Progress in the Production of Virus-Like Particles for Vaccination against Hepatitis E Virus.

Hepatitis E virus (HEV), a pathogen that causes acute viral hepatitis, is a small icosahedral, quasi-enveloped, positive ssRNA virus. Its genome has three open reading frames (ORFs), with ORF1 and ORF3 encoding for nonstructural and regulatory proteins, respectively, while ORF2 is translated into the structural, capsid protein. ORF2 is most widely used for vaccine development in viral hepatitis. Hepatitis E virus-like particles (VLPs) are potential vaccine candidates against HEV infection. VLPs are composed of capsid subunits mimicking the natural configuration of the native virus but lack the genetic material needed for replication. As a result, VLPs are unable to replicate and cause disease, constituting safe vaccine platforms. Currently, the recombinant VLP-based vaccine Hecolin® against HEV is only licensed in China. Herein, systematic information about the expression of various HEV ORF2 sequences and their ability to form VLPs in different systems is provided.

A Recombinant HAV Expressing a Neutralization Epitope of HEV Induces Immune Response against HAV and HEV in Mice.

Hepatitis A virus (HAV) and hepatitis E virus (HEV) are causative agents of acute viral hepatitis transmitted via the fecal-oral route. Both viruses place a heavy burden on the public health and economy of developing countries. To test the possibility that HAV could be used as an expression vector for the development of a combination vaccine against hepatitis A and E infections, recombinant HAV-HEp148 was created as a vector to express an HEV neutralization epitope (HEp148) located at aa 459-606 of the HEV capsid protein. The recombinant virus expressed the HEp148 protein in a partially dimerized state in HAV-susceptible cells. Immunization with the HAV-HEp148 virus induced a strong HAV- and HEV-specific immune response in mice. Thus, the present study demonstrates a novel approach to the development of a combined hepatitis A and E vaccine.

Induction of a dwarf phenotype with IBH1 may enable increased production of plant-made pharmaceuticals in plant factory conditions.

Year-round production in a contained, environmentally controlled 'plant factory' may provide a cost-effective method to produce pharmaceuticals and other high-value products. However, cost-effective production may require substantial modification of the host plant phenotype; for example, using dwarf plants can enable the growth of more plants in a given volume by allowing more plants per shelf and enabling more shelves to be stacked vertically. We show here that the expression of the chimeric repressor for Arabidopsis AtIBH1 (P35S:AtIBH1SRDX) in transgenic tobacco plants (Nicotiana tabacum) induces a dwarf phenotype, with reduced cell size. We estimate that, in a given volume of cultivation space, we can grow five times more AtIBH1SRDX plants than wild-type plants. Although, the AtIBH1SRDX plants also showed reduced biomass compared with wild-type plants, they produced about four times more biomass per unit of cultivation volume. To test whether the dwarf phenotype affects the production of recombinant proteins, we expressed the genes for anti-hepatitis B virus antibodies (anti-HBs) in tobacco plants and found that the production of anti-HBs per unit fresh weight did not significantly differ between wild-type and AtIBH1SRDX plants. These data indicate that P35S:AtIBH1SRDX plants produced about fourfold more antibody per unit of cultivation volume, compared with wild type. Our results indicate that AtIBH1SRDX provides a useful tool for the modification of plant phenotype for cost-effective production of high-value products by stably transformed plants in plant factory conditions.

Mucoadhesive glycol chitosan nanoparticles for intranasal delivery of hepatitis B vaccine: enhancement of mucosal and systemic immune response.

In this study, for the first time, glycol chitosan (GC) nanoparticles (NPs) were prepared and evaluated to obtain systemic and mucosal immune responses against nasally administered hepatitis B surface antigen (HBsAg). Size, zeta potential and morphology of the NPs were investigated as a function of preparation method. NPs with high loading efficacy ( > 95%) and positively charged surface were obtained with an average particle size of approximately 200 nm. The structural integrity of HBsAg in NPs was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and further confirmed by measuring the in vitro antigenicity using an enzyme immunoassay. During in vivo studies, GC NPs showed the lowest nasal clearance rate and better mucosal uptake when compared with chitosan (CS) NPs. The immunogenicity of NPs-based delivery system(s) was assessed by measuring anti-HBsAg antibody titer in mice serum and secretions after intranasal administration. The alum-based HBsAg vaccine injected subcutaneously was used as positive control. Results indicated that alum-based HBsAg induced strong humoral but negligible mucosal immunity. However, GC NPs induced stronger immune response at both of the fronts as compared to generated by CS NPs. This study demonstrates that this newly developed system has potential for mucosal administration of vaccines.

