Production and Purification of Adeno-Associated Viral Vectors for the Development of Immune-Competent Mouse Models of Persistent Hepatitis B Virus Replication.

Mice infected with a recombinant adeno-associated virus carrying a replication-competent hepatitis B virus genome (rAAV-HBV) via the intravenous route establish a persistent HBV replication in hepatocytes and develop immune tolerance. They serve as models to evaluate antiviral immunity and to assess potential therapeutic approaches for chronic HBV infection. Combining selected HBV variants and different mouse genotypes allows for addressing a broad spectrum of research questions. This chapter describes the basic principles of the rAAV-HBV mouse model, rAAV-HBV production and purification methods, and finally, the in vivo application.

A liver immune rheostat regulates CD8 T cell immunity in chronic HBV infection.

Chronic hepatitis B virus (HBV) infection affects 300 million patients worldwide1,2, in whom virus-specific CD8 T cells by still ill-defined mechanisms lose their function and cannot eliminate HBV-infected hepatocytes3-7. Here we demonstrate that a liver immune rheostat renders virus-specific CD8 T cells refractory to activation and leads to their loss of effector functions. In preclinical models of persistent infection with hepatotropic viruses such as HBV, dysfunctional virus-specific CXCR6+ CD8 T cells accumulated in the liver and, as a characteristic hallmark, showed enhanced transcriptional activity of cAMP-responsive element modulator (CREM) distinct from T cell exhaustion. In patients with chronic hepatitis B, circulating and intrahepatic HBV-specific CXCR6+ CD8 T cells with enhanced CREM expression and transcriptional activity were detected at a frequency of 12-22% of HBV-specific CD8 T cells. Knocking out the inhibitory CREM/ICER isoform in T cells, however, failed to rescue T cell immunity. This indicates that CREM activity was a consequence, rather than the cause, of loss in T cell function, further supported by the observation of enhanced phosphorylation of protein kinase A (PKA) which is upstream of CREM. Indeed, we found that enhanced cAMP-PKA-signalling from increased T cell adenylyl cyclase activity augmented CREM activity and curbed T cell activation and effector function in persistent hepatic infection. Mechanistically, CD8 T cells recognizing their antigen on hepatocytes established close and extensive contact with liver sinusoidal endothelial cells, thereby enhancing adenylyl cyclase-cAMP-PKA signalling in T cells. In these hepatic CD8 T cells, which recognize their antigen on hepatocytes, phosphorylation of key signalling kinases of the T cell receptor signalling pathway was impaired, which rendered them refractory to activation. Thus, close contact with liver sinusoidal endothelial cells curbs the activation and effector function of HBV-specific CD8 T cells that target hepatocytes expressing viral antigens by means of the adenylyl cyclase-cAMP-PKA axis in an immune rheostat-like fashion.

CARs derived from broadly neutralizing, human monoclonal antibodies identified by single B cell sorting target hepatitis B virus-positive cells.

To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs.

The lack of HBsAg secretion does neither facilitate induction of antiviral T cell responses nor Hepatitis B Virus clearance in mice.

Immune tolerance to the hepatitis B virus (HBV) is crucial for developing chronic hepatitis B, and the HBV surface antigen (HBsAg) produced and secreted in high amounts is regarded as a key contributor. HBsAg is expressed in HBV-infected hepatocytes and those carrying an HBV integration. Whether either HBsAg secretion or the high antigen amount expressed in the liver determines its immunomodulatory properties, however, remains unclear. We, therefore, developed a novel HBV animal model that allowed us to study the role of secreted HBsAg. We introduced a previously described HBs mutation, C65S, abolishing HBsAg secretion into a replication-competent 1.3-overlength HBV genome and used adeno-associated virus vectors to deliver it to the mouse liver. The AAV-HBV established a carrier state of wildtype and C65S mutant HBV, respectively. We investigated antiviral B- and T-cell immunity in the HBV-carrier mice after therapeutic vaccination. Moreover, we compared the effect of a lacking HBsAg secretion with that of an antiviral siRNA. While missing HBsAg secretion allowed for higher levels of detectable anti-HBs antibodies after therapeutic vaccination, it did neither affect antiviral T-cell responses nor intrahepatic HBV gene expression, irrespective of the starting level. A treatment with HBV siRNA restricting viral antigen expression within hepatocytes, however, improved the antiviral efficacy of therapeutic vaccination, irrespective of the ability of HBV to secrete HBsAg. Our data indicate that clearing HBsAg from blood cannot significantly impact HBV persistence or T-cell immunity. This indicates that a restriction of hepatic viral antigen expression will be required to break HBV immunotolerance.

