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

The second session of the symposium included two sections, one on the mechanisms of viral oncogenicity, and the other, on viral infections, cancer and chronic inflammation.

The 1st section on Mechanisms of viral oncogenicity included the plenary lecture of Prof Maria Lina Tornesello, Istituto Tumori – IRCCS “Fondazione Pascale”, Napoli, Italy) on Reversible and Irreversible Activation of Telomerases in HPV-Related Cancers presented at symposium opening. The lecture highlighted the higher telomerase activity in most stages of HPV-related cancers, which is however functionally associated to the high levels of the viral E6 oncogene in the early stages of cancer progression, and later to the acquisition of mutations in the TERT promoter region, as preliminarily reported in HPV-related cancers [4] and in Hepathocellular Carcinoma (HCC) [5, 6]. These results indicate that promoter mutations have a much stronger effect on TERT activation in cervical neoplasia than the increased TERT expression associated with the HPV E6 oncoprotein. Moreover, this suggests that (i) Telomerase expression is reversibly regulated by viral E6 protein in the early stages of tumorigenesis, and (ii) Telomerase is irreversibly and highly activated by genetic alterations and promoter mutations in TERT gene in progressing cervical neoplasia. The identification of such two stages is critical for the molecular characterization of HPV-related cancers, along with their different prognosis and therapeutic responsivity. In particular, anti-HPV therapeutic vaccines could be highly effective within the first stage, while new actionable targets should be identified for SCC of the lower genital tract and in general for TERT mutated tumors.

Modulation of promoter activity underlying oncogenesis was also tackled in the presentation Activity of Long Control Region of HPV as Determinant of Oncogenicity by Prof Felicity Burt (Department of Medical Microbiology and Virology, University of the Free State, Bloemfontein, South Africa). Her data demonstrated that reporter gene activity downstream of an HPV6-derived LCR region with a 170bp duplication was significantly higher than the activity obtained with constructs made with LCR control containing no duplication. Similarly, enhanced transcriptional activity was observed for a reporter gene system constructed using HPV 31 derived LCR with nucleotide variations in the p97 promotor region. Enhanced transcriptional activity was observed with the mutant that possessed a single nucleotide change within the YY1 transcription binding site. Such data confirmed the knowledge that sequence variation within the LCR may have a functional effect on the activity of viral promotor, and that mutations in the non-coding region would represent potential biomarkers of an aggressive disease.

An oral presentation by Dudorova Alesja and Avdoshina Daria (Paul Stradins University Hopsital, and Riga Stradins University, Riga, Latvia; Chumakov Federal Scientific Center for Research and Developement of Immune- and Biological Products of Russian Academy of Science, Moscow, Russia) on Comparative Characteristics of TC-1 and Novel 4TL-Based Cell Lines Expressing HPV 16 Oncoproteins E6 and E7 in Ability to Reproduce HPV-Associated Carcinogenesis in a Mouse Model reported construction of adenocarcinoma 4T1 based cells expressing oncoproteins E6 and E7 of HPV 16, and comparison of the resulting subclones with a known model of HPV-associated cancer exploiting TC-1 cell line (https://www.atcc.org/products/crl-2493). TC-1 carry over 500, and 4T1luc2-based subclones only one copy of the E6/E7 genes. Nine 4T1 subclones were obtained that expressed different levels of E6/E7 mRNA. Interestingly, as in human cancer, expression of E6/E7 mRNA in 4T1luc2 induced expression of TERT. E6/E7-expressing 4T1luc2 demonstrated signs of G0/G1 arrest with diminished population of cells in S-phase. For TC-1, on the contrary, cells accumulated in the S-phase, the process correlated with expression of E7 mRNA. Both TC-1 and E6/E7-expressing 4T1luc2 subclones exhibited γ-H2AX foci indicating dsDNA damage, normally attributed to the production of ROS. Implanted into mice, both TC-1 and E6/E7-expressing 4T1luc2 cells formed solid tumors of similar size and growth rate (p>0.1). Ex vivo BLI demonstrated preferable infiltration of tumor cells into lungs in 4T1- and into spleen in TC-1 models, and similar infiltration into the liver, forming similar numbers of liver metastasis. Tumor size, organ infiltration by tumor cells, and number of metastasis were not correlated to the level of expression of E6/E7 mRNA. The latter observation corroborates the concept that expression of E6/E7 induces DNA damage that through accumulation of mutations allows HPV-driven cancers to become independent of the expression of viral oncogenes. This phenomenon was described in the plenary lecture presented on the Symposium by Prof Maria Lina Tornesello, and also demonstrated in the independent studies [7]. Overall, the parameters of tumor growth and metastatic activity of E6/E7-expressing 4Tluc2 cells were similar to that of TC-1. New subclones could be useful for testing HPV vaccines, as they allow to perform experiments in BALB/c mice syngenic to 4T1 cells. This model also allows to circumvent the drawback of high load of E6/E7 in TC-1 cells that may stimulate immune rejection of these cells in the E6/E7-immunized mice [8].