Chronically infected wild boar can transmit genotype 3 hepatitis E virus to domestic pigs.

Hepatitis E virus (HEV) causes acute hepatitis E in humans in developing countries, but sporadic and autochthonous cases do also occur in industrialized nations. In Europe, food-borne zoonotic transmission of genotype 3 (gt3) has been associated with the consumption of raw and undercooked products from domestic pig and wild boar. As shown recently, naturally acquired HEV gt3 replicates efficiently in experimentally infected wild boar and is transmissible from a wild boar to domestic pigs. Generally, following an acute infection swine suffer from a transient febrile illness and viremia in connection with fecal virus shedding. However, little is known about sub-acute or chronic HEV infections in swine, and how and where HEV survives the immune response. In this paper, we describe the incidental finding of a chronic HEVgt3 infection in two naturally infected European wild boar which were raised and housed at FLI over years. The wild boar displayed fecal HEV RNA excretion and viremia over nearly the whole observation period of more than five months. The animal had mounted a substantial antibody response, yet without initial clearance of the virus by the immune system. Further analysis indicated a subclinical course of HEV with no evidence of chronic hepatitis. Additionally, we could demonstrate that this chronic wild boar infection was still transmissible to domestic pigs, which were housed together with this animal. Sentinel pigs developed fecal virus shedding accompanied by seroconversion. Wild boar should therefore be considered as an important reservoir for transmission of HEV gt3 in Europe.

Inadequate activation of the HBsAg-specific Th cells by APCs leads to hyporesponsiveness to HBsAg vaccine in B10.S mice.

Hepatitis B can be effectively prevented by hepatitis B vaccination. However, hyporesponse to the hepatitis B vaccine has been found in both human and inbred mice with particular MHC alleles or haplotypes, but the mechanisms underlying this poor response remains elusive. In the present study, we investigated the mechanisms underlying the hyporesponse to hepatitis B vaccination using B10.S-H2s/SgMcdJ (B10.S, H-2(s), poor responder) and C57BL/10J (B10, H-2(b), good responder) mice. We observed that the B10.S mice displayed a hyporesponse to HBsAg vaccine but a normal response to 3 other foreign antigens (influenza A (H1N1) 2009 monovalent vaccine, tetanus toxoid and ovalbumin). In B10.S mice immunized with HBsAg, the levels of serum anti-HBs IgG, the number of HBsAg-specific IgG-secreting plasma cells and HBsAg-specific Th cells were considerably lower than that in B10 mice. Further, the findings of the insufficient maturation (CD86), co-stimulation (CD40) and migration (CCR7) activities of DCs together with the inadequate activation of the HBsAg-specific Th cells by APCs were identified as part of the reason for the HBsAg hyporesponse in B10.S mice, which supports the hypothesis that measures aimed at promoting the maturation, co-stimulation or migration of APCs to enhance Th cell activation may be a useful strategy for the development of new hepatitis B vaccines.

HCV antigen testing for the diagnosis of hepatitis C infection: a cost-efficient algorithm.

BACKGROUND: In North America, diagnosis of active hepatitis C virus (HCV) infection is currently performed using RNA testing which is highly sensitive and specific but is associated with three major limitations: lability of RNA molecules, higher costs, and longer turn-around time as compared with commercially-available HCV core antigen testing. In the current study, a new HCV core antigen assay product was evaluated for the diagnosis of HCV infection and its cost reducing potential. METHODS: Ninety plasma specimens positive for HCV RNA along with 25 negative HCV specimens were used for HCV antigen assay. Twenty-four specimens positive for a panel of agents were used for possible cross-reactivity. Sixty-four HCV antibody-positive specimens with negative HCV RNA and indeterminate HCV immunoblot results were also employed. RESULTS: In the first group, 78/90 (86.6%) tested positive for HCV antigen with regression analysis showing no significant deviation from linearity. None of the prenatal specimens tested positive for HCV antigen. Non-specific reactions were not observed. In the HCV antibody-indeterminate group, only 2/64 (3.1%) were antigen positive. In the last group, none of the HCV antibody very-low-positive specimens tested positive for HCV antigen. Both inter- and intra-run reproducibility of 100% were noted. The cost analysis showed a minimum of 52.13% reduction in costs associated with qualitative RNA testing. CONCLUSIONS: Considering the complexity of HCV infection diagnosis and the significant cost and turn-around time burden it imposes on clinical laboratories, HCV antigen testing seems an attractive adjunct to the current battery of laboratory diagnosis that demands more attention.