Introducing adjuvant-loaded particulate hepatitis B core antigen as an alternative therapeutic hepatitis B vaccine component.

Background & Aims: Particulate hepatitis B core antigen (HBcoreAg) is a potent immunogen used as a vaccine carrier platform. HBcoreAg produced in E. coli encapsidates random bacterial RNA (bRNA). Using the heterologous protein-prime, viral-vector-boost therapeutic hepatitis B vaccine TherVacB, we compared the properties of different HBcoreAg forms. We explored how the content of HBcoreAg modulates antigen stability, immunogenicity, and antiviral efficacy. Methods: bRNA was removed from HBcoreAg by capsid disassembly, followed by reassembly in the absence or presence of specific nucleic acid-based adjuvants poly I:C or CpG. The morphology and structure of empty, bRNA-containing and adjuvant-loaded HBcoreAg were monitored by electron microscopy and nuclear magnetic resonance spectroscopy. Empty, bRNA-containing or adjuvant-loaded HBcoreAg were applied together with HBsAg and with or without nucleic acid-based external adjuvants within the TherVacB regimen in both wild-type and HBV-carrier mice. Results: While HBcoreAg retained its structure upon bRNA removal, its stability and immunogenicity decreased significantly. Loading HBcoreAg with nucleic acid-based adjuvants re-established stability of the capsid-like antigen. Immunization with poly I:C- or CpG-loaded HBcoreAg induced high antibody titers against co-administered HBsAg. When applied within the TherVacB regimen, they activated vigorous HBcoreAg- and HBsAg-specific T-cell responses in wild-type and HBV-carrier mice, requiring a significantly lower dose of adjuvant compared to externally added adjuvant. Finally, immunization with adjuvant-loaded HBcoreAg mixed with HBsAg led to long-term control of persistent HBV replication in the HBV-carrier mice. Conclusion: Adjuvant-loaded HBcoreAg retained capsid integrity and stability, was as immunogenic in vivo as externally adjuvanted HBcoreAg, requiring lower adjuvant levels, and supported immunity against co-administered, non-adjuvanted HBsAg. Thus, adjuvant-loaded HBcoreAg represents a promising novel platform for vaccine development. Impact and implications: Hepatitis B core antigen (HBcoreAg) recapitulates the capsid of the HBV that hosts the viral genome. Produced recombinantly, it is not infectious but emerges as a potent immunogen in vaccine development. In this preclinical study, we show that loading HBcoreAg with defined nucleic-acid-based adjuvants on the one hand stabilizes the HBcoreAg with standardized capsid content and, on the other hand, efficiently promotes the immunity of HBcoreAg and a co-administered antigen, allowing for reduced adjuvant doses. Therefore, adjuvant-loaded HBcoreAg not only serves as an encouraging option for therapeutic hepatitis B vaccines, but could also act as an efficient adjuvant delivery system for other types of vaccine.

Exploring the use of nanofluids in pump-free systems for solar thermal applications.

By using nanofluids as a working fluid in pump-free designs, thermal energy systems can become more efficient and have reduced maintenance costs, ultimately extending the system's lifespan. In this paper, our goal is to investigate unsteady phenomena in the irradiation process and highlight their significance. To accomplish this, we conducted a series of experiments using a square loop of glass pipes filled with carbon black nanofluids and irradiated with a halogen lamp to simulate solar irradiation. The resulting convective motion of the nanofluids allowed us to observe the performance of different concentrations of carbon black, with 0.005-0.01 wt.% proving to be the most effective. Additionally, we identified unsteady processes that occur at the beginning of the process or when the irradiation changes. Finally, we employed computational fluid dynamics simulations to gain further insight into these phenomena.