The oral presentation by Karen Kyuregyan (Russian Medical Academy of Continous Professional Education, and Peoples Friendship University, Moscow, Russia) on Silent HDV Epidemics Culminates in High Levels of Liver Cirrhosis and Hepatocellular Carcinoma in the Population Despite 20 Years of HBV Vaccination reported on the high frequency of HDV infection in Tuva. Bayesian analysis has shown that HBV had a long history of circulation in Tuva with the time to most common ancestor (MRCA) for predominant genotype HBV-D dating back over 1000 years. HDV circulation in Tuva started much later and was the consequence of two successive introductions of HDV genotype 1 (HDV-1). First wave was associated with HDV-1 sequences from the Central Asia and dates back to 1811 (95% HPD: 1741 – 1834). The second wave was associated with strains from Russia, its TMCA dates back to 1960s (95% HPD: 1953 – 1979). SkyGrid reconstruction of population dynamics showed an increase in the intensity of HDV spread since the 1990s peaking in 2010s. The reproduction number (Re) for HDV in Tuva calculated based on the population dynamics predicted using Birth-Death Skyline analysis increased rapidly after 2010s, reaching the plateau of about 3 cases of infection from one source in recent years. This group also observed the rise in predicted Re values for HBV in Tuva, from less than 1 before 2000s to 5 after year 2000. At the same time, a serosurvey of healthy volunteers has shown the average detection rate of HBsAg with anti-HDV to be 1.0%, which was significantly lower compared to data from a similar serosurvey done in 2008 (2.3%, p = 0.0218). Importantly, a serosurvey done in 2019 detected no anti-HDV positive samples among participants under 30 years. HBsAg/anti-HDV positivity rate peaked at 7.4% in the age group of 50-59 years, prevalence of HBV/HDV in this population group has increased since 2008 five-fold. Authors associate the increase in the intensity of the spread of HDV in Tuva with HBV circulation in non-vaccinated adults, with a large number of people living with HBV who are susceptible to HDV superinfection. HDV epidemics revealed by Kyuregyan K et al. explain the high rates of liver cirrhosis and hepatocellular carcinoma observed in Tuva. These data demonstrate that a massive HBV vaccination program for newborns does not limit the spread of HBV and HDV infections in populations dominated by non-vaccinated middle-age and elderly people and urges for implementation of massive HBV vaccination, HDV screening and chemo- and immunotherapy of HBV-infected aimed at HBV eradication as the healthcare policy for endemic regions.

