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The introduction of directly acting antiviral agents (DAAs) has produced significant improvements in the ability to cure chronic hepatitis C infection. However, with over 2% of the world’s population infected with HCV, complications arising from the development of cirrhosis of the liver, chronic hepatitis C infection remains the leading indication for liver transplantation. Several modelling studies have indicated that DAAs alone will not be sufficient to eliminate HCV, but if combined with an effective vaccine this regimen would provide a significant advance towards achieving this critical World Health Organisation goal. We have previously generated a genotype 1a, 1b, 2a, 3a HCV virus like particle (VLP) quadrivalent vaccine. The HCV VLPs contain the core and envelope proteins (E1 and E2) of HCV and the vaccine has been shown to produce broad humoral and T cell immune responses following vaccination of mice. In this report we further advanced this work by investigating vaccine responses in a large animal model. We demonstrate that intradermal microneedle vaccination of pigs with our quadrivalent HCV VLP based vaccine produces long-lived multi-genotype specific and neutralizing antibody (NAb) responses together with strong T cell and granzyme B responses and normal Th1 and Th2 cytokine responses. These responses were achieved without the addition of adjuvant. Our study demonstrates that our vaccine is able to produce broad immune responses in a large animal that, next to primates, is the closest animal model to humans. Our results are important as they show that the vaccine can produce robust immune responses in a large animal model before progressing the vaccine to human trials.

Background: Duration and quality of immunity to SARS-CoV-2 have significant implications for the management of COVID-19 pandemic. Here, we present a comprehensive set of immunological data from a cohort of individuals (n=43), 12 months after mild COVID-19 disease and in the absence of virus re-exposure. Methods: Serum and PBMC were collected from mild-COVID-19 convalescents 12 months after the COVID-19 positive PCR (n=43) and from healthy SARS-CoV-2-seronegative controls (n=15). Serum titers of SARS-CoV-2-specific immunoglobulins were quantified by ELISA and virus neutralisation activity was assessed using SARS-CoV-2-Spike pseudovirus particles. Frequencies of Spike and RBD-specific memory B cells were quantified by flow cytometry. Magnitude of memory T cell responses was quantified and phenotyped with an activation-induced marker assay. Findings: In the absence of re-exposure to SARS-CoV-2 Spike- and RBD-specific antibodies were present in 90% of COVID-19 convalescents 12 months post-infection. RBD-specific IgG + memory B cells were maintained in 88.9% of patients, while 62% of patients had serum neutralising activity. Functionally mature memory CD4 + and CD8 + T cells were maintained at frequencies previously reported for earlier time points post-COVID-19, indicating substantial maintenance of durable T cell responses. Interpretations: Immunity to SARS-CoV-2 persists for 12 months in mild COVID-19 convalescent patients that retain high Spike-specific antibody titres, virus neutralisation capacity and circulating RBD-specific memory B cells. Significantly, T cell immunity remained stable 12 months post-infection. This study offers vital information on the duration of natural COVID-19 immunity and its potential protective effect against SARS-CoV-2 reinfection and clinical disease, with clear implications for the ongoing management of the global pandemic. Funding Statement: This work was funded by project grants from The Hospital Research Foundation and Women’s and Children’s Foundation, Adelaide, Australia. This work has been supported by NIH contract 75N9301900065 (A.S, D.W). Declaration of Interests: A.S. is currently a consultant for Gritstone, Flow Pharma, Arcturus, Epitogenesis, Oxfordimmunotech, Caprion and Avalia. LJI has filed for patent protection for various aspects of T cell epitope and vaccine design work. Authors PGV, CMH, MGM, AELY, HB, ZAM, ZAD, AA, DA, JG, CF, SO, EMM, DJL, GM, EJG, BAJR, DS, CKL, MRB, DW, RAB, SCB and BGB declare no conflict of interest. Ethics Approval Statement: Study protocols were approved by the Central Adelaide Clinical Human Research Ethics Committee (#13050) and the Women’s and Children’s Health Network Human research ethics (protocol HREC/19/WCHN/65), Adelaide, Australia.

A vaccine that induces potent, broad and sustained cell-mediated immunity, resulting in effective memory has the potential to restrict hepatitis C (HCV) virus infection. Early, multi-functional CD4+ and CD8+ T cell responses against non-structural protein 3 (NS3) have been associated with HCV clearance. Necrotic cells generate strong immune responses and represent a major antigenic source used by dendritic cells (DC) for processing and presentation, but there is conflicting evidence as to their immunogenicity in vaccination. Immunization with DC loaded with viral antigens has been done in the past, but to date the immunogenicity of live vs. necrotic DC vaccines has not been investigated. We developed a DC2.4 cell line stably expressing HCV NS3, and compared the NS3-specific responses of live vs. necrotic NS3 DC. Vaccination of mice with necrotic NS3 DC increased the breadth of T-cell responses and enhanced the production of IL-2, TNF-α, and IFN-γ by effector memory CD4+ and CD8+T cells, compared to mice vaccinated with live NS3 DC. A single dose of necrotic NS3 DC vaccine induced a greater influx and activation of cross-presenting CD11c+ CD8α+ DC and necrosis-sensing Clec9A+ DC in the draining lymph nodes. Furthermore, using a hydrodynamic challenge model necrotic NS3 DC vaccination resulted in enhanced clearance of NS3-positive hepatocytes from the livers of vaccinated mice. Taken together, the data demonstrate that necrotic DC represent a novel and exciting vaccination strategy capable of inducing broad and multifunctional T cell memory.

A novel T cell–based ZIKV vaccine, encoding NS1 protein, confers protection against systemic infection., The causal association of Zika virus (ZIKV) with microcephaly, congenital malformations in infants, and Guillain-Barré syndrome in adults highlights the need for effective vaccines. Thus far, efforts to develop ZIKV vaccines have focused on the viral envelope. ZIKV NS1 as a vaccine immunogen has not been fully explored, although it can circumvent the risk of antibody-dependent enhancement of ZIKV infection, associated with envelope antibodies. Here, we describe a novel DNA vaccine encoding a secreted ZIKV NS1, that confers rapid protection from systemic ZIKV infection in immunocompetent mice. We identify novel NS1 T cell epitopes in vivo and show that functional NS1-specific T cell responses are critical for protection against ZIKV infection. We demonstrate that vaccine-induced anti-NS1 antibodies fail to confer protection in the absence of a functional T cell response. This highlights the importance of using NS1 as a target for T cell–based ZIKV vaccines.

