anti-HEV Titers Research Articles

Chitosan-functionalized graphene oxide as adjuvant in HEV P239 vaccine

Searching appropriate adjuvants for vaccine is a potent method to intense the immune efficacy. In the present study, we developed a novel Hepatitis E virus (HEV) vaccine by utilizing chitosan modified nano-graphene oxide (GO-CS) as an adjuvant to support HEV antigen P239 protein (GO/CS/P239). The characterization of GO/CS/P239 was observed by atomic force microscope. The safety of GO/CS/P239 was measured by CCK-8 method, hemolysis test and acute challenge test. The anti-HEV titers and cytokines production were analyzed by double antibody sandwich ELISA. As the results showed, by contrast with a vaccine that contained only the P239 protein, GO/CS/P239 vaccine can promote immune cells to produce more IgG antibodies and cytokines, which were able to stimulate the organism to produce stronger both cellular and humoral immunity. Collectively, GO/CS/P239 particles have been demonstrated to be safe both in vitro and in vivo, and can facilitate sufficient immune response to protect organisms from virus infection, which suggested that our exploration offers a promising alternative vaccine that can control HEV infection.

Seroprevalence of hepatitis E virus among blood donors on Corsica, France, 2017.

BackgroundHepatitis E virus (HEV) is an emerging zoonotic pathogen and an important cause of acute viral hepatitis in European countries. Corsica Island has been previously identified as a hyperendemic area for HEV.AimOur aim was to characterise the prevalence and titres of IgG antibodies to HEV among blood donors on Corsica and establish a model of the annual force of infection.MethodsBetween September 2017 and January 2018, 2,705 blood donations were tested for anti-HEV IgG using the Wantai HEV IgG enzyme immunoassay.ResultsThe overall seroprevalence was 56.1%. In multivariate analysis, seroprevalence was higher in men than in women (60.0% vs 52.2%; p < 0.01), increased with age and was significantly higher among donors born on Corsica (60.6% vs 53.2%; p < 0.01). No significant difference was observed between the five districts of the island. IgG anti-HEV titres were mostly low (70% of positive donors had titres < 3 IU/mL). In Corsican natives, increasing seroprevalence by age could be explained by models capturing a loss of immunity (annual probability of infection: 4.5%; duration of immunity: 55 years) or by age-specific probabilities of infection (3.8% for children, 1.3% for adults).ConclusionWe confirmed the high HEV seroprevalence on Corsica and identified three aspects that should be further explored: (i) the epidemiology in those younger than 18 years, (ii) common sources of contamination, in particular drinking water, that may explain the wide exposure of the population, and (iii) the actual protection afforded by the low IgG titres observed and the potential susceptibility to secondary HEV infection.

Differential Immune Responses in Mice Immunized with Recombinant Neutralizing Epitope Protein of Hepatitis E Virus Formulated with Liposome and Alum Adjuvants.

In the developing countries, Hepatitis E virus (HEV) is a predominant cause of sporadic acute hepatitis in adults and waterborne epidemics leading to high mortality in pregnant women. Vaccine development mainly focuses on the structural capsid protein open-reading-frame-2 (ORF-2) of the virus. We successfully evaluated liposome-adjuvanted recombinant neutralizing epitope protein (rNEp), a part of ORF-2, 458-607aa, in mice and rhesus macaques. We compared immune response to adjuvants alone, rNEp alone, or adjuvanted with liposome (lipo-rNEp)/alum (al-rNEp) in mice following intramuscular administration of two doses of 5 μg each. IgG anti-HEV titers (enzyme-linked immunosorbent assay), immunophenotyping (flow cytometry, CD3(+)CD4(+), CD3(+)CD8(+), CD11c(+), CD11b(+), CD19(+) cells; costimulatory markers CD80, CD86, MHC-I, MHC-II, and early activation marker CD69), and levels of Th1/Th2 cytokines (IL-2/IFN-γ/IL-4/IL-5 and additionally IL-1β/IL-6/IL-10/TNF for early time points) were determined at early (4/12/24-h postdose-1) and later time points (2 weeks post-both doses). IgG anti-HEV titers were higher in the lipo-rNEp group than al-rNEp post-both doses (p < 0.05). At early time points, cell type proportions were comparable at the site of injection; IL-Iβ levels increased in lipo-rNEp, 24 h, while IL-6 levels rose in lipo-rNEp/al-rNEp/alum-alone groups, 4 h, compared to controls. In the draining lymph nodes (DLNs), CD11c(+)CD86(+) cells increased at 24 h in liposome-alone/lipo-rNEp groups. A rise in the CD11c(+)CD69(+) cells was noted in the lipo-rNEp group compared to other groups (p < 0.05). Cytokine levels in the spleen/sera remained unchanged in all the groups (p > 0.05). At 2 weeks postdose-2, CD11c(+)MHC-II(+)/CD11b(+)MHC-II(+) cells increased in the spleen in the lipo-rNEp and al-rNEp groups, respectively. In the DLNs, CD19(+)MHC-II(+) cells increased in rNEp/al-rNEp/lipo-rNEp groups post-both doses and CD11c(+)CD86(+) cells in the lipo-rNEp group. A balanced Th1/Th2 response was evident in the lipo-rNEp, while a Th2 bias was noted in al-rNEp. Different immune response gene clustering patterns were noted in uncultured spleens from immunized mice and cultured-stimulated splenocytes. In conclusion, lipo-rNEp is a better immunogen, works through dendritic cells, and elicits a balanced Th1/Th2 response, while alum functions through macrophages and induces a Th2 response.

