Glioblastoma cellular cross-talk converges on NF-κB to attenuate EGFR inhibitor sensitivity.

In glioblastoma (GBM), heterogeneous expression of amplified and mutated epidermal growth factor receptor (EGFR) presents a substantial challenge for the effective use of EGFR-directed therapeutics. Here we demonstrate that heterogeneous expression of the wild-type receptor and its constitutively active mutant form, EGFRvIII, limits sensitivity to these therapies through an interclonal communication mechanism mediated by interleukin-6 (IL-6) cytokine secreted from EGFRvIII-positive tumor cells. IL-6 activates a NF-κB signaling axis in a paracrine and autocrine manner, leading to bromodomain protein 4 (BRD4)-dependent expression of the prosurvival protein survivin (BIRC5) and attenuation of sensitivity to EGFR tyrosine kinase inhibitors (TKIs). NF-κB and survivin are coordinately up-regulated in GBM patient tumors, and functional inhibition of either protein or BRD4 in in vitro and in vivo models restores sensitivity to EGFR TKIs. These results provide a rationale for improving anti-EGFR therapeutic efficacy through pharmacological uncoupling of a convergence point of NF-κB-mediated survival that is leveraged by an interclonal circuitry mechanism established by intratumoral mutational heterogeneity.

Prokaryotic Expression, Purification and Immunogenicity in Rabbits of the Small Antigen of Hepatitis Delta Virus.

Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 µg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 µg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 107. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.

Chronic Viral Infections and Cancer, Openings for Therapies and Vaccines.

Infections are responsible for approximately one out of six cases of cancer worldwide [...].

HPV Type Distribution in Benign, High-Grade Squamous Intraepithelial Lesions and Squamous Cell Cancers of the Anus by HIV Status.

The incidence of anal cancer is increasing, especially in high-risk groups, such as PLWH. HPV 16, a high-risk (HR) HPV genotype, is the most common genotype in anal high-grade squamous intraepithelial lesions (HSIL) and squamous cell carcinoma (SCC) in the general population. However, few studies have described the distribution of HR HPV genotypes other than HPV 16 in the anus of PLWH. HPV genotyping was performed by DNA amplification followed by dot-blot hybridization to identify the HR and low-risk (LR) genotypes in benign anal lesions (n = 34), HSIL (n = 30), and SCC (n = 51) of PLWH and HIV-negative individuals. HPV 16 was the most prominent HR HPV identified, but it was less common in HSIL and SCC from PLWH compared with HIV-negative individuals, and other non-HPV 16 HR HPV (non-16 HR HPV) types were more prevalent in samples from PLWH. A higher proportion of clinically normal tissues from PLWH were positive for one or more HPV genotypes. Multiple HPV infection was a hallmark feature for all tissues (benign, HSIL, SCC) of PLWH. These results indicate that the development of anal screening approaches based on HPV DNA testing need to include non-16 HR HPVs along with HPV 16, especially for PLWH. Along with anal cytology, these updated screening approaches may help to identify and prevent anal disease progression in PLWH.

An overview of "Chronic viral infection and cancer, openings for vaccines" virtual symposium of the TechVac Network - December 16-17, 2021.

This is a report on the research activities currently ongoing in virology, oncology and virus-associated cancers and possibilities of their treatment and prevention by vaccines and immunotherapies as outlined at the symposium "Chronic viral infection and cancer, openings for vaccines" virtually held on December 16-17, 2021. Experts from the various disciplines involved in the study of the complex relationships between solid tumors and viruses met to discuss recent developments in the field and to report their personal contributions to the specified topics. Secondary end point was to sustain the TECHVAC Network established in 2016 as a multidisciplinary work group specifically devoted to development of vaccines and immunotherapies against chronic viral infections and associated cancers, with the aim to identify areas of common interest, promote research cooperation, establish collaborative cross-border programs and projects, and to coordinate clinical and research activities.