Toyé Rayana (Cancer Research Center of Lyon, INSERM, CNRS, Lyon, France) in her short presentation on MicroRNAs Profiling in Senegalese HBV-Infected Patients reported profiling, using NanoString’s nCounter® technology, of approximately 800 circulating miRNAs in a retrospective cohort of 34 Senegalese patients, 17 with chronic hepatitis B (CHB) and 17 with HBV-related hepatocellular carcinomas (HCCs). MiRNA are small, non-coding single-stranded RNAs that can modulate target gene expression at the post-transcriptional level. Regulation occurs mainly by binding to complementary sequences in the 3′-untranslated region (UTR) of target mRNAs and then integrating into RNA-induced silencing complexes to suppress translation or to degrade miRNA-bound mRNA transcripts [9]. A total of 404 miRNAs were detected, 58 miRNAs were quantified in at least 75% of patients. Principal component analysis (PCA) detected no differences in representation of circulating miRNAs with age, viral load, HBe status or clinical parameters such as ALT, AST. At the same time, PCA revealed differential expression of nine miRNAs (let-7g-5p, miR-122-5p, miR-181a-3p, miR-210-3p, miR-2682-5p, miR-300, miR-451a, miR-514a-3p and miR-519c-3p) in the HBV-related HCCs as compared to CHB (p < 0.05). Six of them (let-7g-5p, miR-122-5p, miR-210-3p, miR-300, miR-451a and miR-519c-3p) shared a network of regulatory pathways and target genes (e.g MYC, BCL2L1, HIF1A, and BMI1) involved in the control of cell proliferation and immune system. In particular, miR-122-5p, reported to participate in the regulation of various cancers, was strongly downregulated in HCC confirming its onco-suppressor role in HCC [10]. Finding pattern(s) of modified expression of miRNA characteristic to HBV-related HCC has important clinical implications. Such miRNAs, in particular, miR-122-5p, could be used as candidate biomarkers in a composite clinical diagnosis/prognostic score. The authors plan to evaluate the potential of differentially expressed circulating miRNAs they detected in HBV-related HCCs to predict development of HCC in a larger, longitudinal followed cohort of CHB patients.

The 2nd section on Viral infections, cancer and chronic inflammation started with the Plenary Lecture of Nicolas Noel (MCU-PH, Service de Médecine Interne et Immunologie Clinique, GHU Paris Saclay, AP-HP BICÊTRE/CEA 1184, Paris, France) on Chronic Inflammation in HIV-1 Infection, are Elite Controllers Different? Antiretroviral therapy (ART) of human immunodeficiency virus (HIV) infection is usually mandatory to maintain an undetectable viremia and to preserve immune functions. There are, however, HIV-1 elite controllers who harbor replication competent virus, but are able to control its replication without antiretroviral therapy. A hallmark of HIV-induced pathology is immune activation occurring already at the early stages of infection. In his plenary lecture, Dr Noel described multiple sources of immune activation such as: viral replication and release of virions, antigen presentation, microbial translocation of mucosal origin, activation of the interferon pathway, viral co-infections and others. Immune activation is also linked to the control of the HIV reservoir and viral latency, as well as to the risk of clinical events such as cardiovascular or neurocognitive diseases. ART reduces chronic immune activation, but does not necessarily normalize the inflammatory parameters. HIV-1 controllers allow to study the causes and consequences of inflammation. Plenary lecture provided the evidence of persistent immune activation in HIV-controller patients in comparison with controlled patients under ART, and discussed the risk of viral evolution and the research perspectives in this field.