Evaluation of the Effect of CD70 Co-Expression on CD8 T Cell Response in Protein-Prime MVA-Boost Vaccination in Mice.

Here, we investigate the potential of CD70 co-expression during viral vector boost vaccination to improve an antigen-specific T cell response. To determine the chance of activating antigen-specific T cells by CD70, we used the HBV core antigen as a model antigen in a heterologous protein-prime, Modified Vaccinia virus Ankara (MVA) boost vaccination scheme. Both the HBV core and a CD70 expression cassette were co-expressed upon delivery by an MVA vector under the same promoter linked by a P2A site. To compare immunogenicity with and without CD70 co-expression, HBV-naïve, C57BL/6 (wt) mice and HBV-transgenic mice were prime-vaccinated using recombinant HBV core antigen followed by the MVA vector boost. Co-expression of CD70 increased the number of vaccine-induced HBV core-specific CD8 T cells by >2-fold and improved their effector functions in HBV-naïve mice. In vaccinated HBV1.3tg mice, the number and functionality of HBV core-specific CD8 T cells was slightly increased upon CD70 co-expression in low-viremic, but not in high-viremic animals. CD70 co-expression did not impact liver damage as indicated by ALT levels in the serum, but increased the number of vaccine-induced, proliferative T cell clusters in the liver. Overall, this study indicates that orchestrated co-expression of CD70 and a vaccine antigen may be an interesting and safe means of enhancing antigen-specific CD8 T cell responses using vector-based vaccines, although in our study it was not sufficient to break immune tolerance.

Activation of CD4 T cells during prime immunization determines the success of a therapeutic hepatitis B vaccine in HBV-carrier mouse models.

BACKGROUND & AIMS: We recently developed a heterologous therapeutic vaccination scheme (TherVacB) comprising a particulate protein prime followed by a modified vaccinia-virus Ankara (MVA)-vector boost for the treatment of HBV. However, the key determinants required to overcome HBV-specific immune tolerance remain unclear. Herein, we aimed to study new combination adjuvants and unravel factors that are essential for the antiviral efficacy of TherVacB. METHODS: Recombinant hepatitis B surface and core antigen (HBsAg and HBcAg) particles were formulated with different liposome- or oil-in-water emulsion-based combination adjuvants containing saponin QS21 and monophosphoryl lipid A; these formulations were compared to STING-agonist c-di-AMP and conventional aluminium hydroxide formulations. Immunogenicity and the antiviral effects of protein antigen formulations and the MVA-vector boost within TherVacB were evaluated in adeno-associated virus-HBV-infected and HBV-transgenic mice. RESULTS: Combination adjuvant formulations preserved HBsAg and HBcAg integrity for ≥12 weeks, promoted human and mouse dendritic cell activation and, within TherVacB, elicited robust HBV-specific antibody and T-cell responses in wild-type and HBV-carrier mice. Combination adjuvants that prime a balanced HBV-specific type 1 and 2 T helper response induced high-titer anti-HBs antibodies, cytotoxic T-cell responses and long-term control of HBV. In the absence of an MVA-vector boost or following selective CD8 T-cell depletion, HBsAg still declined (mediated mainly by anti-HBs antibodies) but HBV replication was not controlled. Selective CD4 T-cell depletion during the priming phase of TherVacB resulted in a complete loss of vaccine-induced immune responses and its therapeutic antiviral effect in mice. CONCLUSIONS: Our results identify CD4 T-cell activation during the priming phase of TherVacB as a key determinant of HBV-specific antibody and CD8 T-cell responses. IMPACT AND IMPLICATIONS: Therapeutic vaccination is a potentially curative treatment option for chronic hepatitis B. However, it remains unclear which factors are essential for breaking immune tolerance in HBV carriers and determining successful outcomes. Our study provides the first direct evidence that efficient priming of HBV-specific CD4 T cells determines the success of therapeutic hepatitis B vaccination in two preclinical HBV-carrier mouse models. Applying an optimal formulation of HBV antigens that activates CD4 and CD8 T cells during prime immunization provided the foundation for an antiviral effect of therapeutic vaccination, while depletion of CD4 T cells led to a complete loss of vaccine-induced antiviral efficacy. Boosting CD8 T cells was important to finally control HBV in these mouse models. Our findings provide important insights into the rational design of therapeutic vaccines for the cure of chronic hepatitis B.