The Molecular Interplay between Human Oncoviruses and Telomerase in Cancer Development.

Human oncoviruses are able to subvert telomerase function in cancer cells through multiple strategies. The activity of the catalytic subunit of telomerase (TERT) is universally enhanced in virus-related cancers. Viral oncoproteins, such as high-risk human papillomavirus (HPV) E6, Epstein-Barr virus (EBV) LMP1, Kaposi's sarcoma-associated herpesvirus (HHV-8) LANA, hepatitis B virus (HBV) HBVx, hepatitis C virus (HCV) core protein and human T-cell leukemia virus-1 (HTLV-1) Tax protein, interact with regulatory elements in the infected cells and contribute to the transcriptional activation of TERT gene. Specifically, viral oncoproteins have been shown to bind TERT promoter, to induce post-transcriptional alterations of TERT mRNA and to cause epigenetic modifications, which have important effects on the regulation of telomeric and extra-telomeric functions of the telomerase. Other viruses, such as herpesviruses, operate by integrating their genomes within the telomeres or by inducing alternative lengthening of telomeres (ALT) in non-ALT cells. In this review, we recapitulate on recent findings on virus-telomerase/telomeres interplay and the importance of TERT-related oncogenic pathways activated by cancer-causing viruses.

Coverage with Timely Administered Vaccination against Hepatitis B Virus and Its Influence on the Prevalence of HBV Infection in the Regions of Different Endemicity.

Universal hepatitis B vaccination of newborns was implemented in Russia starting from 1998. From 1998 to 2019, the incidence of acute hepatitis B reduced from 43.8 to 0.57 cases per 100,000 population. Here, we assessed the timely coverage of newborns with the birth dose (HepB-BD), second dose (HepB-2nd), and three vaccine doses (HepB3) in two remote regions of Russia with low (Belgorod Oblast) and high (Yakutia) levels of hepatitis B virus (HBV) endemicity. Vaccination data were obtained from the medical records of 1000 children in Yakutia and 2182 children in Belgorod Oblast. Sera of healthy volunteers from Belgorod Oblast (n = 1754) and Yakutia (n = 1072) across all age groups were tested for serological markers of HBV to assess the infection prevalence and herd immunity. Average HepB-BD coverage was 99.2% in Yakutia and 89.4% in Belgorod Oblast (p < 0.0001) and in both regions varied significantly, from 66% to 100%, between medical centers. The principal reason for the absence of HepB-BD was parent refusal, which accounted for 63.5% of cases of non-vaccination (83/123). While timely HepB-2nd coverage was only 55.4%-64.7%: HepB3 coverage by the age of one year exceeded 90% in both study regions. HBV surface antigen (HBsAg) prevalence in the 1998-2019 birth cohort was 0.2% (95% CI: 0.01-1.3%) in Belgorod Oblast and 3.2% (95% CI: 1.9-5.2%) in Yakutia. The proportion of persons testing negative for both antibodies to HBsAg (anti-HBs) and antibodies to HBV core antigen (anti-HBc) in the 1998-2019 birth cohort was 26.2% (125/481) in Belgorod Oblast and 32.3% (162/501) in Yakutia. We also assessed the knowledge of and attitude towards vaccination among 782 students and teachers of both medical and non-medical specialties from Belgorod State University. Only 60% of medical students knew that hepatitis B is a vaccine-preventable disease. Both medical and nonmedical students, 37.8% and 31.3%, respectively, expressed concerns about safety and actual necessity of vaccination. These data indicate the need to introduce a vaccine delivery audit system, improve medical education with respect to vaccination strategies and policies, and reinforce public knowledge on the benefits of vaccination.

Factors Influencing the Prevalence of Resistance-Associated Substitutions in NS5A Protein in Treatment-Naive Patients with Chronic Hepatitis C.

