HAMdetector: a Bayesian regression model that integrates information to detect HLA-associated mutations.

MOTIVATION: A key process in anti-viral adaptive immunity is that the human leukocyte antigen (HLA) system presents epitopes as major histocompatibility complex I (MHC I) protein-peptide complexes on cell surfaces and in this way alerts CD8+ cytotoxic T-lymphocytes (CTLs). This pathway exerts strong selection pressure on viruses, favoring viral mutants that escape recognition by the HLA/CTL system. Naturally, such immune escape mutations often emerge in highly variable viruses, e.g. HIV or HBV, as HLA-associated mutations (HAMs), specific to the hosts MHC I proteins. The reliable identification of HAMs is not only important for understanding viral genomes and their evolution, but it also impacts the development of broadly effective anti-viral treatments and vaccines against variable viruses. By their very nature, HAMs are amenable to detection by statistical methods in paired sequence/HLA data. However, HLA alleles are very polymorphic in the human host population which makes the available data relatively sparse and noisy. Under these circumstances, one way to optimize HAM detection is to integrate all relevant information in a coherent model. Bayesian inference offers a principled approach to achieve this. RESULTS: We present a new Bayesian regression model for the detection of HAMs that integrates a sparsity-inducing prior, epitope predictions and phylogenetic bias assessment, and that yields easily interpretable quantitative information on HAM candidates. The model predicts experimentally confirmed HAMs as having high posterior probabilities, and it performs well in comparison to state-of-the-art models for several datasets from individuals infected with HBV, HDV and HIV. AVAILABILITY AND IMPLEMENTATION: The source code of this software is available at https://github.com/HAMdetector/Escape.jl under a permissive MIT license. The data underlying this article were provided by permission. Data will be shared on request to the corresponding author with permission of the respective co-authors. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Aborted infection of human sodium taurocholate cotransporting polypeptide (hNTCP) expressing woodchuck hepatocytes with hepatitis B virus (HBV).

Due to the limited host range of HBV, research progress has been hindered by the absence of a suitable animal model. The natural history of woodchuck hepatitis virus (WHV) infection in woodchuck closely mirrors that of HBV infection in human, making this species a promising candidate for establishing both in vivo and in vitro HBV infection models. Therefore, this animal may be a valuable species to evaluate HBV vaccines and anti-HBV drugs. A significant milestone in HBV and hepatitis D virus (HDV) infection is the discovery of sodium taurocholate cotransporting polypeptide (NTCP) as the functional receptor. In an effort to enhance susceptibility to HBV infection, we introduced hNTCP into the woodchuck hepatocytes by multiple approaches including transduction of vLentivirus-hNTCP in woodchuck hepatocytes, transfection of p-lentivirus-hNTCP-eGFP plasmids into these cells, as well as transduction of vAdenovirus-hNTCP-eGFP. Encouragingly, our findings demonstrated the successful introduction of hNTCP into woodchuck hepatocytes. However, it was observed that these hNTCP-expressing hepatocytes were only susceptible to HDV infection but not HBV. This suggests the presence of additional crucial factors mediating early-stage HBV infection that are subject to stringent species-specific restrictions.

Mutations in Hepatitis D Virus Allow It to Escape Detection by CD8+ T Cells and Evolve at the Population Level.

