Can Engineered “Designer” T Cells Outsmart Chronic Hepatitis B?

More than 350 million people worldwide are persistently infected with human heptatitis B virus (HBV) and at risk to develop liver cirrhosis and hepatocellular carcinoma making long-term treatment necessary. While a vaccine is available and new antiviral drugs are being developed, elimination of persistently infected cells is still a major issue. Recent efforts in adoptive cell therapy are experimentally exploring immunotherapeutic elimination of HBV-infected cells by means of a biological attack with genetically engineered “designer” T cells

Depletion of T cells via Inducible Caspase 9 Increases Safety of Adoptive T-Cell Therapy Against Chronic Hepatitis B

T-cell therapy with T cells that are re-directed to hepatitis B virus (HBV)-infected cells by virus-specific receptors is a promising therapeutic approach for treatment of chronic hepatitis B and HBV-associated cancer. Due to the high number of target cells, however, side effects such as cytokine release syndrome or hepatotoxicity may limit safety. A safeguard mechanism, which allows depletion of transferred T cells on demand, would thus be an interesting means to increase confidence in this approach. In this study, T cells were generated by retroviral transduction to express either an HBV-specific chimeric antigen receptor (S-CAR) or T-cell receptor (TCR), and in addition either inducible caspase 9 (iC9) or herpes simplex virus thymidine kinase (HSV-TK) as a safety switch. Real-time cytotoxicity assays using HBV-replicating hepatoma cells as targets revealed that activation of both safety switches stopped cytotoxicity of S-CAR- or TCR-transduced T cells within less than one hour. In vivo, induction of iC9 led to a strong and rapid reduction of transferred S-CAR T cells adoptively transferred into AAV-HBV-infected immune incompetent mice. One to six hours after injection of the iC9 dimerizer, over 90% reduction of S-CAR T cells in the blood and the spleen and of over 99% in the liver was observed, thereby limiting hepatotoxicity and stopping cytokine secretion. Simultaneously, however, the antiviral effect of S-CAR T cells was diminished because remaining S-CAR T cells were mostly non-functional and could not be restimulated with HBsAg. A second induction of iC9 was only able to deplete T cells in the liver. In conclusion, T cells co-expressing iC9 and HBV-specific receptors efficiently recognize and kill HBV-replicating cells. Induction of T-cell death via iC9 proved to be an efficient means to deplete transferred T cells in vitro and in vivo containing unwanted hepatotoxicity

Functional classification of memory CD8(+) T cells by CX(3)CR1 expression

Localization of memory CD8(+) T cells to lymphoid or peripheral tissues is believed to correlate with proliferative capacity or effector function. Here we demonstrate that the fractalkine-receptor/CX(3)CR1 distinguishes memory CD8(+) T cells with cytotoxic effector function from those with proliferative capacity, independent of tissue-homing properties. CX(3)CR1-based transcriptome and proteome-profiling defines a core signature of memory CD8(+) T cells with effector function. We find CD62L(hi)CX(3)CR1(+) memory T cells that reside within lymph nodes. This population shows distinct migration patterns and positioning in proximity to pathogen entry sites. Virus-specific CX(3)CR1(+) memory CD8(+) T cells are scarce during chronic infection in humans and mice but increase when infection is controlled spontaneously or by therapeutic intervention. This CX3CR1-based functional classification will help to resolve the principles of protective CD8(+) T-cell memory

Infection with hepatitis B virus carrying novel pre-S/S gene mutations in female siblings vaccinated at birth: two case reports

<p>Abstract</p> <p>Introduction</p> <p>After the initiation of a mass hepatitis B vaccination program in Taiwan, the prevalence of hepatitis B virus infection has declined progressively. However, about 1 percent of the young generation, who received hepatitis B vaccination at birth, remain carriers. Infection with vaccine-escape hepatitis B virus mutants always occurs shortly after birth. Here, we report two female siblings in whom the infection occurred in their adolescence. This report raises the question of whether a booster for hepatitis B vaccination is needed.</p> <p>Case presentation</p> <p>Two 19 and 14-year-old Taiwanese female siblings were born to a mother infected with hepatitis B virus and received a complete course of hepatitis B vaccination at birth. They remained negative for serum hepatitis B surface antigen and positive for serum anti-hepatitis B surface antibody throughout their childhood. However, both were infected with the hepatitis B virus in their adolescence. Hepatitis B virus DNA was extracted from serum samples from the mother and two siblings. Hepatitis B virus pre-S/S sequence was amplified by polymerase chain reaction followed by nucleotide sequencing. When compared with the sequence obtained from the mother, multiple amino acid substitutions located near or in the major hydrophilic region of the surface antigen were identified in the elder sister. Four of these mutations (sL97S, sL98S, sG102R, and sA159P) were novel. A novel in-frame deletion (14 amino acids deleted, pre-S 127-140) was found in the hepatitis B virus pre-S2 region in the younger sister.</p> <p>Conclusions</p> <p>Despite having received hepatitis B vaccination at birth, hepatitis B virus infection can still occur in adolescence with the emergence of novel mutations in the pre-S/S gene. This is a rare event and, to the best of our knowledge, has not been previously reported.</p

