Design of a native-like secreted form of the hepatitis C virus E1E2 heterodimer.

Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.

Efficacy and safety of a nanoparticle therapeutic vaccine in patients with chronic hepatitis B: A randomized clinical trial.

BACKGROUND AND AIM: HBV DNA can be reduced using antiviral drugs in patients with chronic hepatitis B (CHB); however, the rate of HBeAg seroconversion remains low. A clinical trial was conducted to assess the efficacy and safety of a de novo designed liposome-based nanoparticle lipopeptide vaccine, εPA-44, for CHB. APPROACH AND RESULTS: A two-stage phase 2 trial, which included a 76-week, randomized, double-blind, placebo-controlled trial (stage 1) and a 68-week open-label extension (stage 2), was conducted in 15 centers across China (Clinicaltrials.gov No. NCT00869778). In stage 1, 360 human leukocyte antigen A2 (HLA-A2)-positive and HBeAg-positive patients were randomly and equally distributed to receive six subcutaneous injections of 600 µg or 900 µg εPA-44 or placebo at week 0, 4, 8, 12, 20, and 28. In stage 2, 183 patients received extended 900 µg εPA-44, and 26 patients were observed for relapse without further treatment. The primary endpoint was the percentage of patients with HBeAg seroconversion at week 76. At week 76, patients receiving 900 µg εPA-44 achieved significantly higher HBeAg seroconversion rate (38.8%) versus placebo (20.2%) (95% CI, 6.9-29.6%; p = 0.002). With a combined endpoint of HBeAg seroconversion, alanine aminotransferase normalization and HBV DNA < 2,000 IU/mL, both 900 µg (18.1%) and 600 µg (14.3%), resulted in significantly higher rate versus placebo (5.0%) (p = 0.002 and p = 0.02, respectively) at week 76. In stage 2, none (0 of 20) of 900 µg εPA-44-treated patients experienced serologic relapse. The safety profile of εPA-44 was comparable to that of placebo. CONCLUSIONS: Among HLA-A2-positive patients with progressive CHB, a finite duration of 900 µg εPA-44 monotherapy resulted in significantly higher HBeAg seroconversion rate than placebo and sustained off-treatment effect. A phase 3 trial is ongoing (ChiCTR2100043708).

Multimodal investigation of rat hepatitis E virus antigenicity: Implications for infection, diagnostics, and vaccine efficacy.

BACKGROUND & AIMS: Rat hepatitis E virus (Orthohepevirus species C; HEV-C1) is an emerging cause of viral hepatitis in humans. HEV-C1 is divergent from other HEV variants infecting humans that belong to Orthohepevirus species A (HEV-A). This study assessed HEV-C1 antigenic divergence from HEV-A and investigated the impact of this divergence on infection susceptibility, serological test sensitivity, and vaccine efficacy. METHODS: Immunodominant E2s peptide sequences of HEV-A and HEV-C1 were aligned. Interactions of HEV-C1 E2s and anti-HEV-A monoclonal antibodies (mAbs) were modeled. Recombinant peptides incorporating E2s of HEV-A (HEV-A4 p239) and HEV-C1 (HEV-C1 p241) were expressed. HEV-A and HEV-C1 patient sera were tested using antibody enzymatic immunoassays (EIA), antigen EIAs, and HEV-A4 p239/HEV-C1 p241 immunoblots. Rats immunized with HEV-A1 p239 vaccine (Hecolin), HEV-A4 p239 or HEV-C1 p241 peptides were challenged with a HEV-C1 strain. RESULTS: E2s sequence identity between HEV-A and HEV-C1 was only 48%. There was low conservation at E2s residues (23/53; 43.4%) involved in mAb binding. Anti-HEV-A mAbs bound HEV-C1 poorly in homology modeling and antigen EIAs. Divergence resulted in low sensitivity of commercial antigen (0%) and antibody EIAs (10-70%) for HEV-C1 diagnosis. Species-specific HEV-A4 p239/HEV-C1 p241 immunoblots accurately differentiated HEV-A and HEV-C1 serological profiles in immunized rats (18/18; 100%) and infected-patient sera (32/36; 88.9%). Immunization with Hecolin and HEV-A4 p239 was partially protective while HEV-C1 p241 was fully protective against HEV-C1 infection in rats. CONCLUSIONS: Antigenic divergence significantly decreases sensitivity of hepatitis E serodiagnostic assays for HEV-C1 infection. Species-specific immunoblots are useful for diagnosing HEV-C1 and for differentiating the serological profiles of HEV-A and HEV-C1. Prior HEV-A exposure is not protective against HEV-C1. HEV-C1 p241 is an immunogenic vaccine candidate against HEV-C1. LAY SUMMARY: Rat hepatitis E virus (HEV-C1) is a new cause of hepatitis in humans. Using a combination of methods, we showed that HEV-C1 is highly divergent from the usual cause of human hepatitis (HEV-A). This divergence reduces the capacity of existing tests to diagnose HEV-C1 and also indicates that prior exposure to HEV-A (via infection or vaccination) is not protective against HEV-C1.