Direct-acting antivirals (DAAs) revolutionized treatment of hepatitis C virus (HCV) infection. Resistance-associated substitutions (RASs) present at the baseline impair response to DAA due to rapid selection of resistant HCV strains. NS5A is indispensable target of the current DAA treatment regimens. We evaluated prevalence of RASs in NS5A in DAA-naïve patients infected with HCV 1a (n = 19), 1b (n = 93), and 3a (n = 90) before systematic DAA application in the territory of the Russian Federation. Total proportion of strains carrying at least one RAS constituted 35.1% (71/202). In HCV 1a we detected only M28V (57.9%) attributed to a founder effect. Common RASs in HCV 1b were R30Q (7.5%), L31M (5.4%), P58S (4.4%), and Y93H (5.4%); in HCV 3a, A30S (31.0%), A30K (5.7%), S62L (8.9%), and Y93H (2.2%). Prevalence of RASs in NS5A of HCV 1b and 3a was similar to that worldwide, including countries practicing massive DAA application, i.e., it was not related to treatment. NS5A with and without RASs exhibited different co-variance networks, which could be attributed to the necessity to preserve viral fitness. Majority of RASs were localized in polymorphic regions subjected to immune pressure, with selected substitutions allowing immune escape. Altogether, this explains high prevalence of RAS in NS5A and low barrier for their appearance in DAA-inexperienced population.

The Immunogenicity in Mice of HCV Core Delivered as DNA Is Modulated by Its Capacity to Induce Oxidative Stress and Oxidative Stress Response.

HCV core is an attractive HCV vaccine target, however, clinical or preclinical trials of core-based vaccines showed little success. We aimed to delineate what restricts its immunogenicity and improve immunogenic performance in mice. We designed plasmids encoding full-length HCV 1b core and its variants truncated after amino acids (aa) 60, 98, 152, 173, or up to aa 36 using virus-derived or synthetic polynucleotides (core191/60/98/152/173/36_191v or core152s DNA, respectively). We assessed their level of expression, route of degradation, ability to trigger the production of reactive oxygen species/ROS, and to activate the components of the Nrf2/ARE antioxidant defense pathway heme oxygenase 1/HO-1 and NAD(P)H: quinone oxidoreductase/Nqo-1. All core variants with the intact N-terminus induced production of ROS, and up-regulated expression of HO-1 and Nqo-1. The capacity of core variants to induce ROS and up-regulate HO-1 and Nqo-1 expression predetermined their immunogenicity in DNA-immunized BALB/c and C57BL/6 mice. The most immunogenic was core 152s, expressed at a modest level and inducing moderate oxidative stress and oxidative stress response. Thus, immunogenicity of HCV core is shaped by its ability to induce ROS and oxidative stress response. These considerations are important in understanding the mechanisms of viral suppression of cellular immune response and in HCV vaccine design.

A qPCR assay for measuring the post-integrational DNA repair in HIV-1 replication.

The post-integrational gap repair is a critical and poorly studied stage of the lentiviral life cycle. It might be performed by various cellular DNA repair pathways but the exact mechanism of the repair process has not yet been described. One of the reasons for that is the lack of a functional quantitative assay that could precisely measure the amount of integrated viral DNA that has completed the post-integrational gap repair stage. Here, we present an approach that is based on a widely used Alu-specific PCR for the estimation of integrated viral DNA but includes several steps that allow discrimination between integrated-repaired and integrated-unrepaired viral DNA forms. We used the approach for the estimation of the kinetics of gap repair in a viral vector system and showed that the gap repair process starts at 17 h post infection and lasts 10 more hours. We also showed that the addition of Nu7441 - a small molecule inhibitor of DNA-breaks sensor kinase in the non-homologous end joining DNA repair pathway - specifically inhibits the gap repair process while having no influence on the integration itself.

Signal sequences modulate the immunogenic performance of human hepatitis C virus E2 gene.

