Early protective effect of a ("pan") coronavirus vaccine (PanCoVac) in Roborovski dwarf hamsters after single-low dose intranasal administration.

Introduction: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the danger posed by human coronaviruses. Rapid emergence of immunoevasive variants and waning antiviral immunity decrease the effect of the currently available vaccines, which aim at induction of neutralizing antibodies. In contrast, T cells are marginally affected by antigen evolution although they represent the major mediators of virus control and vaccine protection against virus-induced disease. Materials and methods: We generated a multi-epitope vaccine (PanCoVac) that encodes the conserved T cell epitopes from all structural proteins of coronaviruses. PanCoVac contains elements that facilitate efficient processing and presentation of PanCoVac-encoded T cell epitopes and can be uploaded to any available vaccine platform. For proof of principle, we cloned PanCoVac into a non-integrating lentivirus vector (NILV-PanCoVac). We chose Roborovski dwarf hamsters for a first step in evaluating PanCoVac in vivo. Unlike mice, they are naturally susceptible to SARS-CoV-2 infection. Moreover, Roborovski dwarf hamsters develop COVID-19-like disease after infection with SARS-CoV-2 enabling us to look at pathology and clinical symptoms. Results: Using HLA-A*0201-restricted reporter T cells and U251 cells expressing a tagged version of PanCoVac, we confirmed in vitro that PanCoVac is processed and presented by HLA-A*0201. As mucosal immunity in the respiratory tract is crucial for protection against respiratory viruses such as SARS-CoV-2, we tested the protective effect of single-low dose of NILV-PanCoVac administered via the intranasal (i.n.) route in the Roborovski dwarf hamster model of COVID-19. After infection with ancestral SARS-CoV-2, animals immunized with a single-low dose of NILV-PanCoVac i.n. did not show symptoms and had significantly decreased viral loads in the lung tissue. This protective effect was observed in the early phase (2 days post infection) after challenge and was not dependent on neutralizing antibodies. Conclusion: PanCoVac, a multi-epitope vaccine covering conserved T cell epitopes from all structural proteins of coronaviruses, might protect from severe disease caused by SARS-CoV-2 variants and future pathogenic coronaviruses. The use of (HLA-) humanized animal models will allow for further efficacy studies of PanCoVac-based vaccines in vivo.

Epstein-Barr virus, interleukin-10 and multiple sclerosis: A ménage à trois.

Multiple Sclerosis (MS) is an autoimmune disease that is characterized by inflammation and demyelination of nerve cells. There is strong evidence that Epstein-Barr virus (EBV), a human herpesvirus infecting B cells, greatly increases the risk of subsequent MS. Intriguingly, EBV not only induces human interleukin-10 but also encodes a homologue of this molecule, which is a key anti-inflammatory cytokine of the immune system. Although EBV-encoded IL-10 (ebvIL-10) has a high amino acid identity with its cellular counterpart (cIL-10), it shows more restricted and partially weaker functionality. We propose that both EBV-induced cIL-10 and ebvIL-10 act in a temporally and functionally coordinated manner helping the pathogen to establish latency in B cells and, at the same time, to balance the function of antiviral T cells. As a result, the EBV load persisting in the immune system is kept at a constant but individually different level (set point). During this immunological tug of war between virus and host, however, MS can be induced as collateral damage if the set point is too high. Here, we discuss a possible role of ebvIL-10 and EBV-induced cIL-10 in EBV-driven pathogenesis of MS.

HCMV-Mediated Interference of Bortezomib-Induced Apoptosis in Colon Carcinoma Cell Line Caco-2.