Characterizing Hepatitis C Virus-Specific CD4+ T Cells Following Viral-Vectored Vaccination, Directly Acting Antivirals, and Spontaneous Viral Cure.

BACKGROUND AND AIMS: Induction of functional helper CD4+ T cells is the hallmark of a protective immune response against hepatitis C virus (HCV), associated with spontaneous viral clearance. Heterologous prime/boost viral vectored vaccination has demonstrated induction of broad and polyfunctional HCV-specific CD8+ T cells in healthy volunteers; however, much less is known about CD4+ T-cell subsets following vaccination. APPROACH AND RESULTS: We analyzed HCV-specific CD4+ T-cell populations using major histocompatibility complex class II tetramers in volunteers undergoing HCV vaccination with recombinant HCV adenoviral/modified vaccinia Ankara viral vectors. Peptide-specific T-cell responses were tracked over time, and functional (proliferation and cytokine secretion) and phenotypic (cell surface and intranuclear) markers were assessed using flow cytometry. These were compared to CD4+ responses in 10 human leukocyte antigen-matched persons with HCV spontaneous resolution and 21 chronically infected patients treated with directly acting antiviral (DAA) therapy. Vaccination induced tetramer-positive CD4+ T cells that were highest 1-4 weeks after boosting (mean, 0.06%). Similar frequencies were obtained for those tracked following spontaneous resolution of disease (mean, 0.04%). In addition, the cell-surface phenotype (CD28, CD127) memory subset markers and intranuclear transcription factors, as well as functional capacity of peptide-specific CD4+ T-cell responses characterized after vaccination, are comparable to those following spontaneous viral resolution. In contrast, helper responses in chronic infection were infrequently detected and poorly functional and did not consistently recover following HCV cure. CONCLUSIONS: Helper CD4+ T-cell phenotype and function following HCV viral vectored vaccination resembles "protective memory" that is observed following spontaneous clearance of HCV. DAA cure does not promote resurrection of exhausted CD4+ T-cell memory in chronic infection.

In Vivo and In Vitro Potency of Polyphosphazene Immunoadjuvants with Hepatitis C Virus Antigen and the Role of Their Supramolecular Assembly.

Two well-defined synthetic polyphosphazene immunoadjuvants, PCPP and PCEP, were studied for their ability to potentiate the immune response to the hepatitis C virus (HCV) E2 glycoprotein antigen in vivo. We report that PCEP induced significantly higher serum neutralization and HCV-specific IgG titers in mice compared to other adjuvants used in the study: PCPP, Alum, and Addavax. PCEP also shifted the response toward the desirable balanced Th1/Th2 immunity, as evaluated by the antibody isotype ratio (IgG2a/IgG1). The in vivo results were analyzed in the context of antigen-adjuvant molecular interactions in the system and in vitro immunostimulatory activity of formulations. Asymmetric flow field flow fractionation (AF4) and dynamic light scattering (DLS) analysis showed that both PCPP and PCEP spontaneously self-assemble with the E2 glycoprotein with the formation of multimeric water-soluble complexes, which demonstrates the role of polyphosphazene macromolecules as vaccine delivery vehicles. Intrinsic in vitro immunostimulatory activity of polyphosphazene adjuvants, which was assessed using a mouse macrophage cell line, revealed comparable activities of both polymers and did not provide an explanation of their in vivo performance. However, PCEP complexes with E2 displayed greater stability against agglomeration and improved in vitro immunostimulatory activity compared to those of PCPP, which is in line with superior in vivo performance of PCEP. The results emphasize the importance of often neglected antigen-polyphosphazene self-assembly mechanisms in formulations, which can provide important insights on their in vivo behavior and facilitate the establishment of a structure-activity relationship for this important class of immunoadjuvants.