Heterologous Replacement of the Supposed Host Determining Region of Avihepadnaviruses: High In Vivo Infectivity Despite Low Infectivity for Hepatocytes

Hepadnaviruses, including hepatitis B virus (HBV), a highly relevant human pathogen, are small enveloped DNA viruses that replicate via reverse transcription. All hepadnaviruses display a narrow tissue and host tropism. For HBV, this restricts efficient experimental in vivo infection to chimpanzees. While the cellular factors mediating infection are largely unknown, the large viral envelope protein (L) plays a pivotal role for infectivity. Furthermore, certain segments of the PreS domain of L from duck HBV (DHBV) enhanced infectivity for cultured duck hepatocytes of pseudotyped heron HBV (HHBV), a virus unable to infect ducks in vivo. This implied a crucial role for the PreS sequence from amino acid 22 to 90 in the duck tropism of DHBV. Reasoning that reciprocal replacements would reduce infectivity for ducks, we generated spreading-competent chimeric DHBVs with L proteins in which segments 22–90 (Du-He4) or its subsegments 22–37 and 37–90 (Du-He2, Du-He3) are derived from HHBV. Infectivity for duck hepatocytes of Du-He4 and Du-He3, though not Du-He2, was indeed clearly reduced compared to wild-type DHBV. Surprisingly, however, in ducks even Du-He4 caused high-titered, persistent, horizontally and vertically transmissable infections, with kinetics of viral spread similar to those of DHBV when inoculated at doses of 108 viral genome equivalents (vge) per animal. Low-dose infections down to 300 vge per duck did not reveal a significant reduction in specific infectivity of the chimera. Hence, sequence alterations in PreS that limited infectivity in vitro did not do so in vivo. These data reveal a much more complex correlation between PreS sequence and host specificity than might have been anticipated; more generally, they question the value of cultured hepatocytes for reliably predicting in vivo infectivity of avian and, by inference, mammalian hepadnaviruses, with potential implications for the risk assessment of vaccine and drug resistant HBV variants

Toll-Like Receptor 8 Agonist and Bacteria Trigger Potent Activation of Innate Immune Cells in Human Liver

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.The study was supported by a Grant core funding from the Agency for Science Technology and Research (A*STAR, Singapore) and a Singapore Translational Research Investigator Award (NRMC/StaR/013/2012) to AB as well as NIHR Biomedical Centre, Oxford, WT 091663MA, NIAID1U19AI082630-01, Oxford Martin School funding and an NIHR Senior Investigator award to PK

Amino acid substitutions within HLA-B*27- restricted T cell epitopes prevent recognition by hepatitis delta virus-specific CD8+ T cells

Virus-specific CD8 T cell response seems to play a significant role in the outcome of hepatitis delta virus (HDV) infection. However, the HDV-specific T cell epitope repertoire and mechanisms of CD8 T cell failure in HDV infection have been poorly characterized. We therefore aimed to characterize HDV-specific CD8 T cell epitopes and the impacts of viral mutations on immune escape. In this study, we predicted peptide epitopes binding the most frequent human leukocyte antigen (HLA) types and assessed their HLA binding capacities. These epitopes were characterized in HDV-infected patients by intracellular gamma interferon (IFN-γ) staining. Sequence analysis of large hepatitis delta antigen (L-HDAg) and HLA typing were performed in 104 patients. The impacts of substitutions within epitopes on the CD8 T cell response were evaluated experimentally and by in silico studies. We identified two HLA-B*27-restricted CD8 T cell epitopes within L-HDAg. These novel epitopes are located in a relatively conserved region of L-HDAg. However, we detected molecular footprints within the epitopes in HLA-B*27-positive patients with chronic HDV infections. The variant peptides were not cross-recognized in HLA-B*27-positive patients with resolved HDV infections, indicating that the substitutions represent viral escape mutations. Molecular modeling of HLA-B*27 complexes with the L-HDAg epitope and its potential viral escape mutations indicated that the structural and electrostatic properties of the bound peptides differ considerably at the T cell receptor interface, which provides a possible molecular explanation for the escape mechanism. This viral escape from the HLA-B*27-restricted CD8 T cell response correlates with a chronic outcome of hepatitis D infection. T cell failure resulting from immune escape may contribute to the high chronicity rate in HDV infection. © 2018 American Society for Microbiology