Efficient stabilization of therapeutic hepatitis B vaccine components by amino-acid formulation maintains its potential to break immune tolerance.

Background & Aims: Induction of potent, HBV-specific immune responses is crucial to control and finally cure HBV. The therapeutic hepatitis B vaccine TherVacB combines protein priming with a Modified Vaccinia virus Ankara (MVA)-vector boost to break immune tolerance in chronic HBV infection. Particulate protein and vector vaccine components, however, require a constant cooling chain for storage and transport, posing logistic and financial challenges to vaccine applications. We aimed to identify an optimal formulation to maintain stability and immunogenicity of the protein and vector components of the vaccine using a systematic approach. Methods: We used stabilizing amino acid (SAA)-based formulations to stabilize HBsAg and HBV core particles (HBcAg), and the MVA-vector. We then investigated the effect of lyophilization and short- and long-term high-temperature storage on their integrity. Immunogenicity and safety of the formulated vaccine was validated in HBV-naïve and adeno-associated virus (AAV)-HBV-infected mice. Results: In vitro analysis proved the vaccine's stability against thermal stress during lyophilization and the long-term stability of SAA-formulated HBsAg, HBcAg and MVA during thermal stress at 40 °C for 3 months and at 25 °C for 12 months. Vaccination of HBV-naïve and AAV-HBV-infected mice demonstrated that the stabilized vaccine was well tolerated and able to brake immune tolerance established in AAV-HBV mice as efficiently as vaccine components constantly stored at 4 °C/-80 °C. Even after long-term exposure to elevated temperatures, stabilized TherVacB induced high titre HBV-specific antibodies and strong CD8+ T-cell responses, resulting in anti-HBs seroconversion and strong suppression of the virus in HBV-replicating mice. Conclusion: SAA-formulation resulted in highly functional and thermostable HBsAg, HBcAg and MVA vaccine components. This will facilitate global vaccine application without the need for cooling chains and is important for the development of prophylactic as well as therapeutic vaccines supporting vaccination campaigns worldwide. Impact and implications: Therapeutic vaccination is a promising therapeutic option for chronic hepatitis B that may enable its cure. However, its application requires functional cooling chains during transport and storage that can hardly be guaranteed in many countries with high demand. In this study, the authors developed thermostable vaccine components that are well tolerated and that induce immune responses and control the virus in preclinical mouse models, even after long-term exposure to high surrounding temperatures. This will lower costs and ease application of a therapeutic vaccine and thus be beneficial for the many people affected by hepatitis B around the world.

Photothermal conversion of biodegradable fluids and carbon black nanofluids.

The paper is devoted to the topic of direct absorption solar collectors (DASCs). Various kinds of fluids can be used as heat transfer fluid in DASCs, and the main focus of our paper is on comparing nanofluids (water with carbon black nanoparticles, concentrations between 0.25 and 1.00% weight) and biodegradable coffee colloids. At first, these fluids were tested by exposing them to irradiation caused by artificial light in indoor experiments, and the corresponding temperature increase was recorded. The fluids were placed in a beaker with a relatively large size so that most of the fluid was not directly irradiated. In these experiments, the performance of the two studied fluids was similar: the resulting temperature increase varied between 46 and 50 °C. Our next experiments involved a smaller system subjected to irradiation obtained by using a solar collector. As a result, we detected an intense absorption on the nanoparticle surface so that the temperature rise in the nanofluid was higher than in the coffee colloids. Next, the process was analysed using a theoretical analysis that gave good correspondence with the experiments. Finally, we extended the theoretical analysis to a DASC with a flowing fluid. The model was validated against results from the literature, but it also supported our experimental findings.

PD-L1 Silencing in Liver Using siRNAs Enhances Efficacy of Therapeutic Vaccination for Chronic Hepatitis B.