Changing trends in seroprevalence rates of transfusion-transmitted diseases among blood donors in Jordan.

BACKGROUND: Hepatitis B and C infections and transmission are a serious challenge to all healthcare systems. We studied seroprevalence rates of Transfusion Transmitted Diseases (TTD) among blood bank donors in Jordan from 2014 to 2019 as a follow-up study of our previously published work. In addition, we wanted to explore the efficacy of the mandatory vaccination of infants against hepatitis B virus (HBV) which was implemented by the Ministry of Health since 1995 for the eradication of HBV infection in Jordan. METHODS: We reviewed blood bank donors' records at King Hussein Cancer Center (KHCC) from January 1st, 2014, until December 31st, 2019. Results of seropositivity prevalence rates for HBsAg, anti-HBcore, and anti-HCV, using Enzyme-Linked ImmunoSorbent Assay (ELISA) were compared to seropositivity rates from our previously published data. In addition, our results were compared to data obtained from other blood banks in Jordan, as well as compared to published information from blood banks in neighboring countries. RESULTS: The prevalence rates (%) of seropositive blood donors for viral hepatitis for the years 2014, 2015, 2016, 2017, 2018, and 2019, were as follows: HBsAg rates were 0.3386, 0.2108, 0.1801, 0.1898, 0.2068, and 0.2741; anti-HBcore rates were 4.1112, 3.2271, 2.9748, 2.8405, 2.6879 and 3.0986; and anti-HCV rates were 0.1129, 0.0486, 0.0548, 0.0654, 0.0782, and 0.0839, respectively. There was a significant increase in the prevalence of HBsAg, Anti-HBcore and Anti-HCV antibodies in 2019 (one sample z-score test, p < 0.00001). CONCLUSIONS: Prevalence rates of hepatitis B and C infections among Jordanian blood bank donors showed a steady decline between 2009 and 2017, and these rates were much lower in Jordan than in neighboring countries. However, an increase in the prevalence rates of hepatitis B and C infections among blood bank donors was documented in 2019. While the reasons for this increase are not clear yet, these findings highlight the importance of renewed efforts to increase public health awareness of HBV and implement effective measures to prevent the transmission and infection with HBV, including national vaccination programs.

Interventions to reduce acute hepatitis C virus in HIV-positive MSM.

PURPOSE OF REVIEW: The WHO has set ambitious targets for hepatitis C virus (HCV) elimination by 2030. In this review, we explore the possibility of HCV micro-elimination in HIV-positive (+) MSM, discussing strategies for reducing acute HCV incidence and the likely interventions required to meet these targets. RECENT FINDINGS: With wider availability of directly acting antivirals (DAAs) in recent years, reductions in acute HCV incidence have been reported in some cohorts of HIV+ MSM. Recent evidence demonstrates that treatment in early infection is well tolerated, cost effective and may reduce the risk of onward transmission. Modelling studies suggest that to reduce incidence, a combination approach including behavioural interventions and access to early treatment, targeting both HIV+ and negative high-risk groups, will be required. HCV vaccine trials have not yet demonstrated efficacy in human studies, however phase one and two studies are ongoing. SUMMARY: Some progress towards the WHO HCV elimination targets has been reported. Achieving sustained HCV elimination is likely to require a combination approach including early access to DAAs in acute infection and reinfection, validated and reproducible behavioural interventions and an efficacious HCV vaccine.

Prevalence of and risk factors for HBV and HCV among incarcerated people who inject drugs in Iran: A cross sectional study.