Anamnestic immunological response profile in laboratory animals after immunization with recombinant hepatitis B vaccines of different generations.

Hepatitis B vaccines containing preS1 and preS2 fragments are assumed to be more immunogenic than those containing SHBs protein alone, which may be of importance for immunization of people with poorly induced or without any immunological response after vaccination. The aim of this study was to evaluate: The following conclusions can be drawn on the basis of obtained results:

Hepatitis B vaccine antibody response and the risk of clinical AIDS or death.

BACKGROUND: Whether seroresponse to a vaccine such as hepatitis B virus (HBV) vaccine can provide a measure of the functional immune status of HIV-infected persons is unknown.This study evaluated the relationship between HBV vaccine seroresponses and progression to clinical AIDS or death. METHODS AND FINDINGS: From a large HIV cohort, we evaluated those who received HBV vaccine only after HIV diagnosis and had anti-HBs determination 1-12 months after the last vaccine dose. Non-response and positive response were defined as anti-HBs <10 and ≥ 10 IU/L, respectively. Participants were followed from date of last vaccination to clinical AIDS, death, or last visit. Univariate and multivariable risk of progression to clinical AIDS or death were evaluated with Cox regression models. A total of 795 participants vaccinated from 1986-2010 were included, of which 41% were responders. During 3,872 person-years of observation, 122 AIDS or death events occurred (53% after 1995). Twenty-two percent of non-responders experienced clinical AIDS or death compared with 5% of responders (p<0.001). Non-response to HBV vaccine was associated with a greater than 2-fold increased risk of clinical AIDS or death (HR 2.47; 95% CI, 1.38-4.43) compared with a positive response, after adjusting for CD4 count, HIV viral load, HAART use, and delayed type hypersensitivity skin test responses (an in vivo marker of cell-mediated immunity). This association remained evident among those with CD4 count ≥ 500 cells/mm³ (HR 3.40; 95% CI, 1.39-8.32). CONCLUSIONS: HBV vaccine responses may have utility in assessing functional immune status and risk stratificating HIV-infected individuals, including those with CD4 count ≥ 500 cells/mm³.

Sleep after vaccination boosts immunological memory.

Sleep regulates immune functions. We asked whether sleep can influence immunological memory formation. Twenty-seven healthy men were vaccinated against hepatitis A three times, at weeks 0, 8, and 16 with conditions of sleep versus wakefulness in the following night. Sleep was recorded polysomnographically, and hormone levels were assessed throughout the night. Vaccination-induced Th cell and Ab responses were repeatedly monitored for 1 y. Compared with the wake condition, sleep after vaccination doubled the frequency of Ag-specific Th cells and increased the fraction of Th1 cytokine-producing cells in this population. Moreover, sleep markedly increased Ag-specific IgG1. The effects were followed up for 1 y and were associated with high sleep slow-wave activity during the postvaccination night as well as with accompanying levels of immunoregulatory hormones (i.e., increased growth hormone and prolactin but decreased cortisol release). Our findings provide novel evidence that sleep promotes human Th1 immune responses, implicating a critical role for slow-wave sleep in this process. The proinflammatory milieu induced during this sleep stage apparently acts as adjuvant that facilitates the transfer of antigenic information from APCs to Ag-specific Th cells. Like the nervous system, the immune system takes advantage of the offline conditions during sleep to foster adaptive immune responses resulting in improved immunological memory.

Generation of full-length functional antibody against preS2 of hepatitis B virus in hepatic cells in vitro from bicistrons mediated by gutless adenovirus.