Upregulation of the Tim-3/galectin-9 pathway of T cell exhaustion in chronic hepatitis B virus infection.

The S-type lectin galectin-9 binds to the negative regulatory molecule Tim-3 on T cells and induces their apoptotic deletion or functional inactivation. We investigated whether galectin-9/Tim-3 interactions contribute to the deletion and exhaustion of the antiviral T cell response in chronic hepatitis B virus infection (CHB). We found Tim-3 to be expressed on a higher percentage of CD4 and CD8 T cells from patients with CHB than healthy controls (p<0.0001) and to be enriched on activated T cells and those infiltrating the HBV-infected liver. Direct ex vivo examination of virus-specific CD8 T cells binding HLA-A2/peptide multimers revealed that Tim-3 was more highly upregulated on HBV-specific CD8 T cells than CMV-specific CD8 T cells or the global CD8 T cell population in patients with CHB (p<0.001) or than on HBV-specific CD8 after resolution of infection. T cells expressing Tim-3 had an impaired ability to produce IFN-γ and TNF-α upon recognition of HBV-peptides and were susceptible to galectin-9-triggered cell death in vitro. Galectin-9 was detectable at increased concentrations in the sera of patients with active CHB-related liver inflammation (p = 0.02) and was strongly expressed by Kupffer cells within the liver sinusoidal network. Tim-3 blockade resulted in enhanced expansion of HBV-specific CD8 T cells able to produce cytokines and mediate cytotoxicity in vitro. Blocking PD-1 in combination with Tim-3 enhanced the number of patients from whom functional antiviral responses could be recovered and/or the strength of responses, indicating that these co-inhibitory molecules play a non-redundant role in driving T cell exhaustion in CHB. Patients taking antivirals able to potently suppress HBV viraemia continued to express Tim-3 on their T cells and respond to Tim-3 blockade. In summary, both Tim-3 and galectin-9 are increased in CHB and may contribute to the inhibition and deletion of T cells as they infiltrate the HBV-infected liver

The third signal cytokine IL-12 rescues the anti-viral function of exhausted HBV-specific CD8 T cells.

Optimal immune activation of naïve CD8 T cells requires signal 1 mediated by the T cell receptor, signal 2 mediated by co-stimulation and signal 3 provided by pro-inflammatory cytokines. However, the potential for signal 3 cytokines to rescue anti-viral responses in functionally exhausted T cells has not been defined. We investigated the effect of using third signal cytokines IL-12 or IFN-α to rescue the exhausted CD8 T cell response characteristic of patients persistently infected with hepatitis B virus (HBV). We found that IL-12, but not IFN-α, potently augmented the capacity of HBV-specific CD8 T cells to produce effector cytokines upon stimulation by cognate antigen. Functional recovery mediated by IL-12 was accompanied by down-modulation of the hallmark inhibitory receptor PD-1 and an increase in the transcription factor T-bet. PD-1 down-regulation was observed in HBV but not CMV-specific T cells, in line with our finding that the highly functional CMV response was not further enhanced by IL-12. IL-12 enhanced a number of characteristics of HBV-specific T cells important for viral control: cytotoxicity, polyfunctionality and multispecificity. Furthermore, IL-12 significantly decreased the pro-apoptotic molecule Bim, which is capable of mediating premature attrition of HBV-specific CD8 T cells. Combining IL-12 with blockade of the PD-1 pathway further increased CD8 functionality in the majority of patients. These data provide new insights into the distinct signalling requirements of exhausted T cells and the potential to recover responses optimised to control persistent viral infections