BACKGROUND: This study aimed at determining the prevalence of and risk factors for hepatitis B virus (HBV) and hepatitis C virus (HCV) among incarcerated people who inject drugs (PWID) in Iran in 2015-16. METHODS: The required data was collected from a database provided by Iranian national bio-behavioral surveillance surveys (BBSSs) on 11,988 prisoners selected from among 55 prisons in 19 provinces in 2015-16. The data on demographics and behavioral variables were collected through interviews and the status of exposure to HBV and HCV were determined using ELISA blood test. A total of 1387 individuals with a history of drug injection in their lifetime were enrolled into the study. Data were analyzed using the survey package in Stata/SE software, Version 14.0. Univariate and multivariate logistic regression tests were used to investigate the relationships between risk factors and outcomes. RESULTS: The mean age of the incarcerated PWID was 36.83 ± 8.13 years. Of all the studied subjects, 98.46% were male and 50.97% were married. The prevalence of HCV and HBV among the subjects were 40.52 and 2.46%, respectively. The prevalence of HCV was associated with age ≥ 30 years, being single, illiteracy and low level of education, prison term> 5 years, history of piercing, and extramarital sex in lifetime (P < 0.05). CONCLUSIONS: The prevalence of HCV is alarmingly high. In general, it is recommended to adopt measures to screen and treat patients with HCV and vaccinat incarcerated PWID without a history of vaccination against HBV.

Adult Vaccination Rates in the Mentally Ill Population: An Outpatient Improvement Project.

Background: Adults who suffer with severe and persistent mental illness (SPMI) rarely access medical care to receive preventive vaccines. Aims: To increase the rate of vaccines among the SPMI population in an outpatient community mental health center (CMHC). Methods: A review of the literature identified a gap between the general population and SPMI clients in receiving preventive vaccinations. An initial mixed-method convenience survey of SPMI clients (n = 392) provided information on current vaccination status, demographics, beliefs, and interest in receiving vaccines. A vaccination program was developed to address identified barriers and increase vaccination rates. Postintervention data were collected through a mixed-method convenience survey of SPMI clients (n = 60) who participated in immunizations clinics to evaluate client satisfaction. A partnership between the health department and CMHC was developed to deliver vaccines in a nontraditional site. Vaccines administered included annual influenza; hepatitis A; hepatitis B; herpes zoster; measles, mumps, and rubella; pneumococcal; and tetanus, diphtheria, and pertussis (Tdap). Results: More than 1,000 vaccines were administered in the first 8 months, with a significant increase in vaccination rates over baseline for individual vaccines ranging from 18.75% to 83%. Postintervention survey results found a 95% satisfaction rate. Conclusions: Implementation of a vaccination program in a nontraditional site that facilitates access for SPMI clients can promote an overwhelming increase in the vaccination rates for this underserved population. Results suggest that the integration of mental health and CMHC services can have a profound positive effect on SPMI population health.

Interrogation of Antigen Display on Individual Vaccine Nanoparticles for Achieving Neutralizing Antibody Responses against Hepatitis C Virus.

Elicitation of neutralizing antibody responses against hepatitis C virus (HCV) has been a challenging goal. While the E2 subunit of the HCV envelope glycoprotein complex is a promising target for generating cross-genotype neutralizing antibodies, vaccinations with soluble E2 immunogens generally induce weak neutralizing antibody responses. Here, E2 immunogens (i.e., E2.661 and E2c.661) were loaded into lipid-based nanovaccines and examined for induction of neutralizing antibody responses. Compared with soluble E2 immunogens, E2 nanoparticles elicited 6- to 20-fold higher E2-specific serum IgG titers in mice. Importantly, E2 vaccine nanoparticles analyzed at a single particle level with a flow cytometry-based method revealed interesting dynamics between epitope display on the surfaces of nanoparticles in vitro and induction of neutralizing antibody responses in vivo. E2c.661 nanoparticles that are preferentially bound by a broadly neutralizing antibody, HCV1, in vitro elicit neutralizing antibody responses against both autologous and heterologous HCV virions in vivo. In stark contrast, E2.661 nanoparticles with reduced HCV1-antibody binding in vitro mainly induce autologous neutralizing antibody responses in vivo. These results show that rationale antigen design coupled with interrogation of epitope display on vaccine nanoparticles at a single particle level may aid in vaccine development toward achieving neutralizing antibody responses in vivo.