Envelope protein E2 of human hepatitis C virus (HCV) is an attractive component of a prototype HCV vaccine. Delivered by DNA immunogens, E2 evokes specific immune response of Th1-type, failing to induce either considerable antibody production, or T-helper cell proliferation. We aimed at modulating the immunogenic performance of E2 gene by changing the mode of protein expression in eukaryotic cells. Plasmids were constructed encoding full-length E2 and nonstructural protein 1 (p7) fused to either 13 or 38 C-terminal amino acids (aa) of HCV E1 that contain second hydrophobic segment of E1 stop-transfer signal, or a complete E1 stop-transfer signal with duplicated second hydrophobic segment. Injected into BALB/c mice, E2/p7 genes induced potent antibody and T-helper cell response targeted against hypervariable region 1, aa 472-586 of E2, and a novel epitope at aa 774-796 of p7. Profile of cytokines secreted by proliferating mouse splenocytes stimulated in vitro with E2- and p7-derived peptides, indicated mixed Th1/Th2 type of immune response. Thus, the full-length E2 and p7 genes supplied in one cassette were both immunogenic. E2/p7 containing a complete E1 stop-transfer signal with prolonged membrane spanning domain was superior to the shorter E2/p7 version in terms of both antibody and cellular immunogenicity. Optimal performance of HCV E2 could thus be achieved without the aid of external/heterologous signals by easing, through modification of the E2 signal sequence, the release of E2 from the rough ER while retaining full-length E2 and p7 sequences. This finding may help to improve the Th2 performance of HCV envelope genes as prototype vaccines.

Oxidative stress, a trigger of hepatitis C and B virus-induced liver carcinogenesis.

Virally induced liver cancer usually evolves over long periods of time in the context of a strongly oxidative microenvironment, characterized by chronic liver inflammation and regeneration processes. They ultimately lead to oncogenic mutations in many cellular signaling cascades that drive cell growth and proliferation. Oxidative stress, induced by hepatitis viruses, therefore is one of the factors that drives the neoplastic transformation process in the liver. This review summarizes current knowledge on oxidative stress and oxidative stress responses induced by human hepatitis B and C viruses. It focuses on the molecular mechanisms by which these viruses activate cellular enzymes/systems that generate or scavenge reactive oxygen species (ROS) and control cellular redox homeostasis. The impact of an altered cellular redox homeostasis on the initiation and establishment of chronic viral infection, as well as on the course and outcome of liver fibrosis and hepatocarcinogenesis will be discussed The review neither discusses reactive nitrogen species, although their metabolism is interferes with that of ROS, nor antioxidants as potential therapeutic remedies against viral infections, both subjects meriting an independent review.

Hepatitis C virus core protein transforms murine fibroblasts by promoting genomic instability.

The oncogenic potential of hepatitis C virus (HCV) core protein has been demonstrated, but the precise mechanism of cell transformation triggered by HCV core is still unclear. This study shows that constitutive expression of HCV core protein (core) in NIH 3T3 murine fibroblasts triggers malignant transformation. At the preneoplastic stage, clones that expressed HCV core constitutively demonstrated genomic instability seen as disruption of the mitotic spindle cell checkpoint leading to increased ploidy. Transformation was completed by the loss of DNA and resistance to apoptosis induced by serum starvation. Simultaneously, cells acquired a capacity for anchorage independent growth and absence of contact inhibition. Inoculation of these transformed cells into severe combined immune deficiency (SCID) mice led to formation of solid core-expressing tumors. Transformation and tumorigenicity of core-expressing cell lines coincided with a 5- to 10-fold repression of endogenous p53 transactivation. Thus, long-term HCV core expression alone is sufficient for complete transformation of immortal fibroblasts that can then induce tumors in a susceptible host. This data suggests that malignant transformation by HCV core may occur through primary stress, induction of genomic instability, and further HCV core-induced rescue of surviving mutated cells.

Oxidative Stress during HIV Infection: Mechanisms and Consequences.