Hepatitis C virus (HCV) is a significant contributor to the global disease burden, and development of an effective vaccine is required to eliminate HCV infections worldwide. CD4(+) and CD8(+) T cell immunity correlates with viral clearance in primary HCV infection, and intrahepatic CD8(+) tissue-resident memory T (T(RM)) cells provide lifelong and rapid protection against hepatotropic pathogens. Consequently, we aimed to develop a vaccine to elicit HCV-specific CD4(+) and CD8(+) T cells, including CD8(+) T(RM) cells, in the liver, given that HCV primarily infects hepatocytes. To achieve this, we vaccinated wild-type BALB/c mice with a highly immunogenic cytolytic DNA vaccine encoding a model HCV (genotype 3a) nonstructural protein (NS5B) and a mutant perforin (pVAX-NS5B-PRF), as well as a recombinant adeno-associated virus (AAV) encoding NS5B (rAAV-NS5B). A novel fluorescent target array (FTA) was used to map immunodominant CD4(+) T helper (T(H)) cell and cytotoxic CD8(+) T cell epitopes of NS5B in vivo, which were subsequently used to design a K(d)NS5B(451-459) tetramer and analyze NS5B-specific T cell responses in vaccinated mice in vivo. The data showed that intradermal prime/boost vaccination with pVAX-NS5B-PRF was effective in eliciting T(H) and cytotoxic CD8(+) T cell responses and intrahepatic CD8(+) T(RM) cells, but a single intravenous dose of hepatotropic rAAV-NS5B was significantly more effective. As a T-cell-based vaccine against HCV should ideally result in localized T cell responses in the liver, this study describes primary observations in the context of HCV vaccination that can be used to achieve this goal. IMPORTANCE There are currently at least 71 million individuals with chronic HCV worldwide and almost two million new infections annually. Although the advent of direct-acting antivirals (DAAs) offers highly effective therapy, considerable remaining challenges argue against reliance on DAAs for HCV elimination, including high drug cost, poorly developed health infrastructure, low screening rates, and significant reinfection rates. Accordingly, development of an effective vaccine is crucial to HCV elimination. An HCV vaccine that elicits T cell immunity in the liver will be highly protective for the following reasons: (i) T cell responses against nonstructural proteins of the virus are associated with clearance of primary infection, and (ii) long-lived liver-resident T cells alone can protect against malaria infection of hepatocytes. Thus, in this study we exploit promising vaccination platforms to highlight strategies that can be used to evoke highly functional and long-lived T cell responses in the liver for protection against HCV.

DNA vaccines present one of the most cost-effective platforms to develop global vaccines, which have been tested for nearly three decades in preclinical and clinical settings with some success in the clinic. However, one of the major challenges for the development of DNA vaccines is their poor immunogenicity in humans, which has led to refinements in DNA delivery, dosage in prime/boost regimens and the inclusion of adjuvants to enhance their immunogenicity. In this review, we focus on adjuvants that can enhance the immunogenicity of DNA encoded antigens and highlight the development of a novel cytolytic DNA platform encoding a truncated mouse perforin. The application of this innovative DNA technology has considerable potential in the development of effective vaccines.

DNA vaccines are ideal candidates for global vaccination purposes because they are inexpensive and easy to manufacture on a large scale such that even people living in low-income countries can benefit from vaccination. However, the potential of DNA vaccines has not been realized owing mainly to the poor cellular uptake of DNA in vivo resulting in the poor immunogenicity of DNA vaccines. In this review, we discuss the benefits and shortcomings of several promising and innovative non-biological methods of DNA delivery that can be used to increase cellular delivery and efficacy of DNA vaccines.

A method for the large-scale production of a quadrivalent mammalian cell derived hepatitis C virus like particles (HCV VLPs) is described. The HCV core El and E2 coding sequences of genotype 1a, 1b, 2a or 3a were co-expressed in Huh7 cell factories using a recombinant adenoviral expression system. The structural proteins self-assembled into VLPs that were purified from Huh7 cell lysates by iodixanol ultracentrifugation and Stirred cell ultrafiltration. Electron microscopy, revealed VLPs of the different genotypes that are morphologically similar. Our results show that it is possible to produce large quantities of individual HCV genotype VLPs with relative ease thus making this approach an alternative for the manufacture of a quadrivalent mammalian cell derived HCV VLP vaccine. (C) 2016 Published by Elsevier B.V.

Human immunodeficiency virus (HIV)-1 and hepatitis C virus (HCV) are major contributors to the global disease burden with many experts recognizing the requirement of an effective vaccine to bring a durable end to these viral epidemics. The most promising vaccine candidates that have advanced into pre-clinical models and the clinic to eliminate or provide protection against these chronic viruses are viral vectors [e.g., recombinant cytomegalovirus, Adenovirus, and modified vaccinia Ankara (MVA)]. This raises the question, is there a need to develop DNA vaccines against HIV-1 and HCV? Since the initial study from Wolff and colleagues which showed that DNA represents a vector that can be used to express transgenes durably in vivo, DNA has been regularly evaluated as a vaccine vector albeit with limited success in large animal models and humans. However, several recent studies in Phase I-IIb trials showed that vaccination of patients with recombinant DNA represents a feasible therapeutic intervention to even cure cervical cancer, highlighting the potential of using DNA for human vaccinations. In this review, we will discuss the limitations and the strategies of using DNA as a vector to develop prophylactic T cell-mediated vaccines against HIV-1 and HCV. In particular, we focus on potential strategies exploiting DNA vectors to elicit protective localized CD8+ T cell immunity in the liver for HCV and in the cervicovaginal mucosa for HIV-1 as localized immunity will be an important, if not critical component, of an efficacious vaccine against these viral infections.

A universal hepatitis C virus (HCV) vaccine should elicit multiantigenic, multigenotypic responses, which are more likely to protect against challenge with the range of genotypes and subtypes circulating in the community. A vaccine cocktail and vaccines encoding consensus HCV sequences are attractive approaches to achieve this goal. Consequently, in a series of mouse vaccination studies, we compared the immunogenicity of a DNA vaccine encoding a consensus HCV nonstructural 5B (NS5B) protein to that of a cocktail of DNA plasmids encoding the genotype 1b (Gt1b) and Gt3a NS5B proteins. To complement this study, we assessed responses to a multiantigenic cocktail regimen by comparing a DNA vaccine cocktail encoding Gt1b and Gt3a NS3, NS4, and NS5B proteins to a single-genotype NS3/4/5B DNA vaccine. To thoroughly evaluate in vivo cytotoxic T lymphocyte (CTL) and T helper (Th) cell responses against Gt1b and Gt3a HCV peptide-pulsed target cells, we exploited a novel fluorescent-target array (FTA). FTA and enzyme-linked immunosorbent spot (ELISpot) analyses collectively indicated that the cocktail regimens elicited higher responses to Gt1b and Gt3a NS5B proteins than those with the consensus vaccine, while the multiantigenic DNA cocktail significantly increased the responses to NS3 and NS5B compared to those elicited by the single-genotype vaccines. Thus, a DNA cocktail vaccination regimen is more effective than a consensus vaccine or a monovalent vaccine at increasing the breadth of multigenotypic T cell responses, which has implications for the development of vaccines for communities where multiple HCV genotypes circulate. IMPORTANCE Despite the development of highly effective direct-acting antivirals (DAA), infections with hepatitis C virus (HCV) continue, particularly in countries where the supply of DAA is limited. Furthermore, patients who eliminate the virus as a result of DAA therapy can still be reinfected. Thus, a vaccine for HCV is urgently required, but the heterogeneity of HCV strains makes the development of a universal vaccine difficult. To address this, we developed a novel cytolytic DNA vaccine which elicits robust cell-mediated immunity (CMI) to the nonstructural (NS) proteins in vaccinated animals. We compared the immune responses against genotypes 1 and 3 that were elicited by a consensus DNA vaccine or a DNA vaccine cocktail and showed that the cocktail induced higher levels of CMI to the NS proteins of both genotypes. This study suggests that a universal HCV vaccine can most readily be achieved by use of a DNA vaccine cocktail.