BACKGROUND: Monoclonal antibodies (mAbs) have been developed as effective therapeutics for a wide variety of diseases. Delivery of mAbs by gene transfer provides an option for overcoming the difficulties in mAb production and manufacturing processes. However, for the polymeric structure of full-length mAbs, it is important to design an optimal gene transfer system for mAb generation. METHODS: Gutless adenovirus and liver-specific promoter transthyretin (TTR) were combined to deliver bicistronic mAb genes in human hepatic cell lines. In order to optimize the bicistrons for mAb generation, four bicistrons were designed and compared, and the most efficient one was selected. ELISA and Western blot were conducted to evaluate mAb products in the supernatants. RESULTS: Our data showed that all of four gutless adenoviruses elicited liver-specific mAb production in HepG2 and Hep3B hepatic cell lines. It was observed that the L2AH bicistron construct (comprising an immunoglobulin light-chain cDNA situated 5' of a heavy-chain cDNA, with a foot-and-mouth disease virus 2A cleavage site in the middle, subcloned into the helper-dependent adenovirus plasmid pGL) could induce the highest level expression of mAb (about 5.0 microg/mL in Hep3B) among these four constructs. Importantly, the mAb products by gene transfer methods retained specific antigen-binding activity. CONCLUSION: Our studies gave further evidence that it was feasible to produce active full-length mAb in human hepatic cell lines in vitro by a special gene delivery system. Moreover, we developed an optimized bicistron gene transfer system for future gene therapy research, which may also be of use in industrial mAb production.

Evaluation of ISCOMs for immunization against hepatitis B.

Immune stimulating complexes (ISCOMs) incorporating recombinant hepatitis B surface antigen (rHBsAg) were prepared for induction of humoral and cellular immunity by subcutaneous administration. Prepared ISCOMs were characterized for their size, shape, incorporation efficiency, zeta potential, antigen integrity, antigen conformation and immunogenicity by biophysical and immunological techniques including transmission electron microscopy (TEM), Dynamic light scattering (DLS), SDS-PAGE, fluorescence spectroscopy, in vitro potency test and in vivo humoral and cellular immune stimulatory efficacy in Balb/c mice. Prepared ISCOM particles show characteristic cage like morphology with average size of 44 approximately nm, polydispersity index 0.1, negative zeta potential (-21.7 mV) and antigen association efficiency approximately 39%. Tryptophan emission fluorescence and in vitro potency assay data suggest that association of rHBsAg with ISCOMs results in local electrostatic interactions, motional restriction of tryptophan residues of the protein resulting in reduction of anti-rHBsAg monoclonal antibodies binding affinity. Immunization with rHBsAg ISCOMs resulted in upregulation of specific cellular (IFN-gamma and IL-2) as well as IgG response (IgG2a isotype biased) humoral response in Balb/c mice. Immune responses were significantly higher than those produced by of alum-adsorbed antigen (alum-rHBsAg) after (one booster) (p < 0.001). These data demonstrate that although the conformation of rHBsAg after incorporation into ISCOMs was moderately altered but due to strong adjuvant ability, rHBsAg ISCOMs were highly immunogenic as compared to marketed rHBsAg formulations by subcutaneous route of administration.

Induction of multispecific Th-1 type immune response against HCV in mice by protein immunization using CpG and Montanide ISA 720 as adjuvants.

Recent studies demonstrate that Th1-type immune responses against a broad spectrum of hepatitis C virus (HCV) gene products are crucial to the resolution of acute HCV infection. We investigated new vaccine approaches to augment the strength of HCV-specific Th1-type immune responses. ELISPOT assay revealed that single or multiple protein immunization using both CpG ODN and Montanide ISA 720 as adjuvants induced much stronger IFN-gamma-producing Th1 responses against core, NS3 and NS5b targets than did the formulation without these adjuvants. Protein vaccination using CpG ODN and Montanide ISA 720 as adjuvants also greatly enhanced humoral responses to HCV core, E1/E2 and NS3. When specific IgG isotypes were assayed, protein immunization using CpG ODN and Montanide ISA 720 as adjuvants produced higher titers of IgG2a dominant antibodies than did protein immunization alone, indicating a more Th1-biased pathway. This increase in IgG2a is consistent with the induction of Th1 cells secreting IFN-gamma demonstrated by ELISPOT assay. In conclusion, protein immunization using CpG ODN and Montanide ISA 720 as adjuvants greatly enhanced cellular (Th1 type) as well as humoral immune responses against HCV in Balb/c mice. The use of adjuvants appears critical to the induction of Th1 immune responses during HCV vaccination with recombinant proteins.