[Epidemiological analysis of viral hepatitis E in China, 2004-2017].

Objective: To analyze the changing epidemiological characteristics of hepatitis E cases in China, in order to promote in preventing and controlling hepatitis E. Methods: Data of hepatitis E and outbreaks reported through national notifiable diseases reporting system were analyzed from 2004 to 2017, but data of Hongkong, Macau and Taiwan were not included. Data of hepatitis E were divided into three phases as 2004-2007, 2008-2011 and 2012-2017, representing eight years before, four years before and years after the postmarketing of hepatitis E vaccine. Linear regression was used for analyzing the trend of hepatitis E, improved muster method was used for analyzing the seasonal intensity. Results: From 2004 to 2017, 329 519 hepatitis E cases were reported and the annual incidence were increasing from 1.27/100 000 to 2.10/100 000 (t=6.87, P<0.001). The concentrations of hepatitis E during 2004-2007, 2008-2011 and 2012-2017 were 17.43, 16.06, 11.17, respectively, with low seasonal intensity. Number of cases reported by Jiangsu, Guangdong and Zhejiang accounted for 31.54% of national cases. The incidence were lower in central (1.45/100 000) and western (1.11/100 000) region than that in eastern region (2.67/100 000), but were increasing continuously. There was an increasing trend of incidence with growing ages (t=7.85, P<0.001). The incidence was higher than 2/100 000 among cases aged ≥40, and was the highest (5.22/100 000) in the age group of 65-69 years old. Farmers, retired persons, houseworkers and unemployees accounted for 67.46% of total cases. A total of 7 outbreaks were reported, among which 3 were in nursing homes. Conclusion: The incidence of hepatitis E in central and western regions were increasing continuously and the surveillance should be strengthened. There was higher risk among middle-aged population, farmers and nursing homes, so strategy for immunization among those population was in great need.

Structure-Based Design of Hepatitis C Virus Vaccines That Elicit Neutralizing Antibody Responses to a Conserved Epitope.

Despite recent advances in therapeutic options, hepatitis C virus (HCV) remains a severe global disease burden, and a vaccine can substantially reduce its incidence. Due to its extremely high sequence variability, HCV can readily escape the immune response; thus, an effective vaccine must target conserved, functionally important epitopes. Using the structure of a broadly neutralizing antibody in complex with a conserved linear epitope from the HCV E2 envelope glycoprotein (residues 412 to 423; epitope I), we performed structure-based design of immunogens to induce antibody responses to this epitope. This resulted in epitope-based immunogens based on a cyclic defensin protein, as well as a bivalent immunogen with two copies of the epitope on the E2 surface. We solved the X-ray structure of a cyclic immunogen in complex with the HCV1 antibody and confirmed preservation of the epitope conformation and the HCV1 interface. Mice vaccinated with our designed immunogens produced robust antibody responses to epitope I, and their serum could neutralize HCV. Notably, the cyclic designs induced greater epitope-specific responses and neutralization than the native peptide epitope. Beyond successfully designing several novel HCV immunogens, this study demonstrates the principle that neutralizing anti-HCV antibodies can be induced by epitope-based, engineered vaccines and provides the basis for further efforts in structure-based design of HCV vaccines.IMPORTANCE Hepatitis C virus is a leading cause of liver disease and liver cancer, with approximately 3% of the world's population infected. To combat this virus, an effective vaccine would have distinct advantages over current therapeutic options, yet experimental vaccines have not been successful to date, due in part to the virus's high sequence variability leading to immune escape. In this study, we rationally designed several vaccine immunogens based on the structure of a conserved epitope that is the target of broadly neutralizing antibodies. In vivo results in mice indicated that these antigens elicited epitope-specific neutralizing antibodies, with various degrees of potency and breadth. These promising results suggest that a rational design approach can be used to generate an effective vaccine for this virus.

Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG.

A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography. IMPORTANCE: A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fc-tagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.

Evaluation of autophagy induction on HEV 239 vaccine immune response in a mouse model.

Hepatitis E virus (HEV) infection remains a serious threat to life and productivity in developing world. Vaccine seems to be an effective, safe, and affordable approach to address HEV disease burden. The HEV genome consists of three open reading frames (ORFs). Of these, ORF2 encodes a single structural protein (pORF2) for the HEV capsid which has been studied extensively as vaccine candidates. Recently, it has been recognized that autophagy plays an important role in innate and adaptive immunity defense against intracellular pathogens. This mechanism could therefore promote a protective immune response by inducing CD4+ and CD8+ T cells. In this study, HEV 239 and Beclin1 proteins were expressed in prokaryotic host cell [Escherichia coli (BL21)]. HEV 239 protein with different formulations (+Alum, +Beclin1, and +Alum-Beclin1) were used as candidate vaccines and administrated subcutaneously in BALB/c mice on 0, 14, and 28 days. Finally, elicited cellular and humoral immunity were evaluated. Taken together, although our results indicated that mice immunized with HEV 239 protein formulated with Alum, Beclin1, and Alum + Beclin1 displayed humoral and cellular response that was not significant in comparison with each other (P > 0.05); whereas they were significant while compared with control groups (P < 0.05). A comprehensive understanding of the intricate interplay between autophagy and immune response remains to be unraveled. Further study will clear the detailed impact of autophagy manipulation to enhance vaccine efficacy and boost the immune responses against the disease. © 2018 IUBMB Life, 70(3):207-214, 2018.

Development of a recombinant murine tumour model using hepatoma cells expressing hepatitis C virus nonstructural antigens.

Hepatitis C virus (HCV) chronically infects 2%-3% of the world's population, causing liver disease and cancer with prolonged infection. The narrow host range of the virus, being restricted largely to human hepatocytes, has made the development of relevant models to evaluate the efficacy of vaccines a challenge. We have developed a novel approach to accomplish this by generating a murine hepatoma cell line stably expressing nonstructural HCV antigens which can be used in vitro or in vivo to test HCV vaccine efficacies. These HCV-recombinant hepatoma cells formed large solid-mass tumours when implanted into syngeneic mice, allowing us to test candidate HCV vaccines to demonstrate the development of an HCV-specific immune response that limited tumour growth. Using this model, we tested the therapeutic potential of recombinant anti-HCV-specific vaccines based on two fundamentally different attenuated pathogen vaccine systems-attenuated Salmonella and recombinant adenoviral vector based vaccine. While attenuated Salmonella that secreted HCV antigens limited growth of the HCV-recombinant tumours when used in a therapeutic vaccination trial, replication-competent but noninfectious adenovirus expressing nonstructural HCV antigens showed overall greater survival and reduced weight loss compared to non-replicating nondisseminating adenovirus. Our results demonstrate a model with anti-tumour responses to HCV nonstructural (NS) protein antigens and suggest that recombinant vaccine vectors should be explored as a therapeutic strategy for controlling HCV and HCV-associated cancers.

Molecular evolution and phylodynamics of hepatitis B virus infection circulating in Iran.

Previous local and national Iranian publications indicate that all Iranian hepatitis B virus (HBV) strains belong to HBV genotype D. The aim of this study was to analyze the evolutionary history of HBV infection in Iran for the first time, based on an intensive phylodynamic study. The evolutionary parameters, time to most recent common ancestor (tMRCA), and the population dynamics of infections were investigated using the Bayesian Monte Carlo Markov chain (BMCMC). The effective sample size (ESS) and sampling convergence were then monitored. After sampling from the posterior distribution of the nucleotide substitution rate and other evolutionary parameters, the point estimations (median) of these parameters were obtained. All Iranian HBV isolates were of genotype D, sub-type ayw2. The origin of HBV is regarded as having evolved first on the eastern border, before moving westward, where Isfahan province then hosted the virus. Afterwards, the virus moved to the south and west of the country. The tMRCA of HBV in Iran was estimated to be around 1894, with a 95% credible interval between the years 1701 and 1957. The effective number of infections increased exponentially from around 1925 to 1960. Conversely, from around 1992 onwards, the effective number of HBV infections has decreased at a very high rate. Phylodynamic inference clearly demonstrates a unique homogenous pattern of HBV genotype D compatible with a steady configuration of the decreased effective number of infections in the population in recent years, possibly due to the implementation of blood donation screening and vaccination programs. Adequate molecular epidemiology databases for HBV are crucial for infection prevention and treatment programs.

