Phenotypic CD8 T cell profiling in chronic hepatitis B to predict HBV-specific CD8 T cell susceptibility to functional restoration in vitro.

OBJECTIVE: Exhausted hepatitis B virus (HBV)-specific CD8 T cells in chronic HBV infection are broadly heterogeneous. Characterisation of their functional impairment may allow to distinguish patients with different capacity to control infection and reconstitute antiviral function. DESIGN: HBV dextramer+CD8 T cells were analysed ex vivo for coexpression of checkpoint/differentiation markers, transcription factors and cytokines in 35 patients with HLA-A2+chronic hepatitis B (CHB) and in 29 control HBsAg negative CHB patients who seroconverted after NUC treatment or spontaneously. Cytokine production was also evaluated in HBV peptide-stimulated T cell cultures, in the presence or absence of antioxidant, polyphenolic, PD-1/PD-L1 inhibitor and TLR-8 agonist compounds and the effect on HBV-specific responses was further validated on additional 24 HLA-A2 negative CHB patients. RESULTS: Severely exhausted HBV-specific CD8 T cell subsets with high expression of inhibitory receptors, such as PD-1, TOX and CD39, were detected only in a subgroup of chronic viraemic patients. Conversely, a large predominance of functionally more efficient HBV-specific CD8 T cell subsets with lower expression of coinhibitory molecules and better response to in vitro immune modulation, typically detected after resolution of infection, was also observed in a proportion of chronic viraemic HBV patients. Importantly, the same subset of patients who responded more efficiently to in vitro immune modulation identified by HBV-specific CD8 T cell analysis were also identified by staining total CD8 T cells with PD-1, TOX, CD127 and Bcl-2. CONCLUSIONS: The possibility to distinguish patient cohorts with different capacity to respond to immune modulatory compounds in vitro by a simple analysis of the phenotypic CD8 T cell exhaustion profile deserves evaluation of its clinical applicability.

Deregulated intracellular pathways define novel molecular targets for HBV-specific CD8 T cell reconstitution in chronic hepatitis B.

BACKGROUND & AIMS: In chronic HBV infection, elevated reactive oxygen species levels derived from dysfunctional mitochondria can cause increased protein oxidation and DNA damage in exhausted virus-specific CD8 T cells. The aim of this study was to understand how these defects are mechanistically interconnected to further elucidate T cell exhaustion pathogenesis and, doing so, to devise novel T cell-based therapies. METHODS: DNA damage and repair mechanisms, including parylation, CD38 expression, and telomere length were studied in HBV-specific CD8 T cells from chronic HBV patients. Correction of intracellular signalling alterations and improvement of antiviral T cell functions by the NAD precursor nicotinamide mononucleotide and by CD38 inhibition was assessed. RESULTS: Elevated DNA damage was associated with defective DNA repair processes, including NAD-dependent parylation, in HBV-specific CD8 cells of chronic HBV patients. NAD depletion was indicated by the overexpression of CD38, the major NAD consumer, and by the significant improvement of DNA repair mechanisms, and mitochondrial and proteostasis functions by NAD supplementation, which could also improve the HBV-specific antiviral CD8 T cell function. CONCLUSIONS: Our study delineates a model of CD8 T cell exhaustion whereby multiple interconnected intracellular defects, including telomere shortening, are causally related to NAD depletion suggesting similarities between T cell exhaustion and cell senescence. Correction of these deregulated intracellular functions by NAD supplementation can also restore antiviral CD8 T cell activity and thus represents a promising potential therapeutic strategy for chronic HBV infection. IMPACT AND IMPLICATIONS: Correction of HBV-specific CD8 T cell dysfunction is believed to represent a rational strategy to cure chronic HBV infection, which however requires a deep understanding of HBV immune pathogenesis to identify the most important targets for functional T cell reconstitution strategies. This study identifies a central role played by NAD depletion in the intracellular vicious circle that maintains CD8 T cell exhaustion, showing that its replenishment can correct impaired intracellular mechanisms and reconstitute efficient antiviral CD8 T cell function, with implications for the design of novel immune anti-HBV therapies. As these intracellular defects are likely shared with other chronic virus infections where CD8 exhaustion can affect virus clearance, these results can likely also be of pathogenetic relevance for other infection models.