The development of an effective preventative hepatitis C virus (HCV) vaccine will reside, in part, in its ability to elicit neutralizing antibodies (NAbs). We previously reported a genotype 1a HCV virus like particle (VLP) vaccine that produced HCV specific NAb and T cell responses that were substantially enhanced by Toll-like receptor 2 (TLR2) agonists. We have now produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine and tested the ability of two TLR2 agonists, R4Pam2Cys and E8Pam2Cys, to stimulate the production of NAb. We now show that our vaccine with R4Pam2Cys or E8Pam2Cys produces strong antibody and NAb responses in vaccinated mice after just two doses. Total antibody titers were higher in mice inoculated with vaccine plus E8Pam2Cys compared to HCV VLPs alone. However, the TLR2 agonists did not result in stronger NAb responses compared to vaccine without adjuvant. Such a vaccine could provide a substantial addition to the overall goal to eliminate HCV.

Hepatitis C Virus (HCV) infects 2% of the world’s population and is the leading cause of liver disease and liver transplantation. It poses a serious and growing worldwide public health problem that will only be partially addressed with the introduction of new antiviral therapies. However, these treatments will not prevent re-infection particularly in high risk populations. The introduction of a HCV vaccine has been predicted, using simulation models in a high risk population, to have a significant effect on reducing the incidence of HCV. A vaccine with 50 to 80% efficacy targeted to high-risk intravenous drug users could dramatically reduce HCV incidence in this population. Virus like particles (VLPs) are composed of viral structural proteins which self-assemble into non-infectious particles that lack genetic material and resemble native viruses. Thus, VLPs represent a safe and highly immunogenic vaccine delivery platform able to induce potent adaptive immune responses. Currently, many VLP-based vaccines have entered clinical trials, while licensed VLP vaccines for hepatitis B virus (HBV) and human papilloma virus (HPV) have been in use for many years. The HCV core, E1 and E2 proteins can self-assemble into immunogenic VLPs while inclusion of HCV antigens into heterogenous (chimeric) VLPs is also a promising approach. These VLPs are produced using different expression systems such as bacterial, yeast, mammalian, plant, or insect cells. Here, this paper will review HCV VLP-based vaccines and their immunogenicity in animal models as well as the different expression systems used in their production.

The significant public health problem of Hepatitis C virus (HCV) has been partially addressed with the advent of directly acting antiviral agents (DAAs). However, the development of an effective preventative vaccine would have a significant impact on HCV incidence and would represent a major advance towards controlling and possibly eradicating HCV globally. We previously reported a genotype 1a HCV viral-like particle (VLP) vaccine that produced neutralizing antibodies (NAb) and T cell responses to HCV. To advance this approach, we produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine to produce broader immune responses. We show that this quadrivalent vaccine produces antibody and NAb responses together with strong T and B cell responses in vaccinated mice. Moreover, selective neutralizing human monoclonal antibodies (HuMAbs) targeting conserved antigenic domain B and D epitopes of the E2 protein bound strongly to the HCV VLPs, suggesting that these critical epitopes are expressed on the surface of the particles. Our findings demonstrate that a quadrivalent HCV VLP based vaccine induces broad humoral and cellular immune responses that will be necessary for protection against HCV. Such a vaccine could provide a substantial addition to highly active antiviral drugs in eliminating HCV.

Human immunodeficiency virus (HIV)-1 has infected >75 million individuals globally, and, according to the UN, is responsible for ~2.1 million new infections and 1.1 million deaths each year. Currently, there are ~37 million individuals with HIV infection and the epidemic has already resulted in 35 million deaths. Despite the advances of anti-retroviral therapy (ART), a cost-effective vaccine remains the best long-term solution to end the HIV-1 epidemic especially given that the vast majority of infected individuals live in poor socio-economic regions of the world such as Sub-Saharan Africa which limits their accessibility to ART. The modest efficacy of the RV144 Thai trial provides hope that a vaccine for HIV-1 is possible, but as markers for sterilizing immunity are unknown, the design of an effective vaccine is empirical, although broadly cross-reactive neutralizing antibodies (bNAb) that can neutralize various quasispecies of HIV-1 are considered crucial. Since HIV-1 transmission often occurs at the genito-rectal mucosa and is cell-associated, there is a need to develop vaccines that can elicit CD8+ T cell immunity with the capacity to kill virus infected cells at the genito-rectal mucosa and the gut. Here we discuss the recent progress made in developing T cell-mediated vaccines for HIV-1 and emphasize the need to elicit mucosal tissue-resident memory CD8+ T (CD8+ Trm) cells. CD8+ Trm cells will likely form a robust front-line defense against HIV-1 and eliminate transmitter/founder virus-infected cells which are responsible for propagating HIV-1 infections following transmission in vast majority of cases.