In chronic hepatitis B virus (HBV) infection, virus-specific T cells are scarce and partially dysfunctional. Therapeutic vaccination is a promising strategy to induce and activate new virus-specific T cells. In long-term or high-level HBV carriers, however, therapeutic vaccination by itself may not suffice to cure HBV. One reason is the impairment of antiviral T cells by immune checkpoints. In this study, we used small-interfering RNA (siRNA) in combination with a heterologous prime-boost therapeutic vaccination scheme (TherVacB) to interfere with a major immune checkpoint, the interaction of programmed death protein-1 (PD-1) and its ligand (PDL-1). In mice persistently replicating HBV after infection with an adeno-associated virus harboring the HBV genome, siRNA targeting PD-L1 resulted in a higher functionality of HBV-specific CD8+ T cells after therapeutic vaccination, and allowed for a more sustained antiviral effect and control of HBV in peripheral blood and in the liver. The antiviral effect was more pronounced if PD-L1 was down-regulated during prime than during boost vaccination. Thus, targeting PD-L1 using siRNA is a promising approach to enhance the efficacy of therapeutic vaccination and finally cure HBV.

Improving Therapeutic Vaccination against Hepatitis B-Insights from Preclinical Models of Immune Therapy against Persistent Hepatitis B Virus Infection.

Chronic hepatitis B affects more than 250 million individuals worldwide, putting them at risk of developing liver cirrhosis and liver cancer. While antiviral immune responses are key to eliminating hepatitis B virus (HBV) infections, insufficient antiviral immunity characterized by failure to eliminate HBV-infected hepatocytes is associated with chronic hepatitis B. Prophylactic vaccination against hepatitis B successfully established protective immunity against infection with the hepatitis B virus and has been instrumental in controlling hepatitis B. However, prophylactic vaccination schemes have not been successful in mounting protective immunity to eliminate HBV infections in patients with chronic hepatitis B. Here, we discuss the current knowledge on the development and efficacy of therapeutic vaccination strategies against chronic hepatitis B with particular emphasis on the pathogenetic understanding of dysfunctional anti-viral immunity. We explore the development of additional immune stimulation measures within tissues, in particular activation of immunogenic myeloid cell populations, and their use for combination with therapeutic vaccination strategies to improve the efficacy of therapeutic vaccination against chronic hepatitis B.

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.

Immunogenicity and Antiviral Response of Therapeutic Hepatitis B Vaccination in a Mouse Model of HBeAg-Negative, Persistent HBV Infection.

During the natural course of chronic hepatitis B virus (HBV) infection, the hepatitis B e antigen (HBeAg) is typically lost, while the direct transmission of HBeAg-negative HBV may result in fulminant hepatitis B. While the induction of HBV-specific immune responses by therapeutic vaccination is a promising, novel treatment option for chronic hepatitis B, it remains unclear whether a loss of HBeAg may influence its efficacy or tolerability. We therefore generated an adeno-associated virus (AAV)-vector that carries a 1.3-fold overlength HBV genome with a typical stop-codon mutation in the pre-core region and initiates the replication of HBeAg(-) HBV in mouse livers. Infection of C57BL/6 mice established persistent HBeAg(-) HBV-replication without any detectable anti-HBV immunity or liver damage. HBV-carrier mice were immunized with TherVacB, a therapeutic hepatitis B vaccine that uses a particulate HBV S and a core protein for prime vaccination, and a modified vaccinia Ankara (MVA) for boost vaccination. The TherVacB immunization of HBeAg(+) and HBeAg(-) HBV carrier mice resulted in the effective induction of HBV-specific antibodies and the loss of HBsAg but only mild liver damage. Intrahepatic, HBV-specific CD8 T cells induced in HBeAg(-) mice expressed more IFNγ but showed similar cytolytic activity. This indicates that the loss of HBeAg improves the performance of therapeutic vaccination by enhancing non-cytolytic effector functions.

Intramolecular recombination enables the formation of hepatitis B virus (HBV) cccDNA in mice after HBV genome transfer using recombinant AAV vectors.