BACKGROUND & AIMS: Hepatitis D virus (HDV) superinfection in patients with hepatitis B virus (HBV) is associated with rapid progression to liver cirrhosis and hepatocellular carcinoma. Treatment options are limited, and no vaccine is available. Although HDV-specific CD8+ T cells are thought to control the virus, little is known about which HDV epitopes are targeted by virus-specific CD8+ T cells or why these cells ultimately fail to control the infection. We aimed to define how HDV escapes the CD8+ T-cell-mediated response. METHODS: We collected plasma and DNA samples from 104 patients with chronic HDV and HBV infection at medical centers in Europe and the Middle East, sequenced HDV, typed human leukocyte antigen (HLA) class I alleles from patients, and searched for polymorphisms in HDV RNA associated with specific HLA class I alleles. We predicted epitopes in HDV that would be recognized by CD8+ T cells and corresponded with the identified virus polymorphisms in patients with resolved (n = 12) or chronic (n = 13) HDV infection. RESULTS: We identified 21 polymorphisms in HDV that were significantly associated with specific HLA class I alleles (P < .005). Five of these polymorphisms were found to correspond to epitopes in HDV that are recognized by CD8+ T cells; we confirmed that CD8+ T cells in culture targeted these HDV epitopes. HDV variant peptides were only partially cross-recognized by CD8+ T cells isolated from patients, indicating that the virus had escaped detection by these cells. These newly identified HDV epitopes were restricted by relatively infrequent HLA class I alleles, and they bound most frequently to HLA-B. In contrast, frequent HLA class I alleles were not associated with HDV sequence polymorphisms. CONCLUSIONS: We analyzed sequences of HDV RNA and HLA class I alleles that present epitope peptides to CD8+ T cells in patients with persistent HDV infection. We identified polymorphisms in the HDV proteome that associate with HLA class I alleles. Some variant peptides in epitopes from HDV were only partially recognized by CD8+ T cells isolated from patients; these could be mutations that allow HDV to escape the immune response, resulting in persistent infection. HDV escape from the immune response was associated with uncommon HLA class I alleles, indicating that HDV evolves, at the population level, to evade recognition by common HLA class I alleles.

TLR2 Stimulation Strengthens Intrahepatic Myeloid-Derived Cell-Mediated T Cell Tolerance through Inducing Kupffer Cell Expansion and IL-10 Production.

Hepatic APCs play a critical role in promoting immune tolerance in the liver. Recently, we have demonstrated that TLR2 stimulation on liver sinusoidal endothelial cells reverted their suppressive properties to induce T cell immunity. However, there is a paucity of information about how TLR2 stimulation modulates the immunological function of other hepatic APCs. In the current study, we investigated whether TLR2 stimulation influences the function of intrahepatic myeloid-derived cells (iMDCs) and elucidated the mechanisms involved in iMDC-induced T cell immunity. We could show that iMDCs from C57BL/6 mice can potently suppress T cell activation in a cell contact-independent manner. Ag presentation by iMDCs leads to naive CD8 T cell tolerance. To our surprise, instead of inducing cell functional maturation, TLR2 ligand palmitoyl-3-cysteine-serine-lysine-4 (P3C) stimulation further strengthens the suppressive and tolerogenic properties of iMDCs. After P3C administration, the population of Kupffer cells (KCs) of iMDCs dramatically increased. Mechanism analysis shows that KCs are essential for the enhanced inhibition of T cell activation by P3C-stimulated iMDCs. The iMDC-mediated CD8 T cell inhibition was mediated by soluble mediators, one of which was IL-10 secreted by KCs after P3C stimulation. IL-10 blockade could partially abolish iMDC-mediated T cell inhibition. Moreover, hepatitis B virus particle stimulation on iMDCs could also induce IL-10 production by the cells in a TLR2-dependent way. Our results have implications for our understanding of liver-specific tolerance and for the development of strategies to overcome T cell tolerance in situations such as chronic viral liver infections.

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.IMPORTANCE Hepatitis delta virus (HDV) causes severe chronic hepatitis, which affects 20 million people worldwide. Only a small number of patients are able to clear the virus, possibly mediated by a virus-specific T cell response. Here, we performed a systematic screen to define CD8 epitopes and investigated the role of CD8 T cells in the outcome of hepatitis delta and how they fail to eliminate HDV. Overall the number of epitopes identified was very low compared to other hepatotropic viruses. We identified, two HLA-B*27-restricted epitopes in patients with resolved infections. In HLA-B*27-positive patients with chronic HDV infections, however, we detected escape mutations within these identified epitopes that could lead to viral evasion of immune responses. These findings support evidence showing that HLA-B*27 is important for virus-specific CD8 T cell responses, similar to other viral infections. These results have implications for the clinical prognosis of HDV infection and for vaccine development.

Genetic diversity of hepatitis D virus genotype-1 in Europe allows classification into subtypes.