Construction and characterization of the chimeric antibody 8C11 to the hepatitis E virus.

Homodimers of the truncated hepatitis E virus (HEV) capsid proteins, E2 and p239, were conformed to model the dominant antigenic determinants of HEV. Using E2 as an immunogen, two neutralizing monoclonal antibodies (mAbs), namely 8C11 and 8H3, were produced. We constructed a mouse-human chimeric antibody derived from 8C11 and its expression in Chinese hamster ovary (CHO) cells. cDNAs encoding variable regions of heavy and light chains were isolated from hybridoma cells and inserted into mammalian expression vectors containing cDNA of human gamma-1 and kappa constant regions, respectively. The vectors were then cotransfected into CHO cells, and a stable cell line was established. Results from indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the chimeric antibody was assembled correctly to the native IgG molecule and could be secreted from the cells. Similar to the original mAb, the expressed chimeric antibody displayed HEV antigen-binding activity and an enhancement effect on 8H3 binding to HEV antigen. The chimeric antibody could specifically inhibit the binding of p239 to HepG2 cells and compete with HEV IgG in positive serum by antibody-competitive ELISA. The chimeric antibody is expected to be less immunogenic in human and more suitable for antibody therapy of hepatitis E.

Engineering of N-glycosylation of hepatitis C virus envelope protein E2 enhances T cell responses for DNA immunization.

N-linked oligosaccharides are known to be important in modulating immunogenicity of some viral proteins. However, the roles of N-glycans of hepatitis C virus (HCV) E2 envelope glycoprotein in specific cellular immune responses remain elusive. Previous studies showed that the epitopes aa481-500 and aa551-570 of E2 might be important for immunoreactivity and that the binding site of E2 for hCD81 is located at aa480-493 and aa544-551 within the E2 protein. Here, we made plasmids containing genes encoding either wild type or mutant E2 proteins in which N-glycosylation sites (N560NT and N576ST) close to these important regions were mutated separately or in combination. The immunogenicities of wild type E2 and three mutated E2 proteins were analyzed in BALB/c mice using DNA vaccination. The E2-M2 mutant (at N576ST) significantly enhanced (compared to control) E2-specific CTL activity (P<0.05), expression of IFN-gamma (P<0.05) and suppression of tumor growth (P<0.05). Also, high IgG2a/IgG1 ratios were elicited in a Th1-type response. These results indicate that engineering of the N-glycosylation site N576ST of HCV E2 protein enhances specific cellular immune responses, providing insights into the development of E2-based DNA vaccines with enhanced immunogenicity.

TLR agonists as vaccine adjuvants: comparison of CpG ODN and Resiquimod (R-848).

TLR ligands that mimic pathogen associated molecular patterns and activate immune cells via Toll-like receptors (TLRs) are being developed for use in humans as therapy against a variety of diseases as well as vaccine adjuvants. These include imidazoquinoline compounds such as Imiquimod and Resiquimod (R-848) that bind to TLR7 and 8, as well as CpG oligodeoxynucleotides (CpG ODN) that bind to TLR9. This study was aimed at comparing CpG ODN and R-848 for their potential use as vaccine adjuvants and to determine whether there are additive or synergistic effects when they are used together. Using HBsAg as a model antigen in mice, we show CpG ODN to be superior to R-848 for augmenting both humoral and cell mediated immune responses.

[Effects of cytokines on the immunogenic properties of hepatitis A vaccine].

Cytokines were found, in experiments with guinea pigs, to have a stimulating action on the immunogenic potency of hepatitis A vaccine (Hep-A-in-Vac). The most pronounced effects were produced by rhIL-1b, rhTNF-alpha, thymosin-a1, the "neothym" hybrid protein and immunophan. Injections of cytokines as of adjuvants stimulated the formation of antibodies titers that exceeded 2-10-fold those observed in control animals immunized by Hep-A-in-Vac alone. Immunization of guinea pigs made alongside with injections of the above cytokines ensured a 100% seroconversion in animals after the administration of drugs was completed. The number of seropositive animals in the control group was 75-89%.