Traditional vaccine strategies that induce antibody responses have failed to protect against HIV infection in clinical trials, and thus cell-mediated immunity is now an additional criterion. Recent clinical trials that aimed to induce strong T cell responses failed to do so. Therefore, to enhance induction of protective T cell responses, it is crucial that the optimum antigen combination is chosen. Limited research has been performed into the number of antigens selected for an HIV vaccine. This study aimed to compare DNA vaccines encoding either a single HIV antigen or a combination of two antigens, using intradermal vaccination of C57BL/6 mice. Immune assays were performed on splenocytes, and in vivo protection was examined by challenge with a chimeric virus, EcoHIV, able to infect mouse but not human leukocytes, at 10 days (short term) and 60 days (long term) post final vaccination. At 60 days there was significantly lower frequency of induced antigen-specific CD8+ T cells in the spleens of pCMVgag-pol-vaccinated mice compared with mice which received pCMVgag only. Most importantly, short term viral control of EcoHIV was similar for pCMVgag and pCMVgag-pol-vaccinated mice at day 10, but only the pCMVgag-vaccinated significantly controlled EcoHIV at day 60 compared with pCMV-vaccinated mice, showing that control was reduced with the inclusion of the HIV pol gene. Refereed/Peer-reviewed

In humans, DNA vaccines have failed to demonstrate the equivalent levels of immunogenicity that were shown in smaller animals. Previous studies have encoded adjuvants, predominantly cytokines, within these vaccines in an attempt to increase antigen-specific immune responses. However, these strategies have lacked breadth of innate immune activation and have led to disappointing results in clinical trials. Damage associated molecular patterns (DAMPs) have been identified as pattern recognition receptor (PRR) agonists. DAMPs can bind to a wide range of PRRs on dendritic cells (DCs) and thus our studies have aimed to utilize this characteristic to act as an adjuvant in a DNA vaccine approach. Specifically, HSP70 has been identified as a DAMP, but has been limited by its lack of accessibility to PRRs in and on DCs. Here, we discuss the promising results achieved with the inclusion of membrane-bound or secreted HSP70 into a DNA vaccine encoding HIV gag as the model immunogen. Refereed/Peer-reviewed

An effective hepatitis C virus (HCV) vaccine should elicit robust humoral and cell mediated immunity (CMI). A small animal challenge model is required to assess the efficacy of vaccines which elicit CMI. In this study, HCV proteins were expressed in hepatocytes of immunocompetent mice after hydrodynamic injection of a plasmid encoding the HCV NS3/4A protein. This vector, constructed as the "challenge", was optimized for long term, specific gene expression in hepatocytes. To monitor HCV antigen expression in transfected hepatocytes, the plasmid also encoded secreted alkaline phosphatase (SEAP), which was detected in the mouse serum. The design of this novel challenge plasmid was based on studies using luciferase and SEAP as reporter molecules to examine the kinetics of the proteins expressed in hepatocytes and secreted into blood. We designed two constructs to control SEAP expression. In one construct, SEAP expression was controlled by the EMCV IRES, while in the other, a SEAP and luciferase polyprotein was cleaved by the FMDV2A proteinase. We found that SEAP expressed after FMDV2A self cleavage was more sensitive and showed a higher correlation with luciferase expressed in liver. The NSIMA challenge model using the FMDV2A design provided a window period of 50 days to monitor changes in SEAP expression after hydrodynamic injection of DNA. In a challenge experiment, mice which received an adenovirus-based HCV vaccine showed accelerated clearance of SEAP and thus, of NS3/4A positive hepatocytes compared with a mock vaccinated group, that coincided with an increased number of CD8(+) lymphocytes in the liver. Refereed/Peer-reviewed

Expression vectors that are based on live human rhinoviruses (HRVs) are attractive, yet often overlooked in vaccine development due to their limited capacity for foreign gene inserts and poor genetic stability. This chapter describes a novel methodology to engineer a replication-competent genetically stable recombinant HRV (rHRV) without affecting viral replication capability. We have previously used these methods to generate live, genetically stable recombinant HRVs encoding HIV Gag and Tat proteins (rHRV-Gag-Tat), a potential mucosally targeted HIV vaccine.

Mucosal immunity is deemed crucial to control sexual transmission of human immunodeficiency virus (HIV). Herein we report the efficacy of a mucosal HIV vaccine strategy comprising intranasal (IN) vaccination with a cocktail of live recombinant human rhinoviruses (HRVs) encoding overlapping fragments of HIV Gag and full length Tat (rHRV-Gag/Tat) followed by intradermal (ID) vaccination with DNA vaccines encoding HIV Gag and Tat (pVAX-Gag-Tat). This heterologous prime-boost strategy will be referred to hereafter as rHRV-DNA. As a control, IN vaccination with wild type (wt)-HRV-A1 followed by a single ID dose of pVAX (wt-HRV-A1/pVAX vaccination) was included. rHRV-DNA vaccination elicited superior multi-functional CD8+T cell responses in lymphocytes harvested from mesenteric lymph nodes and spleens, and higher titres of Tat-specific antibodies in blood and vaginal lavages, and reduced the viral load more effectively after challenge with EcoHIV, a murine HIV challenge model, in peritoneal macrophages, splenocytes and blood compared compared with wt-HRV-A1/pVAX vaccination or administration of 3 ID doses of pVAX-Gag-Tat (3X pVAX-Gag-Tat vaccination). These data provide the first evidence that a rHRV-DNA vaccination regimen can induce HIV-specific immune responses in the gut, vaginal mucosa and systemically, and supports further testing of this regimen in the development of an effective mucosally-targeted HIV-1 vaccine.

Summary The p7 protein of hepatitis C virus (HCV) is a small, integral membrane protein that plays a critical role in virus replication. Recently, we reported two intergenotypic JFH1 chimeric viruses encoding the partial or full-length p7 protein of the HCV-A strain of genotype 1b (GT1b; Virology; 2007; 360:134). In this study, we determined the consensus sequences of the entire polyprotein coding regions of the wild-type JFH1 and the revertant chimeric viruses and identified predominant amino acid substitutions in core (K74M), NS2 (T23N, H99P) and NS5A (D251G). Forward genetic analysis demonstrated that all single mutations restored the infectivity of the defective chimeric genomes suggesting that the infectious virus production involves the association of p7 with specific regions in core, NS2 and NS5A. In addition, it was demonstrated that the NS2 T23N facilitated the generation of infectious intergenotypic chimeric virus encoding p7 from GT6 of HCV.

The p7 protein of hepatitis C virus (HCV) is a viroporin that is dispensable for viral genome replication but plays a critical role in virus morphogenesis. In this study, we generated a JFH1-based intergenotypic chimeric genome that encoded a heterologous genotype 1b (GT1b) p7. The parental intergenotypic chimeric genome was nonviable in human hepatoma cells, and infectious chimeric virions were produced only when cells transfected with the chimeric genomes were passaged several times. Sequence analysis of the entire polyprotein-coding region of the recovered chimeric virus revealed one predominant amino acid substitution in nonstructural protein 2 (NS2), T23N, and one in NS5B, K151R. Forward genetic analysis demonstrated that each of these mutations per se restored the infectivity of the parental chimeric genome, suggesting that interactions between p7, NS2, and NS5B were required for virion assembly/maturation. p7 and NS5B colocalized in cellular compartments, and the NS5B mutation did not affect the colocalization pattern. The NS5B K151R mutation neither increased viral RNA replication in human hepatoma cells nor altered the polymerase activity of NS5B in an in vitro assay. In conclusion, this study suggests that HCV NS5B is involved in virus morphogenesis.