A prospective study of direct-acting antiviral effectiveness and relapse risk in HCV cryoglobulinemic vasculitis by the Italian PITER cohort.

BACKGROUND AND AIMS: Mixed cryoglobulinemia is the most common HCV extrahepatic manifestation. We aimed to prospectively evaluate the cryoglobulinemic vasculitis (CV) clinical profile after a sustained virologic response (SVR) over a medium-term to long-term period. APPROACH AND RESULTS: Direct-acting antiviral-treated cryoglobulinemic patients, consecutively enrolled in the multicentric Italian Platform for the Study of Viral Hepatitis Therapy cohort, were prospectively evaluated. Cumulative incidence Kaplan-Meier curves were reported for response, clinical deterioration, relapse and relapse-free survival rates. Cox regression analysis evaluated factors associated with different outcomes. A clinical response was reported in at least one follow-up point for 373 of 423 (88%) patients with CV who achieved SVR. Clinical response increased over time with a 76% improvement rate at month 12 after the end of treatment. A full complete response (FCR) was reached by 164 (38.8%) patients in at least one follow-up point. CV clinical response fluctuated, with some deterioration of the initial response in 49.6% of patients (median time of deterioration, 19 months). In patients who achieved FCR and had an available follow-up (137 patients) a relapse was observed in 13% and it was transient in 66.7% of patients. The rate of patients without any deterioration was 58% and 41% at 12 and 24 months, respectively. After achieving SVR, a clinical nonresponse was associated with older age and renal involvement; a clinical deterioration/relapse was associated with high pretreatment rheumatoid factor values, and FCR was inversely associated with age, neuropathy, and high cryocrit levels. CONCLUSION: In patients with CV, HCV eradication may not correspond to a persistent clinical improvement, and clinical response may fluctuate. This implies an attentive approach to post-SVR evaluation through prognostic factors and tailored treatment.

Functional reconstitution of HBV-specific CD8 T cells by in vitro polyphenol treatment in chronic hepatitis B.

BACKGROUND & AIMS: In chronic HBV infection, mitochondrial functions and proteostasis are dysregulated in exhausted HBV-specific CD8 T cells. To better characterise the potential involvement of deregulated protein degradation mechanisms in T cell exhaustion, we analysed lysosome-mediated autophagy in HBV-specific CD8 T cells. Bioactive compounds able to simultaneously target both mitochondrial functions and proteostasis were tested to identify optimal combination strategies to reconstitute efficient antiviral CD8 T cell responses in patients with chronic HBV infection. METHODS: Lysosome-mediated degradation pathways were analysed by flow cytometry in virus-specific CD8 T cells from patients with chronic HBV infection. Mitochondrial function, intracellular proteostasis, and cytokine production were evaluated in HBV-peptide-stimulated T cell cultures, in the presence or absence of the polyphenols resveratrol (RSV) and oleuropein (OLE) and their metabolites, either alone or in combination with other bioactive compounds. RESULTS: HBV-specific CD8 T cells from patients with CHB showed impaired autophagic flux. RSV and OLE elicited a significant improvement in mitochondrial, proteostasis and antiviral functions in CD8 T cells. Cytokine production was also enhanced by synthetic metabolites, which correspond to those generated by RSV and OLE metabolism in vivo, suggesting that these polyphenols may also display an effect after transformation in vivo. Moreover, polyphenolic compounds improved the T cell revitalising effect of mitochondria-targeted antioxidants and of programmed cell death protein 1/programmed cell death ligand 1 blockade. CONCLUSIONS: Simultaneously targeting multiple altered intracellular pathways with the combination of mitochondria-targeted antioxidants and natural polyphenols may represent a promising immune reconstitution strategy for the treatment of chronic HBV infection. LAY SUMMARY: In chronic hepatitis B, antiviral T lymphocytes are deeply impaired, with many altered intracellular functions. In vitro exposure to polyphenols, such as resveratrol and oleuropein, can correct some of the deregulated intracellular pathways and improve antiviral T cell function. This effect can be further strengthened by the association of polyphenols with antioxidant compounds in a significant proportion of patients. Thus, the combination of antioxidants and natural polyphenols represents a promising strategy for chronic hepatitis B therapy.