The mouse is not a natural host of hepatitis B virus (HBV) infection and - despite engraftment of hepatocytes with the HBV receptor - does not support formation of HBV covalently closed circular (ccc) DNA serving as a template for viral transcription and permitting persistent infection. In a recent study, cccDNA formation in mouse hepatocytes has been described following an HBV genome delivery by a recombinant, adeno-associated virus vector (rAAV) (Lucifora et al., 2017). The integrity of HBV cccDNA, its origin and functionality, however, remained open. In this study, we investigated the identity, origin, and functionality of cccDNA established in mice infected with rAAV carrying 1.3-fold overlength HBV genomes. We show that replication of HBV genotypes A, B, C and D can be initiated in mouse livers, and that cccDNA derived from all genotypes is detected. Restriction enzyme and exonuclease digestion as well as sequencing analysis of cccDNA amplicons revealed authentic HBV cccDNA without any detectable alteration compared to cccDNA established after HBV infection of human liver cells. Mouse livers transduced with a core protein-deficient HBV using rAAV still supported cccDNA formation demonstrating that the genesis of cccDNA was independent of HBV replication. When mice were infected with an rAAV-HBV1.3 carrying premature stop codons in the 5' but not in the 3' core protein open reading frame, the stop codon was partially replaced by the wild-type sequence. This strongly indicated that intramolecular recombination, based on >900 identical base pairs residing at the both ends of the HBV1.3 transgene was the origin of cccDNA formation. Accordingly, we observed a constant loss of cccDNA molecules from mouse livers over time, while HBeAg levels increased over the first two weeks after rAAV-HBV1.3 infection and remained constant thereafter, suggesting a minor contribution of the cccDNA molecules formed to viral transcription and protein expression. In summary, our results provide strong evidence that intramolecular recombination of an overlength, linear HBV genome, but not HBV genome recycling, enables cccDNA formation in rAAV-HBV mouse models.

Fetomaternal immune cross talk modifies T-cell priming through sustained changes to DC function.

BACKGROUND: Prenatal exposure to infections can modify immune development. These environmental disturbances during early life potentially alter the incidence of inflammatory disorders as well as priming of immune responses. Infection with the helminth Schistosoma mansoni is widely studied for its ability to alter immune responsiveness and is associated with variations in coinfection, allergy, and vaccine efficacy in endemic populations. OBJECTIVE: Exposure to maternal schistosomiasis during early life, even without transmission of infection, can result in priming effects on offspring immune responses to bystander antigenic challenges as related to allergic responsiveness and vaccination, with this article seeking to further clarify the effects and underlying immunologic imprinting. METHODS: Here, we have combined a model of chronic maternal schistosomiasis infection with a thorough analysis of subsequent offspring immune responses to allergy and vaccination models, including viral challenge and steady-state changes to immune cell compartments. RESULTS: We have demonstrated that maternal schistosomiasis alters CD4+ responses during allergic sensitization and challenge in a skewed IL-4/B-cell-dominant response to antigenic challenge associated with limited inflammatory response. Beyond that, we have uncovered previously unidentified alterations to CD8+ T-cell responses during immunization that are dependent on vaccine formulation and have functional impact on the efficacy of vaccination against viral infection in a murine hepatitis B virus model. CONCLUSION: In addition to steady-state modifications to CD4+ T-cell polarization and B-cell priming, we have traced these modified CD8+ responses to an altered dendritic cell phenotype sustained into adulthood, providing evidence for complex priming effects imparted by infection via fetomaternal cross talk.

Extension of the hard-sphere model for particle-flow simulations.

Discrete element methods require appropriate models for particle-particle collisions. Usually, researchers use soft-sphere types of models where the collision dynamics is solved numerically. This makes the simulation computationally expensive. In this paper, however, we show a hard-sphere model that uses ready analytic formulas that relate the pre- and postcollisional velocities of the particles in contact. This hard-sphere model is an extension of an existing model that uses three input parameters. For this, we applied the linear-spring soft-sphere model, where analytic relations can be found. These relations were implemented into the standard hard-sphere model. As a result, we obtain a robust hard-sphere model that is more accurate than the standard one and is still computationally cheap.