The NS2 protein of hepatitis C virus (HCV) plays a critical role in virus morphogenesis and infectivity. The crystal structure of the C-terminus of the NS2 protein (NS2(Pro)) from the H77 strain indicates that NS2(Pro) forms a homodimer. In this study, using computational modelling, we identified residues at the NS2(Pro) dimer interface that have a role in dimerization and confirmed their capacity to influence dimerization by expression studies. Our modelling analysis identified 22 residues at the NS2(Pro) dimer interface that may be important for dimer formation. Based on the free binding energy, we selected the top five ranked mutations (V162A, M170A, I175A, D186A and I201A) for further study. Western blot analysis revealed that M170A, I175A, I201A, D186A and V162A resulted in a 4.0-, 3.2-, 3.0-, 2.8- and 1.5-fold increase, respectively, in the monomer/dimer ratio compared to wild type, confirming a role in homodimer formation or stability. Japanese Fulminant Hepatitis type 1 mutants expressing M170A, I175A, D186A and I201A demonstrated increasing defects in both RNA replication and the production of infectious virus compared to wild type. This study identified residues at the NS2(Pro) dimer interface that modulate NS2(Pro) homodimerization and demonstrated that abrogation of NS2(Pro) homodimerization results in defects in HCV replication and release of infectious virus.

The transmembrane domains (TMDs) of dengue virus type-1 M protein (DENV-1M) were reported to form cation-selective channels in artificial lipid bilayers. We further explored this observation using the two-electrode voltage clamp (TEVC) method on the Xenopus laevis oocytes expressing DENV PrM and M proteins. Using myc epitope tagged M proteins, M was first shown to adopt its predicted native topology in mammalian cells when expressed on its own. The recombinant proteins were then successfully expressed on the surface of Xenopus oocytes. Using influenza A M2 (Inf A/M2) protein as a control, we measured the conductance of oocytes expressing DENV proteins under hyperpolarized or low-pH conditions. Inf A/M2 showed pH-dependent, amantadine-sensitive channel activity that was consistent with previously published reports. However, no activity was detected for DENV proteins. We conclude that DENV PrM and M proteins do not show pH-activated ion channel activity.

GB virus B (GBV-B) is a hepatotropic virus that is closely related to hepatitis C virus (HCV). GBV-B causes acute hepatitis in infected marmosets and tamarins and is therefore a useful small-animal model for the study of HCV. We investigated virus-specific T-cell responses in marmosets infected with GBV-B. Gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay responses in the peripheral blood of two marmosets were assessed throughout the course of GBV-B infection. These T-cell responses were directed against the GBV-B nonstructural proteins 3 (NS3), 4A (NS4A), and 5B (NS5B), and their appearance was temporally associated with clearance of viremia. These marmosets were then rechallenged with GBV-B at least 3 months after clearance of the primary infection to determine if the animals were protected from reinfection. There was no detectable viremia following reinfection, although a sharp increase in T-cell responses against GBV-B proteins was observed. Epitope mapping of T-cell responses to GBV-B was performed with liver and blood samples from both marmosets after rechallenge with GBV-B. Three shared, immunodominant T-cell epitopes within NS3 were identified in animals with multiple common major histocompatibility complex class I alleles. IFN-γ ELISPOT responses were also detected in the livers of two marmosets that had resolved a primary GBV-B infection. These responses were high in frequency and were directed against epitopes within GBV-B NS3, NS4A, and NS5B proteins. These results indicate that virus-specific T-cell responses are detectable in the liver and blood of GBV-B-infected marmosets and that the clearance of GBV-B is associated with the appearance of these responses.

An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8(+) and class II CD4(+) T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm(-3). The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8(+) T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8(+) T-cell responses and will have important implications for the development of a preventative vaccine for HCV.

Chronic hepatitis C virus (HCV) infection represents a major health threat to global population. In India, approximately 15-20% of cases of chronic liver diseases are caused by HCV infection. Although, new drug treatments hold great promise for HCV eradication in infected individuals, the treatments are highly expensive. A vaccine for preventing or treating HCV infection would be of great value, particularly in developing countries. Several preclinical trials of virus-like particle (VLP) based vaccine strategies are in progress throughout the world. Previously, using baculovirus based system, we have reported the production of hepatitis C virus-like particles (HCV-LPs) encoding structural proteins for genotype 3a, which is prevalent in India. In the present study, we have generated HCV-LPs using adenovirus based system and tried different immunization strategies by using combinations of both kinds of HCV-LPs with other genotype 3a-based immunogens. HCV-LPs and peptides based ELISAs were used to evaluate antibody responses generated by these combinations. Cell-mediated immune responses were measured by using T-cell proliferation assay and intracellular cytokine staining. We observed that administration of recombinant adenoviruses expressing HCV structural proteins as final booster enhances both antibody as well as T-cell responses. Additionally, reduction of binding of VLP and JFH1 virus to human hepatocellular carcinoma cells demonstrated the presence of neutralizing antibodies in immunized sera. Taken together, our results suggest that the combined regimen of VLP followed by recombinant adenovirus could more effectively inhibit HCV infection, endorsing the novel vaccine strategy. (C) 2015 Elsevier Ltd. All rights reserved.

Background/Aims Chronic infection with the hepatitis B virus (HBV) is strongly associated with the development of hepatocellular carcinoma but the mechanism by which this occurs is unknown. Numerous studies have focused on the HBV X protein showing that it activates signal transduction pathways while few have investigated these changes in HBV-replicating hepatocytes. Methods We utilized the recombinant adenovirus system to deliver a replication competent HBV genome into Huh7 and primary marmoset hepatocytes (PMH) to examine the effects of active viral replication on the regulation of Ras-ERK signal transduction and related pathways. Results Huh7 cells and PMHs replicating HBV demonstrated significant upregulation in phosphorylated ERK, Akt, c-myc together with increased p53, cyclin B1 and p21 cip1 expression and cell cycle progression to G2 phase in the absence of increased cell proliferation. Phosphorylation of the key cell survival kinase, Akt, was significantly increased, resulting in increased serine phosphorylation of the downstream target, GSK3-β. Conclusions These results demonstrated simultaneous activation of the MAP Kinase and Akt pathways in HBV-replicating hepatocytes that resulted in dysregulation in the control of cell cycle progression and which help explain the early pathogenic mechanisms that underlie malignant transformation associated with chronic hepatitis B infection.