Hepatitis delta virus (HDV) is an RNA virus which leads to both acute and chronic forms of hepatitis. At present, HDV isolates have been classified into eight major genotypes distributed over different geographical regions. Recent increase in HDV sequences in Europe and worldwide has enabled us to revisit the taxonomic classification of HDV. A total of 116 large hepatitis delta antigen (L-HDAg) nucleotide sequences and 13 full-length HDV genome sequences belonging to genotype-1 from our European cohort, as well as 621 L-HDAg nucleotide sequences belonging to genotype-1 to genotype-8 retrieved from the GenBank NCBI were included in this study. All 116 isolates of our cohort and 341 of 621 isolates (60%) account for genotype-1, while the remaining 40% of isolates were unevenly distributed across genotype-2 to genotype-8. Phylogenetic analysis of 98 L-HDAg sequences selected after elimination of redundant sequences of all 737 isolates was performed to identify plausible subtypes within HDV genotype-1. Pairwise genetic distances for L-HDAg sequences were calculated to estimate the inter-genotype and inter-subtype differences. The HDV genotype-1 isolates phylogenetically formed five distinct clusters (genotype 1a-1e), each of them corresponding to a distinct geographic region. Two distinct subtypes for HDV genotype-2 and -4 (ie -2a and -2b; -4a and -4b, respectively) could be identified based on isolate sequences from GenBank. The previously defined genotype-1 to genotype-8 have an inter-genotypic difference of ≥10%, while the newly defined subtypes of genotype-1, -2 and -4 show an inter-subtype difference of ≥3% to <10% from the average diversity. In addition, we identified unique amino acid residues, known as specificity-determining positions, amongst the proposed subtypes.

Kinetics of hepatitis B surface antigen quasispecies during REP 2139-Ca therapy in HBeAg-positive chronic HBV infection.

Chronic HBV infection results in various clinical manifestations due to different levels of immune response. In recent years, hepatitis B treatment has improved by long-term administration of nucleos(t)ide analogues (NUCs) and peg-interferon. Nucleic acid polymers (NAPs; REP 2139-Ca and REP 2139-Mg) are new antiviral drugs that block the assembly of subviral particles, thus preventing the release of HBsAg and allowing its clearance and restoration of functional control of infection when combined with various immunotherapies. In the REP 102 study (NCT02646189), 9 of 12 patients showed substantial reduction of HBsAg and seroconversion to anti-HBs in response to REP 2139-Ca, whereas 3 of 12 patients did not show responses (>1 log reduction of HBsAg and HBV DNA from baseline). We characterized the dynamic changes of HBV quasispecies (QS) within the major hydrophilic region (MHR) of the 'pre-S/S' open reading frame including the 'a' determinant in responders and nonresponders of the REP 102 study and four untreated matched controls. HBV QS complexity at baseline varied slightly between responders and nonresponders (P = .28). However, these responders showed significant decline in viral complexity (P = .001) as REP 2139-Ca therapy progressed but no significant change in complexity was observed among the nonresponders (P = .99). The MHR mutations were more frequently observed in responders than in nonresponders and matched controls. No mutations were observed in 'a' determinant of major QS population which may interfere with the detection of HBsAg by diagnostic assays. No specific mutations were found within the MHR which could explain patients' poor HBsAg response during REP 2139-Ca therapy.

Variants of hepatitis B virus surface antigen observed during therapy with nucleic acid polymer REP 2139-Ca have no influence on treatment outcome and its detection by diagnostic assays.

The treatment of patients suffering from HBeAg-positive chronic hepatitis B with REP 2139-Ca resulted in potent reductions in HBsAg and HBV DNA, seroconversion to anti-HBs and the establishment of functional control of infection. In this cohort of 12 patients, we investigated whether differences between HBsAg sequences might explain the lack of response to REP 2139-Ca observed in 3 of 12 patients. We also assessed if the reduction or complete loss of HBsAg in serum observed during therapy were caused by mutations in the "a" determinant preventing the detection of HBsAg by standard diagnostic assays. The complete pre-S/S open reading frame (ORF) was sequenced and pre-S1, pre-S2 and S amino acid sequences were analysed. We found no major differences between pre-S1, pre-S2 and S sequences in responders and nonresponders correlated with low reduction in HBsAg. In addition, we found no mutations in the "a" determinant that would significantly affect the reactivity of HBsAg in diagnostic assays. These results demonstrate that the amino acid sequence of complete pre-S/S ORF has no direct influence on response to REP 2139-Ca therapy.