Plenary lecture on chronic inflammation in HIV infection was followed by the oral presentation by Benoit Favier (DRF-IBFJ-IDMIT, and CEA-University Paris Saclay-Inserm, Fontenay-aux-Roses, France) on the Dynamics of LILRB2 Immune-Checkpoint in HIV and SIV Infections. Dendritic cells (DCs) play an important role in initiating and regulating adaptive immune responses leading to the control of viral infections. However, HIV infection dysregulates DC functions which may in-part account for viral persistence. Studies by Favier et al. indicate that this dysregulation is induced by the interaction of the inhibitory receptor LILRB2 (leukocyte immunoglobulin-like receptor subfamily B member 2) which acts to suppress the immune system with its MHC-I ligands. Inhibitory receptors, and paired activating receptor siblings, are critical regulators of innate immunity and inflammation [11]. In HIV and SIV infections, interaction of LILRB2 with MHC-I is associated with the rate of progression of the disease. Dr Favier presented the dynamics of the LILRB2/MHC-I inhibitory axis in DCs during different phases of the infection. Primary phase of HIV infection was characterized by a strong increase of LILRB2 and MHC-I expression on the surface of DCs. The dynamics of LILRB2 and MHC-I in the early phase of infection in blood and tissues was further characterized in a macaque model of SIV infection. The study revealed an up-regulation of LILRB2 and MHC-I not only on DCs, but also on macrophages, starting from the first week after the onset of SIV infection. These results identify LILRB2 as a potential target to improve DC functions and thus anti-viral adaptive immune responses in early stage of retroviral infection. The lecture presented the latest tools to assess the in vivo role of LILRB2. In conclusion, Favier et al. proposed LILRB2 as a target of immunotherapy in HIV infection. Interestingly, in endometrial cancer, knockdown of LILRB2 results in a dramatic decrease in the proliferation, colony formation and migration of cancer cells, and in a notable reduction of tumor growth in in vivo xenograft experiments [12] which suggests analogies between directions to cancer and HIV infection cure.

Presentation of Sara Svensson Akusjärvi (Karolinska Institutet, Stockholm, Sweden) on The CD4+CCr6+ T Cell Compartment is Unique in EC Compared to Long-Term ART-Treated Individuals focused on the unique profile of CCR2 and CCR6 in lymphocyte populations of people living with HIV who are elite controllers of HIV infection (PLWHec) as compared to PLWH on ART (PLWHart) and healthy controls (HC). Flow cytometry analysis identified a significant decrease of CCR6 and CCR2 on CD4+ and CD8+ T cells in PLWHEC compared to PLWHART as well as to HC for both CD4+ and CD8+ T cells. In PLWHart, CCR2 on CD8+ T cells was reduced also if compared to HC. From the sorted cell populations, CD4+T cells of PLWHec expressed an enrichment of interferon-α response, mitochondrial oxidative phosphorylation (OXPHOS) and decreased glycolysis as compared to PLWHart (all p values < 0.05). Specifically, CD4+CCR6+ cells demonstrated an enrichment of apoptosis and p53 signaling in PLWHec compared to PLWHart (adj p < 0.05). The phenomenon was not seen in CD4+CCR6- cells. On the overall, flow cytometry analysis revealed a unique expression profile of CCR2 and CCR6 in PLWHec, while the profile in PLWHart was similar to HC. Interestingly, normal cells are characterized by enriched OXPHOS and decreased glycolysis, while in various cancer cells glycolysis is enhanced and OXPHOS capacity is reduced [13]. Furthermore, the authors observed an enrichment of apoptosis and p53 signaling in CD4+CCR6+ cells from PLWHec showing susceptibility to cell death by apoptosis, whereas primary mode of cell death in HIV-1 infected T cells does not involve apoptosis [14]. CD4+CCR6+ T cells have been proposed as highly permissive to HIV-1 and major contributors to the viral reservoir [15]. Svensson S. et al. hypothesized that the reduced frequency of CD4+CCR6+ cells, their susceptibility to cell death by apoptosis and metabolic profile with increased OXPHOS and decreased glycolysis could potentially aid in achieving natural control of HIV-1.