Liver function following hepatitis C virus eradication by direct acting antivirals in patients with liver cirrhosis: data from the PITER cohort.

BACKGROUND: The development of direct-acting antivirals (DAA) for HCV has revolutionized the treatment of HCV, including its treatment in patients with HIV coinfection. The aim of this study was to compare the changes in liver function between coinfected and monoinfected patients with cirrhosis who achieved HCV eradication by DAA. METHODS: Patients with pre-treatment diagnosis of HCV liver cirrhosis, consecutively enrolled in the multicenter PITER cohort, who achieved a sustained virological response 12 weeks after treatment cessation (SVR12) were analysed. Changes in Child-Pugh (C-P) class and the occurrence of a decompensating event was prospectively evaluated after the end of DAA treatment. Cox regression analysis was used to evaluate factors independently associated with changes in liver function following viral eradication. RESULTS: We evaluated 1350 patients, of whom 1242 HCV monoinfected (median follow-up 24.7, range 6.8-47.5 months after viral eradication) and 108 (8%) HCV/HIV coinfected (median follow-up 27.1, range 6.0-44.6). After adjusting for age, sex, HCV-genotype, HBsAg positivity and alcohol use, HIV was independently associated with a more advanced liver disease before treatment (C-P class B/C vs A) (OR: 3.73, 95% CI:2.00-6.98). Following HCV eradication, C-P class improved in 17/20 (85%) coinfected patients (from B to A and from C to B) and in 53/82 (64.6%) monoinfected patients (from B to A) (p = 0.08). C-P class worsened in 3/56 coinfected (5.3%) (from A to B) and in 84/1024 (8.2%) monoinfected patients (p = 0.45) (from A to B or C and from B to C). Baseline factors independently associated with C-P class worsening were male sex (HR = 2.00; 95% CI = 1.18-3.36), platelet count < 100,000/µl (HR = 1.75; 95% CI 1.08-2.85) and increased INR (HR = 2.41; 95% CI 1.51-3.84). Following viral eradication, in 7 of 15 coinfected (46.6%) and in 61 of 133 (45.8%) monoinfected patients with previous history of decompensation, a new decompensating event occurred. A first decompensating event was recorded in 4 of 93 (4.3%) coinfected and in 53 of 1109 (4.8%) monoinfected patients (p = 0.83). CONCLUSIONS: Improvement of liver function was observed following HCV eradication in the majority of patients with cirrhosis; however viral eradication did not always mean cure of liver disease in both monoinfected and coinfected patients with advanced liver disease.

Combined GS-4774 and Tenofovir Therapy Can Improve HBV-Specific T-Cell Responses in Patients With Chronic Hepatitis.

