. Conclusions: These findings (i) support a crucial role of functional, poly-antigenic α-CCCs/SARS-CoV-2 cross-reactive memory CD4+ and CD8+ T cells, induced following previous CCCs seasonal exposures, in protection against subsequent severe COVID-19 disease and (ii) provide critical insights into developing broadly protective, multi-antigen, CD4+, and CD8+ T-cell-based, universal pan-Coronavirus vaccines capable of conferring cross-species protection.

A Broad-Spectrum Multi-Antigen mRNA/LNP-Based Pan-Coronavirus Vaccine Induced Potent Cross-Protective Immunity Against Infection and Disease Caused by Highly Pathogenic and Heavily Spike-Mutated SARS-CoV-2 Variants of Concern in the Syrian Hamster Model.

The first-generation Spike-alone-based COVID-19 vaccines have successfully contributed to reducing the risk of hospitalization, serious illness, and death caused by SARS-CoV-2 infections. However, waning immunity induced by these vaccines failed to prevent immune escape by many variants of concern (VOCs) that emerged from 2020 to 2024, resulting in a prolonged COVID-19 pandemic. We hypothesize that a next-generation Coronavirus (CoV) vaccine incorporating highly conserved non-Spike SARS-CoV-2 antigens would confer stronger and broader cross-protective immunity against multiple VOCs. In the present study, we identified ten non-Spike antigens that are highly conserved in 8.7 million SARS-CoV-2 strains, twenty-one VOCs, SARS-CoV, MERS-CoV, Common Cold CoVs, and animal CoVs. Seven of the 10 antigens were preferentially recognized by CD8+ and CD4+ T-cells from unvaccinated asymptomatic COVID-19 patients, irrespective of VOC infection. Three out of the seven conserved non-Spike T cell antigens belong to the early expressed Replication and Transcription Complex (RTC) region, when administered to the golden Syrian hamsters, in combination with Spike, as nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNP) (i.e., combined mRNA/LNP-based pan-CoV vaccine): (i) Induced high frequencies of lung-resident antigen-specific CXCR5+CD4+ T follicular helper (TFH) cells, GzmB+CD4+ and GzmB+CD8+ cytotoxic T cells (TCYT), and CD69+IFN-γ+TNFα+CD4+ and CD69+IFN-γ+TNFα+CD8+ effector T cells (TEFF); and (ii) Reduced viral load and COVID-19-like symptoms caused by various VOCs, including the highly pathogenic B.1.617.2 Delta variant and the highly transmittable heavily Spike-mutated XBB1.5 Omicron sub-variant. The combined mRNA/LNP-based pan-CoV vaccine could be rapidly adapted for clinical use to confer broader cross-protective immunity against emerging highly mutated and pathogenic VOCs.