Enhanced humoral response in pregnant mice immunized with liposome encapsulated recombinant neutralizing epitope protein of Hepatitis- E virus.

BackgroundPregnant women from developing countries are at high-risk of hepatitis E-associated high mortality and constitute priority population for vaccination. So far, candidate vaccines have not been evaluated during pregnancy. We evaluated our vaccine candidate, recombinant Neutralizing Epitope protein (rNEp) encapsulated in liposomes, in pregnant mice.MethodsA single dose (10 μg) of the formulation was administered intramuscularly on day 7 of pregnancy. Titres of serum IgG antibodies to hepatitis E virus (IgG-anti-HEV), levels of cytokines and biochemical parameters were determined. Spleens were harvested from pregnant and non-pregnant mice for immunophenotyping (flow cytometry), cytokines (cytometric bead array, CBA) and gene expression of immune response genes (Taqman low density array, TLDA). Histopathology studies of spleen, liver, kidneys, brain and muscle was carried out.ResultsThe vaccine was well-tolerated during pregnancy as evidenced by histopathology and serum biochemical parameters. Anti-HEV titres were significantly higher in the pregnant balb/c and C57BL/6 mice (3592 ± 802 and1016 ± 138 respectively, than in non-pregnant groups (634 ± 191 and 320 ± 55 respectively, p < 0.001 for both) suggesting that the higher antibody response in pregnant mice was independent of the genetic makeup of the host but immunogen-driven. Pups receiving vertically transferred antibodies developed lower anti-HEV antibodies (p < 0.05) when immunized with the formulation after seronegativity than in the age-matched mice without such antibodies. In non-pregnant mice, a Th1 response and discordance between splenic and serum cytokines was evident while in pregnancy, a Th2 bias was observed irrespective of immunization. Increased CD19 levels correlated with higher anti-HEV titres in pregnant mice.ConclusionThe single dose of the vaccine was safe and highly immunogenic in pregnant mice. Degree and type of immune response to vaccination during pregnancy is immunogen-driven. In-depth studies are needed to understand the underlying immunologic mechanism(s). These encouraging results for a vaccine intended for use in pregnant women should be confirmed in higher animals.

Characterization of Hepatitis E Virus Recombinant ORF2 Proteins Expressed by Vaccinia Viruses

Hepatitis E virus (HEV), an enterically transmitted pathogen, is one of the major causes of acute hepatitis in humans worldwide, being responsible for outbreaks and epidemics in regions with suboptimal sanitary conditions, in many of which it is endemic. In industrialized countries, hepatitis E is rarely reported, but recent studies have revealed quite high human seroprevalence rates and the possibility of porcine zoonotic transmission. There is currently no specific therapy or licensed vaccine against HEV infection, and little is known about its intracellular growth cycle, as until very recently no efficient cell culture system has been available. In the present study, vaccinia viruses have been used to express recombinant HEV ORF2 proteins, allowing the study of their glycosylation patterns and subcellular localization. Furthermore, the expressed proteins have been shown to be good antigens for diagnostic purposes and to elicit high and long-lasting specific anti-HEV titers of antibodies in mice that are passively transferred to the offspring by both transplacental and lactation routes.

Viral Load, Antibody Titers and Recombinant Open Reading Frame 2 Protein-Induced Th1/Th2 Cytokines and Cellular Immune Responses in Self-Limiting and Fulminant Hepatitis E

Objectives: Hepatitis E virus (HEV) is the predominant cause of water-borne epidemics, sporadic acute viral hepatitis (AVH) in adults and fulminant hepatic failure (FHF) among pregnant women and other adults in India. This preliminary study was designed to examine the association of viral load and certain host immune responses with uneventful recovery or progression to FHF. Methods: Viral load, anti-HEV antibody titers, rORF2p-induced Th1/Th2 cytokines levels and cellular immune responses were assessed in 47 patients with self-limiting hepatitis E and 14 FHF-E cases. The controls included 16 anti-HEV-IgM and IgG-negative healthy individuals. Results: In AVH category, the viral load was 2.4 × 10⁴ ± 1.92 × 10⁴ copies/ml while except for one, all FHF patients were negative for HEV RNA; anti-HEV-IgM and IgG titers were higher in the FHF group. Lymphocyte proliferative response to rORF2p was comparable in both groups. As compared to AVH, significantly higher levels of both Th1 (IFN-γ, IL-2 and TNF-α) and Th2 (IL-10) cytokines were recorded in FHF patients. Analysis of sequential samples differentiated FHF recovered and fatal patients with respect to IFN-γ and IL-12. Conclusion: The results document increased Th1/Th2 responses and anti-HEV titers in FHF patients that warrant in-depth immunological studies.