BACKGROUND & AIMS: One strategy to treat chronic hepatitis B virus (HBV) infection could be to increase the functions of virus-specific T cells. We performed a multicenter phase 2 study to evaluate the safety and efficacy of GS-4774, a yeast-based therapeutic vaccine engineered to express HBV antigens, given with tenofovir disoproxil fumarate (TDF) to untreated patients with chronic HBV infection. METHODS: We performed an open-label study at 34 sites in Canada, Italy, New Zealand, Romania, South Korea, and United States from July 2014 to August 2016. Adults who were positive for HB surface antigen (HBsAg) > 6 months and levels of HBV DNA ≥2000 IU/mL who had not received antiviral treatment for HBV within 3 months of screening were randomly assigned (1:2:2:2) to groups given oral TDF 300 mg daily alone (n = 27; controls) or with 2, 10, or 40 yeast units GS-4774 (n = 168), administered subcutaneously every 4 weeks until week 20 for a total of 6 doses. Blood samples were collected and analyzed and patients received regular physical examinations. Efficacy was measured by decrease in HBsAg from baseline to week 24. Specific responses to HBV (production of interferon gamma [IFNG], tumor necrosis factor [TNF], interleukin 2 [IL2], and degranulation) were measured in T cells derived from 12 HBeAg-negative patients with genotype D infections, after overnight or 10 days of stimulation of peripheral blood mononuclear cells with peptides from the entire HBV proteome. T-regulatory cells were analyzed for frequency and phenotype. Data from studies of immune cells were compared with data on reductions in HBsAg, HBV DNA, and alanine aminotransferase in blood samples from patients. RESULTS: GS-4774 was safe and well tolerated but did not produce significant decreases in levels of HBsAg. Production of IFNG, TNF, and IL2 increased significantly at weeks 24 and 48, compared with baseline, in HBV-specific CD8+ T cells from patients given GS-4774 but not from controls. GS-4774 had greater effects on CD8+ than CD4+ T cells, which were not affected at all or very weakly by TDF with or without GS-4774. GS-4774 did not affect responses of T cells to other viruses tested. HBV core peptides induced the greatest production of IFNG by T cells following overnight stimulation, whereas HBV envelope antigens did not induce a response. Following 10 days of stimulation, production of IFNG and TNF increased with time of exposure to GS-4774; the greatest levels of responses were to HBV envelope antigens followed by core and polymerase peptides. We observed a correlation in patients given GS-4774 between increased T-cell functions and reductions in numbers of T-regulatory cells. CONCLUSIONS: In a phase 2 study of patients with chronic HBV infection given TDF with or without GS-4774, we found that vaccination can increase production of IFNG, TNF, and IL2 by CD8+ T cells exposed to antigenic peptides, with little effect on CD4+ T cells. Although GS-4774 did not reduce levels of HBsAg in patients, its strong immune stimulatory effect on CD8+ T cells might be used in combination with other antiviral agents to boost the antivirus immune response. Clinicaltrials.gov no: NCT02174276.

TLR7 Agonist Increases Responses of Hepatitis B Virus-Specific T Cells and Natural Killer Cells in Patients With Chronic Hepatitis B Treated With Nucleos(T)Ide Analogues.

BACKGROUND & AIMS: The oral Toll-like receptor (TLR) 7 agonist GS-9620 has antiviral effects in woodchuck and chimpanzee models of chronic hepatitis B virus (HBV) infection. We investigated, in a clinical trial, the capacity of this agent to reconstitute protective immunity in patients with chronic HBV infection. METHODS: We performed a prospective study of 28 patients with suppression of HBV infection by nucleos(t)ide analogue therapy and who tested negative for hepatitis B e antigen at 4 medical centers in Italy. Patients were randomly assigned (1:3:3:3) to groups given placebo or different doses of GS-9620 (1, 2, and 4 mg, weekly for 12 weeks). We added data from 8 patients receiving nucleos(t)ide analogue therapy to the placebo group (controls); 13 treatment-naïve patients with chronic HBV infection and 15 subjects who spontaneously recovered from an acute HBV infection served as additional controls. Peripheral blood mononuclear cells were collected at baseline, during administration of GS-9620 or placebo, and 12 weeks afterward. Phenotype and function of natural killer (NK) and HBV-specific T cells were analyzed by flow cytometry. T cells were expanded by incubation with peptides from the entire HBV proteome and studied after overnight or 10 days culture. NK-cell inhibition of T-cell responses was measured by assessing cytokine production by T cells stimulated with peptides in the presence or absence of NK cells. RESULTS: T cells collected at baseline before addition of GS-9620, when patients were receiving only nucleos(t)ide therapy, had greater responses to HBV than T cells from treatment-naïve patients, based on cytokine production in response to HBV peptides. However, during or after administration of GS-9620, T cells produced higher levels of cytokines compared to baseline. NK-cell activation and function increased after patients were given GS-9620, but the ability of NK cells to suppress T-cell responses was lower during GS-9620 therapy than before. Changes in T-cell or NK-cell function did not correlate with levels of hepatitis B surface antigen. Serum levels of hepatitis B surface antigen did not decrease significantly compared to baseline in patients given any dose of GS-9620. CONCLUSIONS: Twelve weeks administration of GS-9620 had no significant effect on serum hepatitis B surface antigen levels, but did appear to increase T-cell and NK-cell responses and reduce the ability of NK to suppress T cells. GS-9620 might therefore be included in therapies to increase the immune response to HBV.

HBV Immune-Therapy: From Molecular Mechanisms to Clinical Applications.