The p7 protein of hepatitis C virus (HCV) functions as an ion channel in planar lipid bilayers, and its function is vital for the virus life cycle. In this study, we replaced either the entire or partial p7 of genotype 2a (strain JFH1), an HCV strain that replicates and produces virus progeny in vitro, with the corresponding regions of the p7 protein from genotype 1b (Australian isolate, HCV-A). Compared to wild type, the chimeric viruses reached their peak of infectivity with a delay but they produced a comparable titer to the wild type virus and the progeny viruses were able to infect naive permissive cells. Amantadine treatment of wild type and chimeric viruses reduced the virus titers by about 50% and 45%, respectively. Therefore, in this study, for the first time, we demonstrated that genotype 2a (JFH1 strain) genome encoding a full-length genotype 1b p7 gene produces infectious particles in vitro. These chimeric viruses are valuable instruments for comparative studies of the p7 proteins.

Two GB virus B (GBV-B) chimeric genomes, GBV-HVR and GBV-HVRh (with a hinge), containing the coding region of the immunodominant hypervariable region 1 (HVR1) of the E2 envelope protein of Hepatitis C virus (HCV) were constructed. Immunoblot analysis confirmed that HVR1 was anchored to the GBV-B E2 protein. To investigate the replication competence and in vivo stability of in vitro-generated chimeric RNA transcripts, two naïve marmosets were inoculated intrahepatically with the transcripts. The GBV-HVR chimeric genome was detectable for 2 weeks post-inoculation (p.i.), whereas GBV-HVRh reverted to wild type 1 week p.i. Sequencing analysis of the HVR1 and flanking regions from GBV-HVR RNA isolated from marmoset serum demonstrated that the HVR1 insert remained unaltered in the GBV-HVR chimera for 2 weeks. Inoculation of a naïve marmoset with serum collected at 1 week p.i. also resulted in viraemia and confirmed that the serum contained infectious particles. All animals cleared the infection by 3 weeks p.i. and remained negative for the remaining weeks. The chimera may prove useful for the in vivo examination of any HCV HVR1-based vaccine candidates.

The mechanism behind the apparent lack of effective antiviral immune responses in chronic hepatitis C virus (HCV) patients is poorly understood. It remains unclear if natural regulatory T cells (Treg) contribute to the induction and maintenance of HCV persistence. We herein report for the first time that CD25(high)IFN-gamma(-)FOXP3(high) Tregs can be rapidly induced by culturing peripheral blood mononuclear cells (PBMCs) of HCV-positive patients with HCV protein-derived peptides. The HCV-specific Tregs, generally CD4(+)CD45RO(+), did not proliferate in response to HCV peptide and failed to produce interferon (IFN)-gamma, in distinct contrast to antiviral effector cells. Stimulation of healthy donor PBMCs with HCV peptides did not result in CD25 and FOXP3 upregulation above non-antigen background. To further investigate the antigen specificity of these potentially disease-associated natural Tregs, CD25(+) cells were isolated from PBMCs, labeled with carboxyfluorescein diacetate succinimidylester and added back to CD25-depleted PBMCs, and the co-cultures were then stimulated with individual peptides derived from the HCV core protein. We found that the actual peptide that can stimulate Treg varied between patients, but within any given subject only a small number of the peptides were able to stimulate Treg, suggesting the existence of dominant Treg epitopes. Although functional experiments for these peptides are ongoing in our laboratory, data presented here suggests that HCV-specific natural Tregs are abundant in infected individuals, in contrast to the extremely low frequency of anti-HCV effector T cells, supporting the view that natural Treg may be implicated in host immune tolerance during HCV infection.

Before carrying out a clinical trial in humans in which a cell-based therapeutic anti-hepatitis C virus lipopeptide vaccine candidate is to be evaluated, a limited toxicological study was carried out. Murine bone marrow-derived dendritic cells (DCs) were loaded with lipopeptides containing HLA A2.1-restricted epitopes recognised by cytotoxic T lymphocytes (CTL) and then injected into C57BL6 mice by intradermal and intravenous routes. No significant behavioural changes, clinical symptoms or changes in body weight were observed when compared with a control group of animals receiving no treatment. One week after the third dose of lipopeptide-pulsed DC, mice were killed and blood samples taken for biochemical and hematological analyses. The liver, spleen and skin at the injection site were also collected and processed for histological analysis. Mild eosinophilia was observed at intradermal injection sites of animals receiving untreated as well as lipopeptide-loaded DCs. Despite a slight decrease in the size of livers of animals receiving lipopeptide-pulsed DCs, there was no evidence of inflammatory infiltrate or histological change. The only biochemical or hematological abnormality associated with the injection of lipopeptide-pulsed DC was a slight reduction in potassium levels. The evidence indicates that the lipopeptide vaccines per se are not cytotoxic and do not induce adverse events. On this basis, the TGA has granted clinical trial by exemption (CTX) approval for the proposed study using HCV lipopeptide-pulsed autologous DC to proceed in humans. This is the first approval of its kind in Australia setting a precedent for somatic cell immunotherapy of infectious disease.

Changes in the hepatitis C virus (HCV) viral load (VL) were assessed in a retrospective study of 50 HIV/HCV-coinfected patients who initiated highly active antiretroviral therapy (HAART). Most patients responded to HAART [during the first 6 months, plasma HIV VL fell by a mean 1.39 log10, becoming undetectable (10 that was associated with a rapid decrease in the HIV VL, whereas an increase in the HCV VL of greater than 1 log10, noted in eight patients, was associated with a baseline CD4+ cell count of less than 200 cells/μl. The increase in the HCV VL was not associated with hepatitis as determined by raised alanine transferase.

Although hepatitis C virus (HCV) is classified in the Hepacivirus genus in the family Flaviviridae, it is unlike most of the other members of this family due to its propensity to cause persistent infections. This persistent infection eventually results in chronic liver disease, cirrhosis and hepatocellular carcinoma in a proportion of infected individuals. It has been difficult to examine correlates of clearance or persistence because most acute phase HCV infections are subclinical or result in symptoms which are non-specific; consequently, acute infections are not generally recognised and patients often present many years later with persistent infection and accompanying chronic liver disease. Nevertheless, seminal studies, performed during the acute phase, have identified a number of factors which are likely to influence the outcome of infection, although it is possible that the mechanism is multifactorial. One of these factors is impairment of dendritic cell function by a mechanism resulting from expression of an HCV protein(s) in these cells. This may be a major factor in the failure of the immune response to expand after HCV infection, leading to persistence. Nevertheless, it may be possible to overcome this defect by autologous transfusion of HCV antigen-loaded, mature dendritic cells and the purpose of this review is to highlight the need and general approaches for developing dendritic cell-based immunotherapy for HCV infection.