Knockdown of Virus Antigen Expression Increases Therapeutic Vaccine Efficacy in High-Titer Hepatitis B Virus Carrier Mice.

BACKGROUND & AIMS: Hepatitis B virus (HBV) infection persists because the virus-specific immune response is dysfunctional. Therapeutic vaccines might be used to end immune tolerance to the virus in patients with chronic infection, but these have not been effective in patients so far. In patients with chronic HBV infection, high levels of virus antigens might prevent induction of HBV-specific immune responses. We investigated whether knocking down expression levels of HBV antigens in liver might increase the efficacy of HBV vaccines in mice. METHODS: We performed studies with male C57BL/6 mice that persistently replicate HBV (genotype D, serotype ayw)-either from a transgene or after infection with an adeno-associated virus that transferred an overlength HBV genome-and expressed HB surface antigen at levels relevant to patients. Small hairpin or small interfering (si)RNAs against the common 3'-end of all HBV transcripts were used to knock down antigen expression in mouse hepatocytes. siRNAs were chemically stabilized and conjugated to N-acetylgalactosamine to increase liver uptake. Control mice were given either entecavir or non-HBV-specific siRNAs and vaccine components. Eight to 12 weeks later, mice were immunized twice with a mixture of adjuvanted HBV S and core antigen, followed by a modified Vaccinia virus Ankara vector to induce HBV-specific B- and T-cell responses. Serum and liver samples were collected and analyzed for HBV-specific immune responses, liver damage, and viral parameters. RESULTS: In both models of HBV infection, mice that express hepatocyte-specific small hairpin RNAs or that were given subcutaneous injections of siRNAs had reduced levels of HBV antigens, HBV replication, and viremia (1-3 log10 reduction) compared to mice given control RNAs. Vaccination induced production of HBV-neutralizing antibodies and increased numbers and functionality of HBV-specific, CD8+ T cells in mice with low, but not in mice with high, levels of HBV antigen. Mice with initially high titers of HBV and knockdown of HBV antigen expression, but not mice with reduced viremia after administration of entecavir, developed polyfunctional, HBV-specific CD8+ T cells, and HBV was eliminated. CONCLUSIONS: In mice with high levels of HBV replication, knockdown of HBV antigen expression along with a therapeutic vaccination strategy, but not knockdown alone, increased numbers of effector T cells and eliminated the virus. These findings indicate that high titers of virus antigens reduce the efficacy of therapeutic vaccination. Anti-HBV siRNAs and therapeutic vaccines are each being tested in clinical trials-their combination might cure chronic HBV infection.

Dynamic, Helminth-Induced Immune Modulation Influences the Outcome of Acute and Chronic Hepatitis B Virus Infection.

BACKGROUND: Chronic hepatitis B develops more frequently in countries with high prevalence of helminth infections. The crosstalk between these 2 major liver-residing pathogens, Schistosoma mansoni and hepatitis B virus (HBV), is barely understood. METHODS: We used state-of-the-art models for both acute and chronic HBV infection to study the pathogen-crosstalk during the different immune phases of schistosome infection. RESULTS: Although liver pathology caused by schistosome infection was not affected by either acute or chronic HBV infection, S mansoni infection influenced HBV infection outcomes in a phase-dependent manner. Interferon (IFN)-γ secreting, HBV- and schistosome-specific CD8 T cells acted in synergy to reduce HBV-induced pathology during the TH1 phase and chronic phase of schistosomiasis. Consequently, HBV was completely rescued in IFN-γ-deficient or in TH2 phase coinfected mice demonstrating the key role of this cytokine. It is interesting to note that secondary helminth infection on the basis of persistent (chronic) HBV infection increased HBV-specific T-cell frequency and resulted in suppression of virus replication but failed to fully restore T-cell function and eliminate HBV. CONCLUSIONS: Thus, schistosome-induced IFN-γ had a prominent antiviral effect that outcompeted immunosuppressive effects of TH2 cytokines, whereas HBV coinfection did not alter schistosome pathogenicity.