Challenge studies in Rhesus monkeys immunized with candidate hepatitis E vaccines: DNA, DNA-prime-protein-boost and DNA-protein encapsulated in liposomes

Complete ORF2 gene (1983bp) of hepatitis E virus (HEV) and the 450bp region within ORF2 containing neutralizing epitope (NE) cloned in pVAX1 and corresponding proteins expressed in baculovirus and prokaryotic systems respectively were evaluated as vaccine candidates. Two doses of liposome encapsulated DNA plus corresponding protein with both ORF2 and NE regions (Lipo-ORF2-DP and Lipo-NE-DP) showed 100% seroconversion and comparable anti-HEV titres in Swiss albino mice. These vaccine candidates were further evaluated as DNA, DNA-prime-protein-boost (DPPB) and liposome formulations in Rhesus monkeys. Monkeys receiving ORF2/NE DNA seroconverted after fourth dose while those immunized employing ORF2-DPPB format seroconverted at 7 weeks post third dose. In view of the delayed weak antibody response, these monkeys were not challenged. Though Lipo-ORF2-DP was immunogenic, 2 of the 4 monkeys developed HEV infection following homologous virus challenge of 100 Monkey Infectious Dose50. Both monkeys immunized with Lipo-NE-DP and 1 of the 2 monkeys immunized with NE-DPPB showed complete protection, the second monkey being protected from hepatitis with limited viral replication. Irrespective of the type of immunogen, all challenged monkeys were protected from hepatitis. The results document Lipo-NE-DP to be a promising vaccine candidate needing further evaluation.

Should HEV infected mothers breast feed?

The aim of this study was to determine by a prospective study: the presence of anti-hepatitis E virus (HEV) and or HEV RNA in the colostrum of HEV infected mothers; transmission of HEV to infants from their mothers by breast-feeding. Ninety-three infected pregnant mothers in the third trimester of pregnancy of which 36 were positive for anti-HEV antibodies and 57 for HEV RNA (index patients) and 90 healthy pregnant mothers (control subjects) were studied. Maternal blood was taken at 7th and 9th of gestation and also within 5 days post-partum, along with colostrum and tested for anti-HEV and HEV RNA. Blood samples were collected from all infants at birth (cord blood) and at 1, 3, and 6 and 9 months of age. There were 12 cesarean sections and eighty full term vaginal deliveries. Anti-HEV antibody and HEV-RNA was present in the colostral samples but in significantly lower levels ( p<0.001) as compared to corresponding maternal levels. Within 2 weeks post-partum, 6 of these 93 index patients, whose infants were anti-HEV antibody and HEV RNA negative at birth, developed acute hepatic disease. These mothers, four of whom delivered by cesarean section, had anti-HEV titers ranging from 1:10,000 to 1:60,000 and HEV RNA ranging from 1.5x10(6) to 2.5x10(4) copies/ml. Due to acute maternal disease their six respective infants were formula fed. Four of these infants were in close maternal contact, frequently kissed and cuddled, and developed symptomatic liver disease by 6-8 weeks of age. Apart from these 6 infants the remaining were exclusively breast-fed for 3.6+/-0.32 months. There was no evidence of HEV infection in the remaining babies. All mother-infant pairs from the control group remained anti-HEV negative throughout this study. Although anti-HEV antibody and HEV-RNA are present in the colostrum of HEV infected mothers, breast-feeding appears to be safe for these infants. However this report must be confirmed by others. Transmission of infection may occur postpartum, through close contact of mother-infant pairs, especially in the presence of acute maternal disease.

Characterization of a recombinant fowlpox virus expressing the native hexon of hemorrhagic enteritis virus.

The structure of the icosahedral adenovirus capsid is highly conserved among Adenoviridae. In its native form, the hexon is the major capsid protein. The nascent hexon requires the 100 kDa folding protein to fold into its native, trimeric form. The hexon and 100 kDa folding protein were co-expressed in a fowlpox virus (FPV) vector and in the recombinant FPVs (rFPVs) in which the hexon and 100 kDa folding protein genes are cloned head to tail, the native hexon could be detected with indirect immunofluorescence and immunoprecipitation using a native hexon monoclonal antibody. The FPV-@X100 construct, in which the 100kDa folding protein gene follows the hexon gene in a head to tail fashion, elicited the best humoral response in chickens. An attenuated HEV commercial vaccine elicited higher and longer lasting anti-HEV titers than FPV-@X100. Humoral immunity was also compared in turkeys inoculated with rFPVs expressing the hexon alone, the 100 kDa folding protein alone, or expressing both genes in different configurations. No anti-HEV humoral immune response was detected in turkeys inoculated with the rFPVs expressing the hexon alone or the 100 kDa folding protein alone.