A comparison of responsiveness to hepatitis B vaccination in patients on hemodialysis and peritoneal dialysis.

The doses of hepatitis B vaccine given to peritoneal dialysis (PD) patients are currently based on responsiveness data from hemodialysis (HD) patients. To determine whether the doses are also appropriate from PD patients, we did a head-to-head comparison of short-term and 2-year responses to hepatitis B vaccination of HD patients and PD patients. We evaluated serum titers of the antibody to hepatitis B surface antigen (anti-HBs) after the patients had completed a course of four consecutive intramuscular vaccinations (40 microg of Engerix-B administered into the deltoid muscle at 0, 1, 2, and 6 months) in 69 dialysis patients (47 HD and 22 PD patients) who were both hepatitis B surface antigen (HBsAg) and anti-HBs negative. No patients had received a hepatitis B vaccination prior to the study. There was no significant difference in response to hepatitis B vaccination between the HD and PD groups (78.7% versus 77.3%, p=0.33). The seroconversion rate defined as anti-HBs > or = 10IU/L was influenced only by age (p=0.011). There was also no significant difference in responsiveness between the HD and PD groups (60% versus 50%, p=0.41) at a 2-year follow-up. We conclude that doses of HBV vaccine being used for HD patients are also appropriate for PD patients and a booster dose of vaccine is required to maintain seroprotection for those who lost protecting anti-HBs.

A truncated ORF2 protein contains the most immunogenic site on ORF2: antibody responses to non-vaccine sequences following challenge of vaccinated and non-vaccinated macaques with hepatitis E virus.

A candidate hepatitis E vaccine is composed of amino acids (aa) 112-607 of the 660-aa protein encoded by open reading frame 2 (ORF2) of hepatitis E virus (HEV). We have studied the antibody response to vaccine-associated epitopes and to epitopes excluded from the vaccine to determine if important epitopes were omitted from the vaccine and if antibody responses to these regions could be used to differentiate between infection and vaccination. ELISAs were developed based on genotype 1 ORF2 peptides, containing aa 112-607 (vaccine), 458-607 (minimum neutralization site), 1-111 (N-terminus) and 607-660 (C-terminus), as well as on ORF3 peptides, containing aa 1-123 (complete) and 91-123 (C-terminus). All naive macaques infected with HEV genotype 1, 2, 3 or 4 produced antibodies to all ORF2 peptides. Anti-ORF3 was detected in both monkeys infected with genotype 1 virus and in one of two infected with genotype 2 virus. These antibody responses were considerably weaker than those directed against the neutralization site. In contrast, vaccinated animals that were challenged with HEV had a diminished or absent immune response to the peptides not included in the vaccine. Thus, only minor epitopes were excluded from the vaccine; they had limited utility for distinguishing between vaccination and infection.

Pregnant women as a sentinel population to target and implement hepatitis B virus (HBV) vaccine coverage: a three-year survey in Palermo, Sicily.

Hepatitis B virus (HBV) vaccine coverage was assessed using serologic patterns of infection (HBsAg, anti HBc) and vaccine-induced immunity (isolated anti HBs) among 3318 pregnant women attending the Obstetrical Unit of the University Hospital in Palermo who were screened over 3 years (2001-2003). Three thousand and eight of them (90.6%) were born in Sicily, whereas 310 (9.4%) were immigrants from non-EU countries. The overall prevalence of HBsAg was 1.1%, and it was significantly higher among immigrant than indigenous women (4.2% versus 0.8%; OR 5.26; p < 0.0001). Serologic evidence of past HBV infection (anti HBc) also was significantly higher in immigrants than in Sicilian women (24.5% versus 5.2%, respectively). Women aged 17-21 in our study were in cohorts that had been targeted since 1991 for mandatory HBV vaccination at age 12. In this targeted age group, 74.2% of the Sicilian women had isolated anti HBs, compared to only 15.0% among immigrants. The results suggest the need to improve HBV immunization of Sicilian adolescents and especially to implement active surveillance and to launch an HBV immunization programme that targets immigrants to Sicily.