Human cytomegalovirus (HCMV) has been implicated in the development of human malignancies, for instance in colon cancer. Proteasome inhibitors were developed for cancer therapy and have also been shown to influence HCMV infection. The aim of this study was to investigate if proteasome inhibitors have therapeutic potential for colon carcinoma and how this is influenced by HCMV infection. We show by immunofluorescence and flow cytometry that the colon carcinoma cell line Caco-2 is susceptible to HCMV infection. Growth curve analysis as well as protein expression kinetics and quantitative genome analysis further confirm these results. HCMV has an anti-apoptotic effect on Caco-2 cells by inhibiting very early events of the apoptosis cascade. Further investigations showed that HCMV stabilizes the membrane potential of the mitochondria, which is typically lost very early during apoptosis. This stabilization is resistant to proteasome inhibitor Bortezomib treatment, allowing HCMV-infected cells to survive apoptotic signals. Our findings indicate a possible role of proteasome inhibitors in colon carcinoma therapy.

Development of a Human Cytomegalovirus (HCMV)-Based Therapeutic Cancer Vaccine Uncovers a Previously Unsuspected Viral Block of MHC Class I Antigen Presentation.

Human cytomegalovirus (HCMV) induces a uniquely high frequency of virus-specific effector/memory CD8+ T-cells, a phenomenon termed "memory inflation". Thus, HCMV-based vaccines are particularly interesting in order to stimulate a sustained and strong cellular immune response against cancer. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with high lethality and inevitable relapse. The current standard treatment does not significantly improve the desperate situation underlining the urgent need to develop novel approaches. Although HCMV is highly fastidious with regard to species and cell type, GBM cell lines are susceptible to HCMV. In order to generate HCMV-based therapeutic vaccine candidates, we deleted all HCMV-encoded proteins (immunoevasins) that interfere with MHC class I presentation. The aim being to use the viral vector as an adjuvant for presentation of endogenous tumor antigens, the presentation of high levels of vector-encoded neoantigens and finally the repurposing of bystander HCMV-specific CD8+ T cells to fight the tumor. As neoantigen, we exemplarily used the E6 and E7 proteins of human papillomavirus type 16 (HPV-16) as a non-transforming fusion protein (E6/E7) that covers all relevant antigenic peptides. Surprisingly, GBM cells infected with E6/E7-expressing HCMV-vectors failed to stimulate E6-specific T cells despite high level expression of E6/E7 protein. Further experiments revealed that MHC class I presentation of E6/E7 is impaired by the HCMV-vector although it lacks all known immunoevasins. We also generated HCMV-based vectors that express E6-derived peptide fused to HCMV proteins. GBM cells infected with these vectors efficiently stimulated E6-specific T cells. Thus, fusion of antigenic sequences to HCMV proteins is required for efficient presentation via MHC class I molecules during infection. Taken together, these results provide the preclinical basis for development of HCMV-based vaccines and also reveal a novel HCMV-encoded block of MHC class I presentation.

Hantavirus-Driven PD-L1/PD-L2 Upregulation: An Imperfect Viral Immune Evasion Mechanism.

Viruses often subvert antiviral immune responses by taking advantage of inhibitory immune signaling. We investigated if hantaviruses use this strategy. Hantaviruses cause viral hemorrhagic fever (VHF) which is associated with strong immune activation resulting in vigorous CD8+ T cell responses. Surprisingly, we observed that hantaviruses strongly upregulate PD-L1 and PD-L2, the ligands of checkpoint inhibitor programmed death-1 (PD-1). We detected high amounts of soluble PD-L1 (sPD-L1) and soluble PD-L2 (sPD-L2) in sera from hantavirus-infected patients. In addition, we observed hantavirus-induced PD-L1 upregulation in mice with a humanized immune system. The two major target cells of hantaviruses, endothelial cells and monocyte-derived dendritic cells, strongly increased PD-L1 and PD-L2 surface expression upon hantavirus infection in vitro. As an underlying mechanism, we found increased transcript levels whereas membrane trafficking of PD-L1 was not affected. Further analysis revealed that hantavirus-associated inflammatory signals and hantaviral nucleocapsid (N) protein enhance PD-L1 and PD-L2 expression. Cell numbers were strongly reduced when hantavirus-infected endothelial cells were mixed with T cells in the presence of an exogenous proliferation signal compared to uninfected cells. This is compatible with the concept that virus-induced PD-L1 and PD-L2 upregulation contributes to viral immune escape. Intriguingly, however, we observed hantavirus-induced CD8+ T cell bystander activation despite strongly upregulated PD-L1 and PD-L2. This result indicates that hantavirus-induced CD8+ T cell bystander activation bypasses checkpoint inhibition allowing an early antiviral immune response upon virus infection.