Lettuce-produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination.

The hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases (e.g. cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV-related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca sativa) using Agrobacterium-mediated transient expression technology. The wild-type dimer (E1E2) and a variant without an N-glycosylation site in the E2 polypeptide (E1E2∆N6) were expressed, and appropriate N-glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell-expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti-HCV IgM for both antigens; however, the E1E2∆N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N-glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti-HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low-cost oral vaccine development.

Missed Opportunities for Hepatitis A Vaccination, National Immunization Survey-Child, 2013.

OBJECTIVES: To quantify the number of missed opportunities for vaccination with hepatitis A vaccine in children and assess the association of missed opportunities for hepatitis A vaccination with covariates of interest. STUDY DESIGN: Weighted data from the 2013 National Immunization Survey of US children aged 19-35 months were used. Analysis was restricted to children with provider-verified vaccination history (n = 13 460). Missed opportunities for vaccination were quantified by determining the number of medical visits a child made when another vaccine was administered during eligibility for hepatitis A vaccine, but hepatitis A vaccine was not administered. Cross-sectional bivariate and multivariate polytomous logistic regression were used to assess the association of missed opportunities for vaccination with child and maternal demographic, socioeconomic, and geographic covariates. RESULTS: In 2013, 85% of children in our study population had initiated the hepatitis A vaccine series, and 60% received 2 or more doses. Children who received zero doses of hepatitis A vaccine had an average of 1.77 missed opportunities for vaccination compared with 0.43 missed opportunities for vaccination in those receiving 2 doses. Children with 2 or more missed opportunities for vaccination initiated the vaccine series 6 months later than children without missed opportunities. In the fully adjusted multivariate model, children who were younger, had ever received WIC benefits, or lived in a state with childcare entry mandates were at a reduced odds for 2 or more missed opportunities for vaccination; children living in the Northeast census region were at an increased odds. CONCLUSIONS: Missed opportunities for vaccination likely contribute to the poor coverage for hepatitis A vaccination in children; it is important to understand why children are not receiving the vaccine when eligible.

Cross-reactivity of hepatitis C virus specific vaccine-induced T cells at immunodominant epitopes.

Viral diversity is a challenge to the development of a hepatitis C virus (HCV) vaccine. Following vaccination of humans with adenoviral vectors, we determined the capacity of T cells to target common viral variants at immundominant epitopes ex vivo. We identified two major variants for epitopes NS3(1073) and NS3(1446), and multiple variants for epitope NS3(1406) that occurred in >5% of genotype 1 and 3 sequences at a population level. Cross-reactivity of vaccine-induced T cells was determined using variant peptides in IFN-γ ELISPOT assays. Vaccine-induced T cells targeted approximately 90% of NS3(1073) genotype 1 sequences and 50% of NS3(1446) genotype 1 and 3 sequences. For NS3(1406), 62% of subtype-1b sequences were targeted. Next, we assessed whether an in vitro priming system, using dendritic cells and T cells from healthy donors, could identify a variant of NS3(1406) that was maximally cross-reactive. In vitro priming assays showed that of those tested the NS3(1406) vaccine variant was the most immunogenic. T cells primed with genotype 1 variants from subtype 1a or 1b were broadly cross-reactive with other variants from the same subtype. We conclude that immunization with candidate HCV adenoviral vaccines generates cross-reactive T cells at immunodominant epitopes. The degree of cross-reactivity varies between epitopes and may be HCV-subtype specific.

A Multiantigenic DNA Vaccine That Induces Broad Hepatitis C Virus-Specific T-Cell Responses in Mice.

UNLABELLED: 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.