Chronic hepatitis B virus (HBV) infection represents a worldwide public health concern with approximately 250 million people chronically infected and at risk of developing liver cirrhosis and hepatocellular carcinoma. Nucleos(t)ide analogues (NUC) are the most widely used therapies for HBV infection, but they often require long-lasting administration to avoid the risk of HBV reactivation at withdrawal. Therefore, there is an urgent need to develop novel treatments to shorten the duration of NUC therapy by accelerating virus control, and to complement the effect of available anti-viral therapies. In chronic HBV infection, virus-specific T cells are functionally defective, and this exhaustion state is a key determinant of virus persistence. Reconstitution of an efficient anti-viral T cell response may thus represent a rational strategy to treat chronic HBV patients. In this perspective, the enhancement of adaptive immune responses by a checkpoint inhibitor blockade, specific T cell vaccines, lymphocyte metabolism targeting, and autologous T cell engineering, including chimeric antigen receptor (CAR) and TCR-redirected T cells, constitutes a promising immune modulatory approach for a therapeutic restoration of protective immunity. The advances of the emerging immune-based therapies in the setting of the HBV research field will be outlined.

The Good and the Bad of Natural Killer Cells in Virus Control: Perspective for Anti-HBV Therapy.

Immune modulatory therapies are widely believed to represent potential therapeutic strategies for chronic hepatitis B infection (CHB). Among the cellular targets for immune interventions, Natural Killer (NK) cells represent possible candidates because they have a key role in anti-viral control by producing cytokines and by exerting cytotoxic functions against virus-infected cells. However, in patients with chronic hepatitis B, NK cells have been described to be more pathogenic than protective with preserved cytolytic activity but with a poor capacity to produce anti-viral cytokines. In addition, NK cells can exert a regulatory activity and possibly suppress adaptive immune responses in the setting of persistent viral infections. Consequently, a potential drawback of NK-cell targeted modulatory interventions is that they can potentiate the suppressive NK cell effect on virus-specific T cells, which further causes impairment of exhausted anti-viral T cell functions. Thus, clinically useful NK-cell modulatory strategies should be not only suited to improve positive anti-viral NK cell functions but also to abrogate T cell suppression by NK cell-mediated T cell killing. This review outlines the main NK cell features with a particular focus on CHB infection. It describes different mechanisms involved in NK-T cell interplay as well as how NK cells can have positive anti-viral effector functions and negative suppressive effects on T cells activity. This review discusses how modulation of their balance can have potential therapeutic implications.

A holistic evaluation of patients with chronic Hepatitis D virus (HDV) infection enrolled in the Italian PITER-B and delta cohort.

BACKGROUND AND AIMS: We aimed to characterize the epidemiologic and comorbidities profiles of patients with chronic Hepatitis D (CHD) followed in clinical practice in Italy and explored their interferon (IFN) eligibility. METHODS: This was a cross-sectional study of the PITER cohort consisting of consecutive HBsAg-positive patients from 59 centers over the period 2019-2023. Multivariable analysis was performed by logistic regression model. RESULTS: Of 5492 HBsAg-positive enrolled patients, 4152 (75.6%) were screened for HDV, 422 (10.2%) were anti-HDV positive. Compared with HBsAg mono-infected, anti-HDV positive patients were more often younger, non-Italians, with a history of drug use, had elevated alanine transaminase (ALT), cirrhosis, or hepatocellular carcinoma (HCC). Compared with Italians, anti-HDV positive non-Italians were younger (42.2% age ≤ 40 years vs. 2.1%; P < 0.001), more often females (males 43.0% vs. 68.6%; P < 0.001) with less frequent cirrhosis and HCC. HDV-RNA was detected in 63.2% of anti-HDV-positive patients, who were more likely to have elevated ALT, cirrhosis, and HCC. Extrahepatic comorbidities were present in 47.4% of anti-HDV positive patients and could affect the eligibility of IFN-containing therapies in at least 53.0% of patients in care. CONCLUSIONS: CHD affects young, foreign-born patients and older Italians, of whom two-thirds had cirrhosis or HCC. Comorbidities were frequent in both Italians and non-Italians and impacted eligibility for IFN.

Increased levels of arginase in patients with acute hepatitis B suppress antiviral T cells.