Induction of herpes simplex virus type 1 cell-to-cell spread inhibiting antibodies by a calcium phosphate nanoparticle-based vaccine.

Herpes simplex viruses 1 and 2 are among the most ubiquitous human infections and persist lifelong in their host. Upon primary infection or reactivation from ganglia, the viruses spread by direct cell-cell contacts (cell-to-cell spread) and thus escape from the host immune response. We have developed a monoclonal antibody (mAb 2c), which inhibits the HSV cell-to-cell spread, thereby protecting from lethal genital infection and blindness in animal models. In the present study we have designed a nanoparticle-based vaccine to induce protective antibody responses exceeding the cell-to-cell spread inhibiting properties of mAb 2c. We used biodegradable calcium phosphate (CaP) nanoparticles coated with a synthetic peptide that represents the conformational epitope on HSV-1 gB recognized by mAb 2c. The CaP nanoparticles additionally contained a TLR-ligand CpGm and were formulated with adjuvants to facilitate the humoral immune response. This vaccine effectively protected mice from lethal HSV-1 infection by inducing cell-to-cell spread inhibiting antibodies.

Low hepatitis B virus-specific T-cell response in males correlates with high regulatory T-cell numbers in murine models.

Hepatitis B virus (HBV) infection shows significant gender-related differences in pathogenesis, disease progression, and development of hepatocellular carcinoma. The gender-associated differences in HBV replication and viral protein levels may be associated with distinct HBV-specific immune responses in the host. In the present study, we examined the impact of gender on HBV-specific immune responses in two different mouse models representing transient and persistent hepadnaviral infection; hydrodynamic injection with the HBV genome mimicked acute HBV infection, whereas the efficacy of therapeutic vaccination was studied in the woodchuck hepatitis virus transgenic mouse model. Consistent with previous reports, significantly higher HBV DNA and protein levels were detected in male compared to female mice. Although hydrodynamic injection with the HBV genome resulted in similar numbers of intrahepatic HBV-specific cluster of differentiation 8-positive (CD8+ ) T cells, their functionality was significantly reduced in males and correlated with higher numbers of intrahepatic regulatory T cells (Tregs). Similar effects were observed in woodchuck hepatitis virus transgenic mice immunized with a DNA prime-recombinant adenovirus boost vaccination protocol. Male mice showed functionally suppressed woodchuck hepatitis virus-specific CD8+ T-cell responses in the liver and significantly higher numbers of intrahepatic Tregs compared to females. Blockade of Treg responses in male mice led to augmented effector functions of specific CD8+ T cells and subsequently improved virus control in both models of transient and persistent hepadnaviral infection. CONCLUSION: The functionality of virus-specific CD8+ T cells in male mice was suppressed by intrahepatic Tregs and inversely correlated with levels of hepadnaviral DNA and viral protein; the induction of intrahepatic Tregs by viral replication and/or protein levels may explain the gender-related differences in the outcomes of HBV infection and limit the success of immunotherapeutic strategies in male patients. (Hepatology 2017;66:69-83).

Enhancing virus-specific immunity in vivo by combining therapeutic vaccination and PD-L1 blockade in chronic hepadnaviral infection.

Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained in vitro by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether in vivo blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PD-L1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. In vivo blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients.

Acute hepatitis C virus infection induces anti-host cell receptor antibodies with virus-neutralizing properties.