It is generally acknowledged that reactive oxygen species (ROS) play crucial roles in a variety of natural processes in cells. If increased to levels which cannot be neutralized by the defense mechanisms, they damage biological molecules, alter their functions, and also act as signaling molecules thus generating a spectrum of pathologies. In this review, we summarize current data on oxidative stress markers associated with human immunodeficiency virus type-1 (HIV-1) infection, analyze mechanisms by which this virus triggers massive ROS production, and describe the status of various defense mechanisms of the infected host cell. In addition, we have scrutinized scarce data on the effect of ROS on HIV-1 replication. Finally, we present current state of knowledge on the redox alterations as crucial factors of HIV-1 pathogenicity, such as neurotoxicity and dementia, exhaustion of CD4+/CD8+ T-cells, predisposition to lung infections, and certain side effects of the antiretroviral therapy, and compare them to the pathologies associated with the nitrosative stress.

HCV core protein uses multiple mechanisms to induce oxidative stress in human hepatoma Huh7 cells.

Hepatitis C virus (HCV) infection is accompanied by the induction of oxidative stress, mediated by several virus proteins, the most prominent being the nucleocapsid protein (HCV core). Here, using the truncated forms of HCV core, we have delineated several mechanisms by which it induces the oxidative stress. The N-terminal 36 amino acids of HCV core induced TGF\(\upbeta\)1-dependent expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 1 and 4, both of which independently contributed to the production of reactive oxygen species (ROS). The same fragment also induced the expression of cyclo-oxygenase 2, which, however, made no input into ROS production. Amino acids 37-191 of HCV core up-regulated the transcription of a ROS generating enzyme cytochrome P450 2E1. Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1\(\upalpha\). The latter triggered efflux of Ca2+ from ER to mitochondria via mitochondrial Ca2+ uniporter, leading to generation of superoxide anions, and possibly also H2O2. Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production. Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein.

DNA immunization efficiently targets conserved functional domains of protease and ATPase/helicase of nonstructural 3 protein (NS3) of human hepatitis C virus.

Nonstructural protein 3 (NS3) of human hepatitis C virus (HCV) is a conserved multi-functional protein essential for replication and translation of viral RNA and polyprotein processing. Early T-cell response against NS3 is capable of restricting viremia. We aimed at characterizing the immunogenicity in gene immunization of the conserved regions of NS3 critical for protein folding and activity. C57BL/6 mice were injected with NS3 gene of Russian HCV 1b isolate 274933RU. Immunization did not exert any overt histological changes and had no long-term effects on the immune status of NS3 gene-recipients. The immune response in NS3 gene-recipients was screened by antibody ELISA, T-cell proliferation test and immune assays for specific cytokine production. T-lymphocytes of NS3 gene-recipients proliferated in response to peptides representing conserved regions of protease and ATPase/helicase. Stimulated T-lymphocytes produced IL-2, and in response to protease-derived peptides, also IFN-gamma. Potent and long-lasting antibody response was raised against conserved NS3 regions including "Greek-key" motif of protease, motifs II, V and polynucleotide-binding domains of ATPase/helicase. Thus, gene immunization effectively targeted conserved regions critical for NS3 protease and helicase function. In type and specificity, immune response of NS3 gene-immunized mice mimicked immunity achieved in the acute self-limiting HCV infection of human and primates and in virus-exposed healthy individuals, indicating promiscuity of NS3 as immunogen.

Mutations conferring drug resistance affect eukaryotic expression of HIV type 1 reverse transcriptase.