BACKGROUND & AIMS: During viral infection, the activities of virus-specific CD8(+) T cells are carefully regulated to prevent severe damage of the infected organs. We investigated the mechanisms that control the functions of activated T cells. METHODS: We measured the size of the population of activated and proliferating CD8(+) T cells and the functional pattern of CD8(+) T cells specific for the entire hepatitis B virus proteome and for selected heterologous virus (Epstein-Barr virus, human cytomegalovirus, and influenza virus) using blood samples from 18 patients with acute hepatitis B. We analyzed the effects of different modulatory mechanisms, such as inhibitory molecules, suppressive cytokines (interleukin-10), and arginase, on the activities of CD8(+) T cells. RESULTS: In patients with acute hepatitis B, the expansion of activated and proliferating (HLA-DR/CD38(+), Ki-67(+)/Bcl-2(low)) CD8(+) T cells did not quantitatively match their specific functions ex vivo; virus-specific CD8(+) T cells had functional impairments that were temporally restricted to the acute phase of viral hepatitis. These impairments in function were not limited to HBV-specific CD8(+) T cells but were also observed in CD8(+) T cells with specificities for other viruses. We investigated possible causes of antigen-independent CD8(+) T cell inhibition and found that the increased levels of arginase observed in patients with acute hepatitis could suppress the function of activated, but not resting, CD8(+) T cells. CONCLUSIONS: The increased level of arginase in patients with acute hepatitis B suppresses the functions of activated CD8(+) T cells. This mechanism might limit the amount of liver damage caused by activated CD8(+) T cells in patients with acute HBV infection.

Restored function of HBV-specific T cells after long-term effective therapy with nucleos(t)ide analogues.

BACKGROUND & AIMS: In patients with chronic hepatitis B virus (HBV) infection, persistent exposure to high concentrations of antigen can disrupt T-cell functions. It is not clear to what extent long-term suppression of HBV by nucleos(t)ide analogues can restore antiviral T-cell functions. We compared HBV-specific T-cell responses of patients treated with nucleos(t)ide analogues with those detected in other conditions of HBV control. METHODS: We analyzed intracellular levels of interferon gamma, interleukin-2, and tumor necrosis factor α in HBV-specific T cells after 10 days of stimulation with peptides covering the overall HBV genotype D sequence and ex vivo with selected CD8 epitopes and the corresponding HLA-A2 dextramers. Findings from patients treated with nucleos(t)ide analogues who had complete (HBV DNA negative/antibody to hepatitis B surface antigen positive) or partial (HBV DNA negative/hepatitis B surface antigen positive) control of their infections were compared with those of patients with spontaneous or interferon alfa-induced resolution of acute or chronic infections, inactive HBV carriers, or untreated hepatitis B e antigen-negative patients with chronic infections. RESULTS: Although HBV-specific T cells from nucleos(t)ide analogue-treated patients with complete control of infection were dysfunctional ex vivo, they had efficient responses after in vitro expansion. These responses were comparable to those of patients who spontaneously resolved acute HBV infection. Nucleos(t)ide analogue-treated patients who were HBV DNA negative but hepatitis B surface antigen positive had lower levels of T-cell responses but responses greater than those of untreated patients with chronic infection. CONCLUSIONS: In vitro reactivity can be restored to T cells from patients with suppressed HBV infection following long-term treatment with nucleos(t)ide analogues, despite prolonged exposure to large loads of antigen. Immune therapies that increase the antiviral T-cell response might increase the likelihood of complete HBV control in patients undergoing long-term nucleos(t)ide analogue treatment.

What Is the Current Status of Hepatitis B Virus Viro-Immunology?

The natural history of hepatitis B virus (HBV) infection is closely dependent on the dynamic interplay between the host immune response and viral replication. Spontaneous HBV clearance in acute self-limited infection is the result of an adequate and efficient antiviral immune response. Instead, it is widely recognized that in chronic HBV infection, immunologic dysfunction contributes to viral persistence. Long-lasting exposure to high viral antigens, upregulation of multiple co-inhibitory receptors, dysfunctional intracellular signaling pathways and metabolic alterations, and intrahepatic regulatory mechanisms have been described as features ultimately leading to a hierarchical loss of effector functions up to full T-cell exhaustion.