UNLABELLED: Hepatitis C virus (HCV) causes persistent infection in the majority of infected individuals. The mechanisms of persistence and clearance are only partially understood. Antibodies (Abs) against host cell entry receptors have been shown to inhibit HCV infection in cell culture and animal models. In this study, we aimed to investigate whether anti-receptor Abs are induced during infection in humans in vivo and whether their presence is associated with outcome of infection. We established an enzyme-linked immunosorbant assay using a recombinant CD81-claudin-1 (CLDN1) fusion protein to detect and quantify Abs directed against extracellular epitopes of the HCV CD81-CLDN1 coreceptor complex. The presence of anti-receptor Abs was studied in serum of patients from a well-defined cohort of a single-source HCV outbreak of pregnant women and several control groups, including uninfected pregnant women, patients with chronic hepatitis B and D virus (HBV/HDV) infection, and healthy individuals. Virus-neutralizing activity of Abs was determined using recombinant cell culture-derived HCV (HCVcc). Our results demonstrate that HCV-infected patients have statistically significantly higher anti-CD81/CLDN1 Ab titers during the early phase of infection than controls. The titers were significantly higher in resolvers compared to persisters. Functional studies using immunoadsorption and HCV cell culture models demonstrate that HCV-neutralizing anti-receptor Abs are induced in the early phase of HCV infection, but not in control groups. CONCLUSION: The virus-neutralizing properties of these Abs suggest a role for control of viral infection in conjunction with antiviral responses. Characterization of these anti-receptor Abs opens new avenues to prevent and treat HCV infection.

Overcoming drug-resistant herpes simplex virus (HSV) infection by a humanized antibody.

Despite the availability of antiviral chemotherapy, herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections remain a severe global health problem. Of particular concern is the growing incidence of drug resistance in immunocompromised patients, which stresses the urgency to develop new effective treatment alternatives. We have developed a humanized monoclonal antibody (mAb hu2c) that completely abrogates viral cell-to-cell spread, a key mechanism by which HSV-1/2 escapes humoral immune surveillance. Moreover, mAb hu2c neutralized HSV fully independent of complement and/or immune effector cell recruitment in a highly efficient manner. Prophylactic and therapeutic administration of mAb hu2c completely prevented infection-related mortality of severely immunodeficient mice being challenged with a lethal dose of HSV-1. The high neutralization capacity of mAb hu2c was fully maintained toward clinical HSV isolates being multiresistant to standard antiviral drugs, and infection was fully resolved in 7/8 nonobese diabetic/SCID mice being infected with a multidrug resistant HSV-1 patient isolate. Immunohistochemical studies revealed no significant cross-reactivity of the antibody toward human tissues. These features warrant further clinical development of mAb hu2c as an immunotherapeutic compound for the management of severe and particularly drug-resistant HSV infections.

Decades after recovery from hepatitis B and HBsAg clearance the CD8+ T cell response against HBV core is nearly undetectable.

BACKGROUND & AIMS: CD8(+) T cells are an essential component of a successful immune response against hepatitis B virus (HBV). Patients who spontaneously clear HBsAg after acute HBV infection have a strong CD8(+) T cell immune response, predominantly directed against the HBV core protein (HBcAg). However, the fate and phenotype of HBcAg-specific CD8(+) T cells after immune control are unclear. METHODS: The CD8(+) T cell immune response against HBV core was determined in 65 patients with chronic HBV infection, 16 patients after recovery from acute HBV infection, and four patients with acute HBV infection utilizing overlapping peptides and HLA class I/peptide-multimers. RESULTS: Patients who had cleared HBsAg >30 years ago had significantly weaker CD8(+) T cell responses after antigen-specific expansion compared to patients who had cleared the virus <10 years ago and patients with HBeAg negative chronic infection and low viral load (<2000 IU/ml; p<0.01). Also directly ex vivo, patients who had cleared the HBsAg >30 years ago had less HBV-specific CD8(+) T cells compared to patients with HBeAg negative chronic infection (p=0.0025). In patients with acute HBV infection, the frequency of HBc-specific CD8(+) T cells continued to decline after clearance of HBV-DNA and HBsAg even at a time when ALT levels had already normalized (p=0.0313). CONCLUSIONS: The frequency of HBcAg-specific CD8(+) T cells continuously declines after HBsAg clearance. In line with clinical observations, this suggests that humoral and not CD8(+) T cell immune responses mainly contribute to prevention of HBV reactivation decades after HBsAg clearance.