Herpesviral capture of immunomodulatory host genes.

Herpesviruses have acquired numerous genes from their hosts. Although these homologs are not essential for viral replication, they often have important immunomodulatory functions that ensure viral persistence in the host. Some of these viral molecules are called virokines as they mimic cellular cytokines of their host such as interleukin-10 (cIL-10). In recent years, many viral homologs of IL-10 (vIL-10s) have been discovered in the genome of members of the order Herpesvirales. For some, gene and protein structure as well as biological activity and potential use in the clinical context have been explored. Besides virokines, herpesviruses have also captured genes encoding membrane-bound host immunomodulatory proteins such as major histocompatibility complex (MHC) molecules. These viral MHC mimics also retain many of the functions of the cellular genes, in particular directly or indirectly modulating the activity of natural killer cells. The mechanisms underlying capture of cellular genes by large DNA viruses are still enigmatic. In this review, we provide an update of the advances in the field of herpesviral gene piracy and discuss possible scenarios that could explain how the gene transfer from host to viral genome was achieved.

Cephalosporin resistant Escherichia coli from cancer patients in Cairo, Egypt.

Cephalosporin-resistant Escherichia coli has been increasingly reported worldwide. In this study, 32 cephalosporin resistant E. coli isolates identified from cancer patients in Cairo, Egypt in 2009-2010 were analyzed. Twenty-three were of phylogenetic group D, seven A and one each B1 and B2. By rep-PCR 15 phylogroup D isolates were grouped in four clusters, one with sequence type (ST) 405 and three ST68. Seventeen isolates showed single patterns. blaCTX-M-15 and aac(6')-Ib-cr were the most common resistance determinants. blaOXA-48 and blaVIM were also detected. Multidrug resistant E. coli seriously affects healthcare, especially in immunocompromised hosts, such as cancer patients.

NDM-1- and OXA-163-producing Klebsiella pneumoniae isolates in Cairo, Egypt, 2012.

Here we describe carbapenem resistance determinants in two Klebsiella pneumoniae isolates recovered from two hospitalised patients in the same intensive care unit of a cancer hospital in Cairo, Egypt. PCR and sequencing were used to detect and characterise ß-lactamase genes. Clonal relationships between the isolates were analysed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The first K. pneumoniae isolate carried the blaNDM-1 gene and the second isolate carried the blaOXA-163 gene. Both isolates co-expressed the extended-spectrum ß-lactamase CTX-M-15. The two isolates belonged to different sequence types (STs), ST11 and ST16, respectively. No history of travel was established for the two patients. The first identification of NDM-1-producing K. pneumoniae in Egypt adds further evidence to the spread of NDM-1-producing Gram-negative micro-organisms in North Africa. The additional detection of blaOXA-163 in a K. pneumoniae isolate confirms its endemic presence in a critical healthcare setting of this geographic area.

OXA-163-producing Klebsiella pneumoniae in Cairo, Egypt, in 2009 and 2010.

Two genetically unrelated OXA-163-carrying Klebsiella pneumoniae strains were identified from two infection cases in June 2009 and May 2010 in Cairo, Egypt. OXA-163-producing Enterobacteriaceae had been previously reported in Argentina only. Both patients had no history of travel abroad. The emergence of this newly recognized OXA-48-related ß-lactamase able to hydrolyze cephalosporins and carbapenems is especially worrying in a geographic area where OXA-48 is endemic and effective surveillance for antibiotic resistance is largely unaffordable.