Sona Chowdhury (University of California, San Francisco, USA) focused her presentation on Programmed Cell Death Protein 1 (Pd-1) and Programmed Cell Death Ligand 1 (Pd-L1) Expression Profile by Immune and Epithelial Cells in Primary and Metastatic Cervical Cancer Tissue. PD-L1 was expressed in epithelial cells of 70% primary squamous cell carcinomas (SCC, n=12), while 30% were negative. PD-L1(+)-SCC samples revealed heterogeneous PD-L1 expression patterns in-between tumors and within each tumor, mostly seen as membranous focal staining, both in the periphery and in the center of invading nests, and also at the tumor-stroma interface. Similar heterogeneous staining was characteristic to proximal lymph nodes (LNs, n=8) affected by metastasis. Stroma of nearly 50% of PD-L1(+)-SCC was rich in PD-L1 positive immune cells (ICs). PD-L1 expression in ICs in the stroma was observed also in tumors negative for PD-L1. Besides, PD-L1(+)-ICs were observed also in the stroma of 50% of metastatic LN samples. Stromal ICs also expressed PD-1. No PD-1 expression was observed in the epithelial cells in either SCC, or HSIL, or LN tissues. Authors semi-quantified the number of PD-1(+) ICs. Over 70% of primary SCC had moderate to high number of PD-1(+) ICs in the stroma. PD-1(+) ICs were observed also in HSIL samples. In the stroma of LNs, levels of PD-1 expression in IC were lower. Levels of PD-1 expression by IC in SCC and stroma were not correlated to the expression of PD-L1 by the epithelial cells. The manual IHC scoring was validated by a bioimage analysis software. In conclusion, varied PD-L1 expression patterns were observed both in primary and metastatic SCC. PD-L1 expression was seen not only in epithelial cells but also in ICs. In contrast, PD-1 expression was restricted to ICs. Level of PD-L1 expression varied in-between SCC samples and within each of the samples. This heterogeneous pattern of PD-L1/PD-1 expression may contribute to the immune-mediated pathogenesis of CC and warrants further investigation.

Ivan Trotsenko (Peoples Friendship University of Russia, Moscow, Russia) focused his presentation on Violaton of Gene Regulation in Colon Cancer (CC) and Adjacent Colon Mucosa Revealed by Comparative Gene Expression and Gene Correlation Analysis. Tissue samples from tumor (T) adjacent mucosa (AM) and normal control (NC) were analyzed for gene expression levels and gene correlation coefficients. Principal component analysis (PCA) of expression profile revealed significant activation of proliferative, matrix metalloproteinases, antiapoptotic and angiogenic pathways in tumor compared to only slight difference observed between NC and AM. On the PCA plot T samples were separated from NC and AM samples and their gene expression evaluated. Correlation of gene expression in NC differed significantly from that in T and AM samples, whereas the latter two groups did not differ. Cluster analysis revealed that correlation scores of genes in NC were significantly higher than correlation scores in T and AM, i.e. gene expression patterns in normal control tissues, but not in T or AM, were strongly coordinated. To conclude, analysis of gene expression profile in CC samples demonstrated high level of proliferative activity, angiogenesis and apoptosis inhibition. Although gene expression patterns and the level of expression of cancer hallmarks in mucosal tissues surrounding the tumors and normal colon epithelium did not differ, interaction of these pathways in adjacent mucosa was as disrupted as in tumors. Thus, gene correlations analysis revealed violations of gene interaction not only in the tumor, but also in the surrounding morphologically unchanged epithelium.

The first day of the Symposium was concluded with the Plenary Lecture of Margaret Liu (Protherimmune, and University of California, San Francisco, USA) on Nucleic Acid Vaccines and their Potential for Improving Non-Specific Immune Responses and Immune Fitness for Chronic Infections and Cancer. Immunotherapies for chronic viral infections and cancer have utilized approaches including monoclonal and bispecific antibodies, immunodulatory agents (cytokines and checkpoint inhibitors), vaccines (utilizing various delivery systems), and adoptive cell therapies. The efficacy of certain antibodies, immunomodulators, and cell therapies for certain cancers have demonstrated their potential. This has led to increased efforts to develop immunotherapeutic vaccines targeting tumor and viral antigens. But challenges remain even after selecting an appropriate antigen to target, whether a viral protein or a tumor antigen. One reason for this is that tumors and chronic infections can affect the immunological milieu resulting in tolerance or immune suppression. The lecture by Margaret Liu described the use of nucleic acid vaccines as potential vectors for therapeutic vaccines for both chronic infections and cancer with the focus on the immune mechanisms stimulated by these vaccines, including innate immunity and how these innate immune responses may contribute to the specific immune responses against an encoded antigen and what non-specific immune benefits they can give including immune fitness.