Novel Woodchuck Hepatitis Virus (WHV) transgene mouse models show sex-dependent WHV replicative activity and development of spontaneous immune responses to WHV proteins.

The woodchuck model is an informative model for studies on hepadnaviral infection. In this study, woodchuck hepatitis virus (WHV) transgenic (Tg) mouse models based on C57BL/6 mice were established to study the pathogenesis associated with hepadnaviral infection. Two lineages of WHV Tg mice, harboring the WHV wild-type genome (lineage 1217) and a mutated WHV genome lacking surface antigen (lineage 1281), were generated. WHV replication intermediates were detected by Southern blotting. DNA vaccines against WHV proteins were applied by intramuscular injection. WHV-specific immune responses were analyzed by flow cytometry and enzyme-linked immunosorbent assays (ELISAs). The presence of WHV transgenes resulted in liver-specific but sex- and age-dependent WHV replication in Tg mice. Pathological changes in the liver, including hepatocellular dysplasia, were observed in aged Tg mice, suggesting that the presence of WHV transgenes may lead to liver diseases. Interestingly, Tg mice of lineage 1281 spontaneously developed T- and B-cell responses to WHV core protein (WHcAg). DNA vaccination induced specific immune responses to WHV proteins in WHV Tg mice, indicating a tolerance break. The magnitude of the induced WHcAg-specific immune responses was dependent on the effectiveness of different DNA vaccines and was associated with a decrease in WHV loads in mice. In conclusion, sex- and age-dependent viral replication, development of autoimmune responses to viral antigens, pathological changes in the liver in WHV Tg mice, and the possibility of breaking immune tolerance to WHV transgenes will allow future studies on pathogenesis related to hepadnaviral infection and therapeutic vaccines.

Dissection of antibody specificities induced by yellow fever vaccination.

The live attenuated yellow fever (YF) vaccine has an excellent record of efficacy and one dose provides long-lasting immunity, which in many cases may last a lifetime. Vaccination stimulates strong innate and adaptive immune responses, and neutralizing antibodies are considered to be the major effectors that correlate with protection from disease. Similar to other flaviviruses, such antibodies are primarily induced by the viral envelope protein E, which consists of three distinct domains (DI, II, and III) and is presented at the surface of mature flavivirions in an icosahedral arrangement. In general, the dominance and individual variation of antibodies to different domains of viral surface proteins and their impact on neutralizing activity are aspects of humoral immunity that are not well understood. To gain insight into these phenomena, we established a platform of immunoassays using recombinant proteins and protein domains that allowed us to dissect and quantify fine specificities of the polyclonal antibody response after YF vaccination in a panel of 51 vaccinees as well as determine their contribution to virus neutralization by serum depletion analyses. Our data revealed a high degree of individual variation in antibody specificities present in post-vaccination sera and differences in the contribution of different antibody subsets to virus neutralization. Irrespective of individual variation, a substantial proportion of neutralizing activity appeared to be due to antibodies directed to complex quaternary epitopes displayed on the virion surface only but not on monomeric E. On the other hand, DIII-specific antibodies (presumed to have the highest neutralizing activity) as well as broadly flavivirus cross-reactive antibodies were absent or present at very low titers. These data provide new information on the fine specificity as well as variability of antibody responses after YF vaccination that are consistent with a strong influence of individual-specific factors on immunodominance in humoral immune responses.

Combination of DNA prime--adenovirus boost immunization with entecavir elicits sustained control of chronic hepatitis B in the woodchuck model.

A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8+ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8+ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4+ and CD8+ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients.

Instructive even after a decade: Complete results of initial virological diagnostics and re-evaluation of molecular data in the German rabies virus "outbreak" caused by transplantations.