Natural heteroclitic-like peptides are generated by SARS-CoV-2 mutations.

Humoral immunity is sensitive to evasion by SARS-CoV-2 mutants, but CD8 T cells seem to be more resistant to mutational inactivation. By a systematic analysis of 30 spike variant peptides containing the most relevant VOC and VOI mutations that have accumulated overtime, we show that in vaccinated and convalescent subjects, mutated epitopes can have not only a neutral or inhibitory effect on CD8 T cell recognition but can also enhance or generate de novo CD8 T cell responses. The emergence of these mutated T cell function enhancing epitopes likely reflects an epiphenomenon of SARS-CoV-2 evolution driven by antibody evasion and increased virus transmissibility. In a subset of individuals with weak and narrowly focused CD8 T cell responses selection of these heteroclitic-like epitopes may bear clinical relevance by improving antiviral protection. The functional enhancing effect of these peptides is also worth of consideration for the future development of new generation, more potent COVID-19 vaccines.

Peginterferon-α does not improve early peripheral blood HBV-specific T-cell responses in HBeAg-negative chronic hepatitis.

BACKGROUND & AIMS: The effect of IFN-α therapy on HBV-specific T-cell responses in HBeAg-negative, genotype D, chronic hepatitis B is largely undefined. Understanding to what extent IFN-α can modulate HBV-specific T-cells is important to define strategies to optimize IFN efficacy and to identify immunological parameters to predict response to therapy. METHODS: HBV-specific T-cell responses were analyzed longitudinally ex vivo and after expansion in vitro in 15 patients with genotype D, HBeAg-negative chronic hepatitis B treated with peginterferon-α-2a. HBV proteins and synthetic peptides were used to stimulate T-cell responses. Analysis of the CD4 and CD8 T-cell functions was performed by ELISPOT, intracellular cytokine and tetramer staining. The effect of anti-PD-L1 on T-cell functions was also analyzed. RESULTS: Ex vivo IFN-γ production by total HBV-specific T-cells was significantly greater before therapy in patients who showed HBV DNA <50 IU/ml at weeks 24 and/or 48 of therapy. No significant improvement of T-cell proliferation, Th1 cytokine production and cytotoxicity was observed during IFN therapy by both ex vivo and in vitro analysis. PD-1/PD-L1 blockade showed a modest improvement of cytokine production in a total of 15% of T-cell lines. CONCLUSIONS: IFN-α did not improve peripheral blood HBV-specific T-cell responses in the first 24 weeks of treatment, consistent either with a predominant antiviral/antiproliferative effect or with an immunomodulatory activity on other arms of the immune system which were not analyzed in our study. A better pre-treatment ex vivo IFN-γ production was associated with better chances to control HBV replication during therapy and represents a promising predictor of IFN efficacy.

Contribution of herpesvirus specific CD8 T cells to anti-viral T cell response in humans.

Herpesviruses infect most humans. Their infections can be associated with pathological conditions and significant changes in T cell repertoire but evidences of symbiotic effects of herpesvirus latency have never been demonstrated. We tested the hypothesis that HCMV and EBV-specific CD8 T cells contribute to the heterologous anti-viral immune response. Volume of activated/proliferating virus-specific and total CD8 T cells was evaluated in 50 patients with acute viral infections: 20 with HBV, 12 with Dengue, 12 with Influenza, 3 with Adenovirus infection and 3 with fevers of unknown etiology. Virus-specific (EBV, HCMV, Influenza) pentamer+ and total CD8 T cells were analyzed for activation (CD38/HLA-DR), proliferation (Ki-67/Bcl-2(low)) and cytokine production. We observed that all acute viral infections trigger an expansion of activated/proliferating CD8 T cells, which differs in size depending on the infection but is invariably inflated by CD8 T cells specific for persistent herpesviruses (HCMV/EBV). CD8 T cells specific for other non-related non persistent viral infection (i.e. Influenza) were not activated. IL-15, which is produced during acute viral infections, is the likely contributing mechanism driving the selective activation of herpesvirus specific CD8 T cells. In addition we were able to show that herpesvirus specific CD8 T cells displayed an increased ability to produce the anti-viral cytokine interferon-gamma during the acute phase of heterologous viral infection. Taken together, these data demonstrated that activated herpesvirus specific CD8 T cells inflate the activated/proliferating CD8 T cells population present during acute viral infections in human and can contribute to the heterologous anti-viral T cell response.