The second day of the Meeting was opened with the Plenary In-Sight Lecture of Fabien Zoulim (Hepatology Department, Hospices Civils de Lyon; Cancer Research Center of Lyon, Lyon, France) on The Path Towards the Cure of Chronic HBV Infection. Despite the implementation of universal vaccination programs, chronic HBV infection remains a major public health problem worldwide. Moreover, existing therapeutic compounds against HBV are limited and mainly include nucleos(t)ide analogues (NUCs) (i.e. entecavir, tenofovir) and pegylated interferon α (Peg-IFN-α). As beneficial as they may be, these treatments do not usually achieve eradication of the virus and HBsAg loss is still rare. Thus, these regimens require indefinite treatment to maintain viral suppression and prevent the virological relapse that usually occurs after treatment discontinuation. Moreover, it is unrealistic to expect all patients to adhere to long-term or lifelong non-curative treatment and there is a strong patient preference for therapy termination. Drug resistance is still a concern in low-income settings that use early generation NUCs and while there is no resistance with IFN treatment, the use of this agent is rare because of problems with tolerability. The cost of lifelong therapy and monitoring is also an important economic issue in highly endemic areas. Thus, the aim of new therapeutic strategies is to achieve a “functional cure” for chronic hepatitis B (CHB), defined as sustained off-treatment loss of HBsAg, undetectable HBV DNA in serum, normalization of liver enzymes and improvement in liver histology. HBsAg loss is a sign of profound suppression of HBV replication and is the only existing indicator for safe treatment discontinuation. Moreover, HBsAg loss is associated with a decreased risk of developing inflammation-driven hepatic complications such as HCC. The lecture highlighted the current and the potential anti-HBV advanced strategies to be developed in the next years. In particular, Prof Zoulim described the novel direct-acting antivirals against HBV infection articulated in a) DAA targeting the HBV replicative cycle (including entry inhibitors, capsid assembly modulators, HBsAg secretion inhibitors and Nucleos(t)ide reverse transcriptase inhibitors) and b) DAA targeting HBV gene expression (i.e. small-interfering RNAs-siRNAs and antisense oligonucleotides -ASOs). In parallel novel host-targeting agents against HBV infection have been developed to stimulate a) the innate immune response (in hepatocytes via retinoic acid-inducible gene I (RIG-I) or in neighboring cells via Toll-like receptor (TLR) and/or b) adaptive immune response (with checkpoint inhibitors or therapeutic vaccines). Finally, combinations of such treatments have been reported (DAA combinations with or without inhibition of HBV expression or with immunotherapy [16].

In order to meet the 2030 deadline for the elimination of viral hepatitis, several further challenges must be addressed. In particular, the development of better animal models for the study of HBV infection and antiviral drug discovery is needed. These models could help overcome the challenges of HBV cccDNA targeting, evaluating immune stimulation and preclinical testing of drug combinations. Innovative therapeutic strategies are on the way as the chimeric antigen receptor (CAR) T cells, HBV T-cell receptor (TCR)-designed CD8 T cells and soluble TCRs, to redirect HBV-specific T cells to infected hepatocytes and gene editing approaches to directly target cccDNA by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based approaches. In summary, HBV eradication will require a thorough understanding of HBV biology, the specificities of the liver microenvironment and their interactions with the immune system. The design of future therapeutic approaches against HBV will need to take these factors into account, as they will probably pave the way for the next generation of antiviral agents and their combinations.

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