Due to their spatial structure virus-like particles (VLPs) generally induce effective immune responses. VLPs derived from the small envelope protein (HBsAg-S) of hepatitis B virus (HBV) comprise the HBV vaccine. Modified HBsAs-S VLPs, carrying the immunodominant hypervariable region (HVR1) of the hepatitis C virus (HCV) envelope protein E2 within the exposed 'a'-determinant region (HBsAg/HVR1-VLPs), elicited HVR1-specific antibodies in mice. A high percentage of the human population is positive for anti-HBsAg antibodies (anti-HBs), either through vaccination or natural infection. We, therefore, determined if pre-existing anti-HBs could influence immunisation with modified VLPs. Mice were immunised with a commercial HBV vaccine, monitored to ensure an anti-HBs response, then immunised with HBsAg/HVR1-VLPs. The resulting anti-HVR1 antibody titre was similar in mice with or without pre-existing anti-HBs. This suggests that HBsAg/HVR1-VLPs induce a primary immune response to HVR1 in anti-HBs positive mice and, hence, they may be used successfully in individuals already immunised with the HBV vaccine.

Cellular immune responses are critical for the clearance of hepatitis C virus. Persistent infection results from a narrow and weak cellular immune response, in direct contrast to the broad, strong response associated with viral clearance in acute infection. The presence of dendritic cells in the liver facilitates presentation of viral antigens to both CD4+ and CD8+ T cell populations. Exploiting the potent antigen presentation capability of dendritic cells for immunotherapy of chronic hepatitis C is attractive; however, infection or transfection of segments or the entire hepatitis C virus genome appears to impair the allostimulation capacity of dendritic cells. If dendritic cell immunotherapy for hepatitis C virus infection is to become a reality, the mechanism behind the defective allostimulatory capacity needs to be deciphered.

Herein we describe the construction of recombinant human rhinoviruses (rHRVs) encoding HIV Gag or Tat by inserting the full length tat gene or regions of the gag gene flanked by sequences encoding the HRV 2A protease cleavage site into the junction between HRV genes encoding structural (P1) and nonstructural (P2) proteins. Most recombinants were unstable, but this was corrected by mutation of the flanking cleavage sites. Thereafter, all rHRV constructs retained the inserts throughout six passages. Such constructs may find utility as vaccine vectors to generate mucosal immunity. Refereed/Peer-reviewed

There are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising “multiantigen” vaccine that elicits robust CMI. IMPORTANCE Since their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans.

An effective hepatitis C virus (HCV) vaccine should elicit robust humoral and cell mediated immunity (CMI). A small animal challenge model is required to assess the efficacy of vaccines which elicit CMI. In this study, HCV proteins were expressed in hepatocytes of immunocompetent mice after hydrodynamic injection of a plasmid encoding the HCV NS3/4A protein. This vector, constructed as the "challenge", was optimized for long term, specific gene expression in hepatocytes. To monitor HCV antigen expression in transfected hepatocytes, the plasmid also encoded secreted alkaline phosphatase (SEAP), which was detected in the mouse serum. The design of this novel challenge plasmid was based on studies using luciferase and SEAP as reporter molecules to examine the kinetics of the proteins expressed in hepatocytes and secreted into blood. We designed two constructs to control SEAP expression. In one construct, SEAP expression was controlled by the EMCV IRES, while in the other, a SEAP and luciferase polyprotein was cleaved by the FMDV2A proteinase. We found that SEAP expressed after FMDV2A self cleavage was more sensitive and showed a higher correlation with luciferase expressed in liver. The NS3/4A challenge model using the FMDV2A design provided a window period of 50 days to monitor changes in SEAP expression after hydrodynamic injection of DNA. In a challenge experiment, mice which received an adenovirus-based HCV vaccine showed accelerated clearance of SEAP and thus, of NS3/4A positive hepatocytes compared with a mock vaccinated group, that coincided with an increased number of CD8(+) lymphocytes in the liver.

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127–128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.

The envelope of hepatitis B virus (HBV) consists of three related proteins known as the large (L), middle (M) and small (S) hepatitis B surface antigens (HBsAg). L-HBsAg has a 108-119 amino acid extension at the N terminus compared with M-HBsAg and contains the preS1 sequence of the HBV envelope. Previous research has identified this region as the likely virus attachment protein which is thought to interact with the cellular receptor for the virus. However, as the receptor has still not been identified unequivocally, we used the preS1 region of L-HBsAg to screen a human liver cDNA library by the yeast two-hybrid system. Several positive clones were isolated which encoded cellular proteins that interacted with the HBV preS1 protein. The specificity was examined in an independent manner in experiments in which baculovirus-derived glutathione S-transferase (GST)-preS1 was incubated with 35S-labelled protein expressed by in vitro translation from the positive clones. The intensity of the interactions using this alternative approach mirrored those observed in the yeast two-hybrid system and two proteins (an unidentified protein and a mitochondrial protein) were selected for further study. The specificity of the binding reaction between the preS1 protein and these two proteins was further confirmed in a competition assay; HBV purified from serum, but not purified HBsAg, was able to compete with preS1 and thus block GST-preS1 binding to the unidentified protein but not to the mitochondrial protein. The unidentified protein was then expressed as a fusion protein with GST and this was able to bind HBV virions in a direct manner.

The failure of traditional protein-based vaccines to prevent infection by viruses such as HIV or hepatitis C highlights the need for novel vaccine strategies. DNA vaccines have shown promise in small animal models, and are effective at generating anti-viral T cell-mediated immune responses; however, they have proved to be poorly immunogenic in clinical trials. We propose that the induction of necrosis will enhance the immune response to vaccine antigens encoded by DNA vaccines, as necrotic cells are known to release a range of intracellular factors that lead to dendritic cell (DC) activation and enhanced cross-presentation of antigen. Here we provide evidence that induction of cell death in DNA vaccine-targeted cells provides an adjuvant effect following intradermal vaccination of mice; however, this enhancement of the immune response is dependent on both the mechanism and timing of cell death after antigen expression. We report that a DNA vaccine encoding the cytolytic protein, perforin, resulted in DC activation, enhanced broad and multifunctional CD8 T-cell responses to the HIV-1 antigen GAG and reduced viral load following challenge with a chimeric virus, EcoHIV, compared with the canonical GAG DNA vaccine. This effect was not observed for a DNA vaccine encoding an apoptosis-inducing toxin, DTa, or when the level of perforin expression was increased to induce cell death sooner after vaccination. Thus, inducing lytic cell death following a threshold level of expression of a viral antigen can improve the immunogenicity of DNA vaccines, whereas apoptotic cell death has an inhibitory effect on the immune response. Refereed/Peer-reviewed

SUMMARY. The mechanism of hepatitis C virus (HCV) replication is unknown, although the classification of HCV in the Flaviviridae has led to the postulation that HCV may adopt a replication strategy similar to that of the flaviviruses. To determine if HCV double-stranded replicative forms, consistent with this strategy, were present in total liver RNA extracted from HCV-infected individuals, HCV-specific RNA was detected by reverse transcription followed by polymerase chain reaction (RT-PCR). Initially, a strand-specific RT-PCR resulting from chemical modification of the 3′ end of the RNA was established using in vitro transcribed HCV RNA. This procedure allowed the specific detection of positive and negative HCV RNA strands in HCV-infected liver tissue. The species of HCV RNA was then examined in RNA extracted from liver tissue from naturally infected individuals; total liver RNA was either: (i) fractionated with 2m LiCl (designed to precipitate single-stranded and partially double-stranded RNA); or (ii) digested with RNase A in high salt conditions (designed to digest single-stranded RNA only). Amplification of positive sense HCV RNA from the LiCl-insoluble fraction, but not from the LiCl-soluble fraction nor in the RNase A-digested sample, was consistent with the interpretation that single-strand, but not double-stranded HCV RNA, was contained in the liver samples. Thus, it is unclear if a double-stranded RNA species is formed during the HCV replication cycle.