Mutations in reverse transcriptase (RT) confer high levels of HIV resistance to drugs. However, while conferring drug resistance, they can lower viral replication capacity (fitness). The molecular mechanisms behind remain largely unknown. The aim of the study was to characterize the effect of drug-resistance mutations on HIV RT expression. Genes encoding AZT-resistant RTs with single or combined mutations D67N, K70R, T215F, and K219Q, and RTs derived from drug-resistant HIV-1 strains were designed and expressed in a variety of eukaryotic cells. Expression in transiently transfected cells was assessed by Western blotting and immunofluorescent staining with RT-specific antibodies. To compare the levels of expression, mutated RT genes were microinjected into the nucleus of the oocytes of Xenopus laevis. Expression of RT was quantified by sandwich ELISA. Relative stability of RTs was assessed by pulse-chase experiments. Xenopus oocytes microinjected with the genes expressed 2-50 pg of RT mutants per cell. The level of RT expression decreased with accumulation of drug-resistance mutations. Pulse-chase experiments demonstrated that poor expression of DR-RTs was due to proteolytic instability. Instability could be attributed to additional cleavage sites predicted to appear in the vicinity of resistance mutations. Accumulation of drug-resistance mutations appears to affect the level of eukaryotic expression of HIV-1 RT by inducing proteolytic instability. Low RT levels might be one of the determinants of impaired replication fitness of drug-resistant HIV-1 strains.

Hepatitis C virus clearance: the enigma of failure despite an impeccable survival strategy.

Infection with human hepatitis C virus (HCV) as a result of a bilateral process of host-virus interactions. There are factors on both sides that contribute to clearance and to chronicity. Virus strategy to survive is built on several basic features. The first, recently recognized, is a wide cell tropism. HCV can infect not only hepatocytes, but also cells of immune system (B-cells, monocytes, macrophages, dendritic cells), epithelium, and immunologically privileged sites such as the central nervous system. Dendritic cells and platelets can also be passive virus carriers. Possibilities of virus clearance or abortive inapparent HCV infection at the stage of adsorption are considered. The second feature is rapid error-prone replication that leads to accumulation within one host of multiple virus variants (quasispecies). Viral heterogeneity could be multiplied by recombination of HCV genomic/subgenomic RNA molecules. Quasispecies nature gives virus an advantage in adaptation to varying host environment including availability of permissive cells, the presence of innate and adaptive immune response, and antiviral treatment. Analysis of HCV polymorphisms and their evolution rates may pinpoint the molecular (sequence) correlates of HCV clearance. The third feature is the capacity to modify or adapt host milieu. HCV core, envelope E2 and nonstructural NS2, NS3, NS5A proteins seem to hold a grip over the host cellular functions by down-regulating processes unfavorable and up-regulating processes favorable for virus replication and persistence. The relevance of the latter interactions to HCV infection outcome remains to be demonstrated. This review discusses recent developments in this area of HCV research.

Host background factors contributing to hepatitis C virus clearance.

This review is an attempt to characterize the host in the earliest events of hepatitis C virus (HCV) infection before the on-set of adaptive immune response. Host meets the replicating HCV with innate immune response in the form of proinflammatory cytokine production, activation of natural killer (NK), NKT and dendritic cells. The potency of innate response is shaped by a wide panel of genetically predetermined constants and acquired variables. Higher rates of HCV clearance associate with white ethnicity and certain HLA haplotypes. Lower clearance rates correlate with genetic immune deficiencies/disorders. Recent findings link infection outcome with variation in the genes for the low-density lipoprotein and complement type 1 receptors. Important though insufficiently characterized is the role of polymorphisms in the genes responsible for induction of antiviral immunity. The outcome of HCV entry and of subsequent acute infection (if that occurs) is pre-determined by the immune competence of the host at the moment of infection. Higher rate of HCV clearance is observed for pediatric patients and young adults. Bad prognostic markers would be post-transplantation immune suppression, transfusion-related immune modulation, alcohol-induced immune imbalance and intoxication. Among host variables is the immune modulation induced by parasitic and viral co-infections. Some of the variables are transient and hard to define in retrospective. These host characteristics set up the potency, kinetics, and profile (Th1/Th2) of subsequent adaptive immune response. Better understanding of the host correlates of viral clearance would be a step towards prophylaxis of infection and an efficient anti-HCV vaccine.