ABSTRACT
Recent clinical observations that some coronavirus infections induced complete remissions in lymphoma patients emphasized again the potential of cancer virotherapy. Infection of cancer cells with oncolytic viruses reshapes the tumor microenvironment by activating anti-viral and anti-tumor immunity. A phase 1 clinical trial using oncolytic adenovirus Delta-24-RGD (DNX-2401) to treat recurrent malignant gliomas demonstrated activation of CD8+ T-cells and significant clinical benefits for a subset of patients. However, both anti-virus and anti-tumor immune responses are contingent on the activation of respective clones of CD8+ T-cells, which compete for clonal expansion. Thus, overexpansion of T-cells against viral antigens reduces the frequency of subdominant clones against tumor antigens. We hypothesized that inducing immune tolerance for viral antigens will decrease anti-viral immunity and in turn derepress anti-tumor immunity, resulting in enhanced efficacy of cancer virotherapy. In this work, we used nanoparticles encapsulating adenoviral antigens E1A, E1B and hexon that distributed to liver resident macrophages (P<0.0001) and induced peripheral immune tolerance. Functional experiments to restimulate immune cells with viral or tumor antigens showed that injection of nanoparticles induced virus-specific immune tolerance and redirected the focus of the immune response towards tumor peptides as measured by interferon-gamma secretion (P<0.0001). Co-culture experiments also showed increased activation of immune cells against fixed tumor cells after nanoparticle treatment (P<0.0001). Reduction of virus-specific T-cells and concurrent expansion of tumor-specific T-cell clones were further confirmed with E1A or OVA tetramers (P<0.05). Flow cytometry analysis suggested increased anti-tumor responses were due to differences in T-cell clones and not due to other immune populations including natural killer cells or myeloid-derived suppressor cells (P=0.3). Importantly, virotherapy in combination with nanoparticle-induced immune tolerance towards viral antigens in tumor-bearing mice increased the overall survival and doubled the percentage of long-term survivors compared to virus treatment alone. Our data should propel the development of a future clinical trial aiming to maximize the potential of anti-tumor immunity during cancer virotherapies.
ABSTRACT
Background: Following natural infection or vaccination, the generation of stem cell-like memory T (Tscm) cells is essential for long-term protective immunity to the virus. Tscm cells have the capacity for self-renewal and multipotency. In SARS-CoV2 infection, the emergence of CD8+ Tscm cells is correlated with the number of symptom-free days. The development of a COVID-19 vaccine able to generate CD8+ Tscm cells is of the utmost importance since the emergence of SARS-CoV2 variants of concerns requires maintaining strong and long-lasting immune responses, 2) as an efficient alternative in immunocompromised people who have difficulties raising humoral immune responses. Methods: We have developed a new Dendritic Cell-based vaccine composed of a humanized αCD40 monoclonal antibody fused to the RBD protein in its C-terminal Fc-domains and three T cell epitopes spanning sequences from S and N proteins in its light chains (αCD40-CoV2). Previous studies have shown that this platform elicited durable and robust T-and B-cell responses and is currently in phase I clinical development in HIV. We tested the capacity of two injections of the vaccine (10υg, i.p) given with or without poly(IC) (50υg, i.p) at 3 weeks apart to i) elicit human (hu) B-, and huT-cell responses in NSG mice reconstituted with a Human Immune System (HIS mice), ii) protect against SARS-CoV2 infection in the hCD40xK18hACE2 transgenic mice. Results: We performed AIM assays and intracellular staining on spleen cells of HIS mice stimulated with overlapping peptide pools spanning the sequences of vaccine antigens. We found that both non-adjuvanted and adjuvanted vaccine efficiently induced SARS-CoV2-specific Th1 huCD4+ and huCD8+ T cells in all vaccinees compared to mock animals. SARS-CoV2-specific huCD4+ T cells were polyfunctional. We confirmed the presence of RBD-specific huCD8+ T cells in the vaccinated animals using HLA-I tetramers. A significant proportion of the multimer+ huCD8+ T cells were Tscm (CD45RA+ CD62L+ CD95+) cells in both vaccinated groups. Besides, we detected significant amounts of spike-IgG+ switched huB cells in all vaccinees. In SARS-CoV2 challenge experiments, we further showed that both vaccination settings significantly protected animals with a survival rate of 100%. Conclusion: We demonstrate that the targeting of SARS-CoV-2 epitopes to CD40 induces significant B and T cells with a long-term memory phenotype in HIS mice and the ability of the vaccine to ensure complete protection against SARS-CoV2 infection.
ABSTRACT
Introduction: The emergence of SARS-CoV-2 virus, which causes COVID-19, is a major global health hazard. Therefore, a comprehensive characterization of the humoral and cellular immune responses to this virus is essential to combat the COVID-19 pandemic. Our goal was to develop reliable methods and tools for the analysis of humoral and cellular B- and T- cell responses, which will facilitate scientific research for prediction of disease progression, long-term immunity and will support vaccine development. Methods: Plasma samples and PBMCs of COVID-19 convalescent and healthy donors were obtained. For the detection of SARS-CoV-2 specific antibodies and identification of antigen-specific B cells, we manufactured recombinant mono-biotinylated protein variants of the Spike (S), Receptor Binding Domain (RBD) and Nucleoprotein (N). To identify antigen-reactive T cells, SARS-CoV-2 peptide pools were synthetized for the S, N and Membrane (M) antigens and used for stimulation. The peptide pools consist of mainly 15-mer peptides having an 11-mer amino acid overlap and thereby overspan a whole protein sequence. Results: To determine the presence of SARS-CoV-2 reactive antibodies a flow-based bead assay using recombinant, mono-biotinylated SARS-CoV-2 antigens loaded onto Streptavidin (SAV)-coated-PMMA beads was set up. The beads were incubated with plasma samples and fluorochrome conjugated anti-human isotype specific antibodies for flow cytometric analysis. All the antigens tested were shown to be suitable for the detection of antibodies to SARS-CoV-2 in COVID-19 convalescent plasma. To assess the feasibility of recombinant antigens for the detection and isolation of antigen-specific B cells, the mono-biotinylated Spike and RBD antigens were tetramerized on fluorochrome-conjugated SAV. These tetramers were used for staining, magnetic enrichment and flow cytometric sorting of B cells specific to SARS-CoV-2 antigens. We were able to demonstrate that our recombinant antigens can be used to assess the presence and enable the phenotyping and isolation of rare antigen-specific B cells. For further characterization of the SARS-CoV-2 reactive T cell immunity PBMCs were short term stimulated with the S, M and N peptide pools. After intracellular staining of IFNg, TNFa, IL-2 and CD154, reactive T cells were detected using flow cytometry. We could demonstrate T cell reactivity towards each peptide pool. However, strengths of T cell responses towards the S, M and N peptide pools were heterogeneous between different COVID-19 convalescent individuals. Conclusion: To support and improve current research activities for the identification and characterization of SARS-CoV-2 reactive humoral and cellular B- and T- cell responses, potent tools and assays were developed. Described here research solutions offer the opportunity to successfully address and contribute to the investigation on healthy and dysfunctional immune reactions towards SARS-CoV-2.