T cell repertoire diversity and clonotype follow-up in vaccination, cancer, infectious and immune diseases represent a major challenge owing to the enormous complexity of the data generated. Here we describe a next generation methodology, which combines 5′RACE PCR, 454 sequencing and, for analysis, IMGT, the international ImMunoGeneTics information system (IMGT), IMGT/HighV-QUEST web portal and IMGT-ONTOLOGY concepts. The approach is validated in a human case study of T cell receptor beta (TRB) repertoire, by chronologically tracking the effects of influenza vaccination on conventional and regulatory T cell subpopulations. The IMGT/HighV-QUEST paradigm defines standards for genotype/haplotype analysis and characterization of IMGT clonotypes for clonal diversity and expression and achieves a degree of resolution for next generation sequencing verifiable by the user at the sequence level, while providing a normalized reference immunoprofile for human TRB., Dynamic changes in T cell repertoire underlie immune responses during infection, allergy, autoimmunity and cancer. Here, Li et al. present a workflow for high throughput sequencing and analysis of T cell receptor sequences, and use it to monitor the T cell response to influenza vaccination in a human patient.

A cell monolayer radioimmunoassay was established to detect hepatitis B virus (HBV)-binding activity. HBV was shown to bind to HepG2 and HuH7 cells, but not to LTK- and HeLa cells. The binding activity was inhibited by peptide 21-47 from the large hepatitis B surface antigen (L-HBsAg) region, but not by other peptides from the L- and middle-HBsAg. A monoclonal antibody to L-HBsAg (MA18/7) and a polyclonal antibody to HBsAg (anti-HBs) also inhibited the binding. However, only the Fab fragment of MA18/7 showed blocking activity, suggesting that these two antibodies may neutralize virus by different mechanisms. Excess HBV was able to saturate the binding activity of HepG2 cells. The virus was also shown to be internalized; virus DNA was detected in the cytoplasmic fraction 1-2 hr postadsorption and was associated with the nuclear fraction 2 hr later. Furthermore, immunoprecipitation experiments showed that the virus DNA remained encapsidated 48 hr after internalization. Nuclear fractionation experiments showed that the encapsidated HBV DNA remained associated with the nuclear membrane. Trypsinization of intact nuclei resulted in disassociation of the nucleocapsid from the nuclear membranes, suggesting that the nuclear membrane presents a barrier to intact HBV virions or nucleocapsids. This may explain why HepG2 cells are refractile to infection although permissive for HBV replication after transfection of viral DNA.

Background: The diagnosis of hepatitis C virus (HCV) infection currently relies on the detection of antibody to HCV (anti-HCV). However, anti-HCV positivity may indicate past infection, current infection or possibly non-specific reactivity. For confirmation of current infection the virus needs to be assayed directly and this is possible by the polymerase chain reaction (PCR). Aims: The aims were to compare HCV RNA and anti-HCV as markers of infection in two groups of individuals: (i) a heterogeneous group with suspected HCV infection and (ii) a small group of blood and bone marrow donors, and their respective recipients. Methods: Serum samples were tested for alanine aminotransferase (ALT) as part of a liver function screen, for anti-HCV by ELISAII, and HCV RNA was detected by PCR. Single round and nested PCR was performed using primers designed from the sequence of the 5′-untranslated region of the HCV genome. Results: Of the 36 subjects in the heterogeneous group, 19/22 anti-HCV-positive patients with chronic non-A non-B hepatitis (NANBH) were viraemic, and the majority (17/19) demonstrated elevated ALT. However, HCV RNA was undetected in seven anti-HCV-positive patients, four of whom suffered autoimmune hepatitis Type I and three were low risk blood donors. Of the remaining subjects (seven/36) who were anti-HCV-negative, three/seven were HCV-RNA-positive and included two with acute post-transfusion (PT) NANBH and a recent needlestick victim who contracted HCV. In the second group, four individuals (donors), including a mother with a history of drug use, were implicated in transmission to three recipients. ALT levels were normal in all donors but raised in two of the recipients. PCR determined which of two anti-HCV-negative blood donors was infectious, confirmed transmission between a bone marrow donor and recipient, and indicated that anti-HCV detected in a newborn child represented passive transfer of antibody. Conclusions: Anti-HCV detected by ELISA II is a useful marker of chronic HCV infection, particularly in association with raised ALT. However, HCV RNA is a superior marker of acute HCV infection, a more reliable predictor of infectivity and is more specific.

Background & Aims HCV patients who fail conventional interferon-based therapy have limited treatment options. Dendritic cells are central to the priming and development of antigen-specific CD4 + and CD8 + T cell immunity, necessary to elicit effective viral clearance. The aim of the study was to investigate the safety and efficacy of vaccination with autologous dendritic cells loaded with HCV-specific cytotoxic T cell epitopes. Methods We examined the potential of autologous monocyte-derived dendritic cells (MoDC), presenting HCV-specific HLA A2.1-restricted cytotoxic T cell epitopes, to influence the course of infection in six patients who failed conventional therapy. Dendritic cells were loaded and activated ex vivo with lipopeptides. In this phase 1 dose escalation study, all patients received a standard dose of cells by the intradermal route while sequential patients received an increased dose by the intravenous route. Results No patient showed a severe adverse reaction although all experienced transient minor side effects. HCV-specific CD8 + T cell responses were enumerated in PBMC by ELIspot for interferon-γ. Patients generated de novo responses, not only to peptides presented by the cellular vaccine but also to additional viral epitopes not represented in the lipopeptides, suggestive of epitope spreading. Despite this, no increases in ALT levels were observed. However, the responses were not sustained and failed to influence the viral load, the anti-HCV core antibody response and the level of circulating cytokines. Conclusions Immunotherapy using autologous MoDC pulsed with lipopeptides was safe, but was unable to generate sustained responses or alter the outcome of the infection. Alternative dosing regimens or vaccination routes may need to be considered to achieve therapeutic benefit.