Antigen Load and T Cell Function: A Challenging Interaction in HBV Infection.

Current treatment for chronic HBV infection is mainly based on nucleos(t)ide analogues, that in most cases need to be administered for a patient's lifetime. There is therefore a pressing need to develop new therapeutic strategies to shorten antiviral treatments. A severe dysfunction of virus-specific T cell responses contributes to virus persistence; hence, immune-modulation to reconstitute an efficient host antiviral response is considered a potential approach for HBV cure. In this perspective, a detailed understanding of the different causes of T cell exhaustion is essential for the design of successful functional T cell correction strategies. Among many different mechanisms which are widely believed to play a role in T cell dysfunction, persistent T cell exposure to high antigen burden, in particular HBsAg, is expected to influence T cell differentiation and function. Definitive evidence of the possibility to improve anti-viral T cell functions by antigen decline is, however, still lacking. This review aims at recapitulating what we have learned so far on the complex T cell-viral antigen interplay in chronic HBV infection.

Radiofrequency thermal ablation for hepatocellular carcinoma stimulates autologous NK-cell response.

BACKGROUND & AIMS: Radiofrequency thermal ablation (RFA) is a minimally invasive technique used as standard local therapy of hepatocellular carcinoma and second-line treatment for metastatic liver tumors. Studies in preclinical models and in patients have shown that thermal destruction of tumor tissue can enhance anti-tumor cellular responses, but our knowledge of its impact on natural killer (NK) cells is still very limited. METHODS: Thirty-seven patients undergoing RFA for hepatocellular carcinoma were studied for peripheral blood lymphocytes counts followed by phenotypic and functional characterization of NK-cell population. RESULTS: Peripheral blood lymphocytes kinetics revealed an increased frequency and absolute number of NK cells expressing higher levels of activatory along with reduced levels of inhibitory NK receptors, and increased functional NK-cell activity. A prevalent expansion of the CD3(-)CD56(dim) NK subset was observed compared to the CD3(-)CD56(bright) counterpart. Interferon-gamma production, anti-K562 cell cytotoxicity, and antibody-dependent cell cytotoxicity, appeared consistently increased in terms of both absolute activity and killing efficiency at 4 weeks after RFA, as compared to baseline. Interestingly, when recurrence-free survival was assessed in 2 groups of patients separated according to higher vs lower enhancement of cytotoxicity and/or interferon-gamma production, a significant difference was observed, thus suggesting a potential predictive role of NK functional assays on efficacy of RFA. CONCLUSIONS: RFA can lead to stimulation of NK cells with a more differentiated and proactivatory phenotypic profile with general increase of functional activities. This observation may be relevant for development of adjuvant immunotherapeutic strategies aimed at enhancing NK-cell responses against primary and metastatic liver tumors.

Unraveling the Multifaceted Nature of CD8 T Cell Exhaustion Provides the Molecular Basis for Therapeutic T Cell Reconstitution in Chronic Hepatitis B and C.

In chronic hepatitis B and C virus infections persistently elevated antigen levels drive CD8+ T cells toward a peculiar differentiation state known as T cell exhaustion, which poses crucial constraints to antiviral immunity. Available evidence indicates that T cell exhaustion is associated with a series of metabolic and signaling deregulations and with a very peculiar epigenetic status which all together lead to reduced effector functions. A clear mechanistic network explaining how intracellular metabolic derangements, transcriptional and signaling alterations so far described are interconnected in a comprehensive and unified view of the T cell exhaustion differentiation profile is still lacking. Addressing this issue is of key importance for the development of innovative strategies to boost host immunity in order to achieve viral clearance. This review will discuss the current knowledge in HBV and HCV infections, addressing how innate immunity, metabolic derangements, extensive stress responses and altered epigenetic programs may be targeted to restore functionality and responsiveness of virus-specific CD8 T cells in the context of chronic virus infections.