Long-term serological follow up and cross-challenge studies in rhesus monkeys experimentally infected with hepatitis E virus

Backround/Aims: The aims of this study were to examine the decline of IgG anti-HEV antibodies over a period of 7 years in rhesus monkeys experimentally infected with hepatitis E virus, and to assess the protectivity of these antibodies by challenging the monkeys with a heterologous i solate of hepatitis E virus, 5 years after the primary inoculation.Methods: Nine rhesus monkeys (six non-pregnant and three pregnant at the time of hepatitis E virus inoculation) were followed serologically and biochemically for 7 years post-inoculation. Based on regression analysis, estimated time for IgG anti-HEV titers to reach 1:100 or 1:50 was calculated. Three of the monkeys inoculated initially with AKL-90 isolate and challenged 2 years later with PUN-85 isolate of hepatitis E virus were rechallenged with KOL-91 isolate of the virus, 5 years post-primary inoculation. Evidence of viral replication was assessed by measuring serum alanine aminotransferase levels, excretion of the virus in feces or bile (reverse-transcription polymerase chain reaction) and rise in IgG anti-HEV titers (ELISA).Results: None of the challenged monkeys showed evidence of disease. In contrast to extensive replication of the virus in anti-HEV-negative control monkeys, limited replication was noted in one of the challenged monkeys. The estimated time for the titers to reach 1:100 or 1:50 varied from 3.15 to 44.9 years (19.4±11.6 years) and 6.9 to 84.3 years (35.4±21.3 years), respectively. Decline in titers was independent of the pregnancy status at the time of infection or reexposure of the monkeys to HEV.Conclusion: The results show persistence of IgG anti-HEV antibodies for a long time and protectivity of low titered antibodies against reinfection, leading to disease even after intravenous exposure to a heterologous isolate of hepatitis E virus.

Antibodies against hepatitis E virus in Old World monkeys.

To examine whether Indian monkeys are infected with hepatitis E virus (HEV) in nature, serum samples from wild rhesus (Macaca mullata), bonnet (M. radiata) and langur (Presbytes entellus) monkeys were screened for anti-HEV IgG antibodies in recombinant antigen-based ELISA assays. The positivity rates were 36.7%, 19.1% and 2% respectively. The protection of such antibodies against human HEV was studied in four rhesus monkeys. Of the two rhesus monkeys with anti-HEV titres of 100 and 1000 respectively which were inoculated with the KOL-91 strain of HEV, the former demonstrated a 10-fold rise in anti-HEV titres. Anti-HEV titre in the second rhesus monkey remained unchanged. Neither of the monkeys showed any rise in serum alanine transaminase (ALT) or presence of virus in the faeces, as tested by polymerase chain reaction (PCR). Two other rhesus monkeys with anti-HEV titres of 10,000 and 100 respectively were inoculated with the AKL-90 strain of HEV. Serum ALT levels and anti-HEV titres remained unchanged in the first monkey. Excretion of virus in faeces was not noted (PCR). The second monkey developed a typical HEV infection. HEV infection could be produced in anti-HEV negative control monkeys inoculated with both strains of HEV. These results show that either human or simian HEV, or a closely related agent, is circulating among Indian macaques. Titre-dependent protection of naturally occurring anti-HEV antibodies supports this view.

ELISA for antibody to hepatitis E virus (HEV) based on complete open-reading frame-2 protein expressed in insect cells: identification of HEV infection in primates.

A recombinant baculovirus containing the complete open-reading frame (ORF)-2 region of the hepatitis E virus (HEV) genome was constructed. The major protein synthesized in insect cells infected with recombinant virus was about the size expected for the complete ORF-2 product. This protein reacted in a Western blot assay with plasma from an HEV-infected chimpanzee. Lysates of the recombinant virus-infected insect cells were used in ELISA to monitor seroconversion of eight primate species (chimpanzees, four species of Old World monkeys, and three species of New World monkeys) inoculated with HEV. Homologous detector anti-immunoglobulin was more sensitive than heterologous anti-immunoglobulin for detecting anti-HEV by ELISA. All primate species except tamarins seroconverted after inoculation with HEV, although anti-HEV titers of Old World monkey species were generally higher than those of New World monkey species. The ELISA with complete ORF-2 antigen appeared to be a sensitive and practical method for detecting anti-HEV.