In 2005, six patients in Germany received solid organs and both corneas from a donor with an unrecognized rabies infection. Initial virological diagnostics with the machinery available at the two national reference laboratories could quickly clarify the situation. Rabies virus antigen was detected in the organ donor's brain. In two of the three recipients with neurological alterations, intra vitam diagnosis was achieved by conventional RT-PCRs. Comparison of the phylogenetic relatedness of the different viral isolates proved transmission from the donor and, consequently, also established the diagnosis for the third patient. As indicated by the titre of neutralizing antibodies, the liver transplant recipient was protected from the lethal infection due to a vaccination against rabies virus, which he had received more than 15 years ago. All samples from the recipients of the corneas were invariably negative. Re-evaluation of the molecular data by real-time PCR did not lead to an improvement of intra vitam diagnosis but provided intriguing insights regarding the relative amounts of rabies virus RNA in different body fluids and peripheral organs. In saliva and skin, they were 250-200,000 times lower than in the infected patient's brains. Furthermore, in saliva samples taken serially from the same patient fluctuations by a factor of 160-500 were recorded. These findings highlight the problems of intra vitam diagnosis of rabies virus infections and make understandable why the virus can escape from all diagnostic attempts. Finally, in this context one should recall an almost trivial fact: Simple and appropriate postexposure prophylaxis could not only have saved the young organ donor's life but would also have prevented the whole transplantation-associated rabies "outbreak" in Germany.

Enhanced immune response to hepatitis B vaccination through immunization with a Pre-S1/Pre-S2/S vaccine.

Efficacy and safety of recombinant yeast-derived hepatitis B vaccines for prevention of hepatitis B have been demonstrated unequivocally worldwide as reflected in reduction in HBsAg carrier rates and hepatocellular carcinoma. A new generation of recombinant HBV vaccines expressed in mammalian cells containing Pre-S/S epitopes has been developed in several countries. Such vaccines are useful in special risk groups, i.e., in non-responders to conventional HBV vaccines including older adults, obese people, health care workers, patients with renal failure and on dialysis, transplant patients, patients with HIV as well as travelers on short notice to HBV endemic regions. The future of such vaccines depends on their enhanced immunogenicity and cost profile. Sci-B-Vac™ is a mammalian cell-derived recombinant Pre-S1/Pre-S2/S hepatitis B vaccine which has been shown to be highly immunogenic, inducing faster and higher seroprotection rates against HBV with higher anti-HBs levels at lower HBsAg doses as compared to conventional yeast-derived vaccines. Recently, it has been suggested that such Pre-S/S vaccines against HBV might be efficacious not only for prevention but also for intervention in persistent HBV infection. Data obtained in a recent clinical trial conducted in Vietnam in patients with chronic hepatitis B suggest that repeated monthly i.m. injections of the Sci-B-Vac™ co-administered with daily oral lamivudine treatment can suppress HBV replication and lead to anti-HBs seroconversion in ~50 % of treated patients. Optimization of protocols and efficacy of such an intervention, intended to bypass T cell exhaustion and immune tolerance to HBV remains to be explored.

Therapeutic vaccination and immunomodulation in the treatment of chronic hepatitis B: preclinical studies in the woodchuck.

Infection with hepatitis B virus (HBV) may lead to subclinical, acute or chronic hepatitis. In the prevaccination era, HBV infections were endemic due to frequent mother to child transmission in large regions of the world. However, there are still estimated 240 million chronic HBV carriers today and ca. 620,000 patients die per year due to HBV-related liver diseases. Recommended treatment of chronic hepatitis B with interferon-α and/or nucleos(t)ide analogues does not lead to satisfactory results. Induction of HBV-specific T cells by therapeutic vaccination or immunomodulation may be an innovative strategy to overcome virus persistence. Vaccination with commercially available HBV vaccines in patients with or without therapeutic reduction of viral load did not result in effective immune control of HBV infection, suggesting that combination of antiviral treatment with new formulations of therapeutic vaccines is needed. The woodchuck (Marmota monax) and its HBV-like woodchuck hepatitis virus are a useful preclinical animal model for developing new therapeutic approaches in chronic hepadnaviral infections. Several innovative approaches combining antiviral treatments using nucleos(t)ide analogues, with prime-boost vaccination using DNA vaccines, new hepadnaviral antigens or recombinant adenoviral vectors were tested in the woodchuck model. In this review, we summarize these encouraging results obtained with these therapeutic vaccines. In addition, we present potential innovations in immunostimulatory strategies by blocking the interaction of the inhibitory programmed death receptor 1 with its ligand in this animal model.