Multiplex Hydrolysis Probe Real-Time PCR for Simultaneous Detection of Hepatitis A Virus and Hepatitis E Virus

Detection of hepatitis viral infections has traditionally relied on the circulating antibody test using the enzyme-linked immunosorbent assay. However, multiplex real-time PCR has been increasingly used for a variety of viral nucleic acid detections and has proven to be superior to traditional methods. Hepatitis A virus (HAV) and hepatitis E virus (HEV) are the major causes of acute hepatitis worldwide; both HAV and HEV infection are a main public health problem. In the present study, a one-step multiplex reverse transcriptase quantitative polymerase chain reaction assay using hydrolysis probes was developed for simultaneously detecting HAV and HEV. This novel detection system proved specific to the target viruses, to be highly sensitive and to be applicable to clinical sera samples, making it useful for rapid, accurate and feasible identification of HAV and HEV

Investigation of the risk factors associated with the failure of hepatitis B vaccination of neonates in Yunnan province, China

Objective: This study aimed to investigate HBsAg positive rates and risk factors of HBV infection among the children less than 15 years old in Yunnan province, a remote southwest part of mainland China. Methods: Multi-stage sampling was used to randomly select study subjects from 9,360,000 individuals. Hepatitis B vaccine inoculation rate and HBsAg positive rate were investigated, and then propensity score and generalized linear mixed model (GLMMs) were applied to the case-control study. Results: The average HBsAg positive rate was 1.81%, with 1.2% in urban areas and 2.4% in rural areas. Rate of first-dose-in-time in urban areas was 77.7%, obviously higher than 49.5% in rural areas (χ2 = 2811.71, P < 0.01). Similarly, 3-dose completion coverage rate in urban areas was 93.7%, also higher than 79.0% in rural areas (χ2 = 1561.43, P < 0.01). Maternal HBeAg positivity and HBsAg positivity were proved to be the main risk factors of children with HBV infection. Moreover, paternal HBeAg positivity, paternal HBsAg positivity, the absence and unknown status of HBV vaccine inoculation were risk factors of children with HBV infection as well. Conclusion: It was very important to improve the HBV vaccine inoculation rates. Delivering babies in hospital and timely inoculation with HBV vaccine were efficient ways to prevent HBV vertical transmission. Keywords: Hepatitis B vaccines, Immune failure, Risk factors, Propensity score, Generalized linear mixed mode

Enhanced mucosal immune responses induced by a combined candidate mucosal vaccine based on Hepatitis A virus and Hepatitis E virus structural proteins linked to tuftsin.

Hepatitis A virus (HAV) and Hepatitis E virus (HEV) are the most common causes of infectious hepatitis. These viruses are spread largely by the fecal-oral route and lead to clinically important disease in developing countries. To evaluate the potential of targeting hepatitis A and E infection simultaneously, a combined mucosal candidate vaccine was developed with the partial open reading frame 2 (ORF2) sequence (aa 368-607) of HEV (HE-ORF2) and partial virus protein 1 (VP1) sequence (aa 1-198) of HAV (HA-VP1), which included the viral neutralization epitopes. Tuftsin is an immunostimulatory peptide which can enhance the immunogenicity of a protein by targeting it to macrophages and dendritic cells. Here, we developed a novel combined protein vaccine by conjugating tuftsin to HE-ORF2 and HA-VP1 and used synthetic CpG oligodeoxynucleotides (ODNs) as the adjuvant. Subsequent experiments in BALB/c mice demonstrated that tuftsin enhanced the serum-specific IgG and IgA antibodies against HEV and HAV at the intestinal, vaginal and pulmonary interface when delivered intranasally. Moreover, mice from the intranasally immunized tuftsin group (HE-ORF2-tuftsin + HA-VP1-tuftsin + CpG) showed higher levels of IFN-γ-secreting splenocytes (Th1 response) and ratio of CD4+/CD8+ T cells than those of the no-tuftsin group (HE-ORF2 + HA-VP1 + CpG). Thus, the tuftsin group generated stronger humoral and cellular immune responses compared with the no-tuftsin group. Moreover, enhanced responses to the combined protein vaccine were obtained by intranasal immunization compared with intramuscular injection. By integrating HE-ORF2, HA-VP1 and tuftsin in a vaccine, this study validated an important concept for further development of a combined mucosal vaccine against hepatitis A and E infection

Immune responses to HBsAg conjugated to protein D of non-typeable Haemophilus influenzae in mice.

BACKGROUND:Hepatitis B vaccine that contains an aluminum hydroxide adjuvant induces apoptotic death of Hepa 1-6 cells. Difficult-to-degrade chemical additives in vaccines effectively enhance vaccine immunogenicity, but also affect the host tissue. Identification of bio-molecules that are readily degraded and compatible in vivo as an adjuvant is important for vaccine research. The hapten-carrier effect suggests that stimulation of helper T (Th) cells by carrier adjuvants is feasible. Protein D (PD) of non-typeable Haemophilus influenzae covalently conjugated to some polysaccharide vaccines has been confirmed to convert T-cell independent (TI) antigens into T-cell dependent (TD) antigens, and elicit strong T-cell responses ultimately. Herein, we would substitube PD for aluminum hydroxide adjuvant in Hepatitis B vaccine. METHODS AND RESULTS:Truncated PD (amino acids 20-364) was expressed in Escherichia coli and purified by (NH4)2SO4 precipitation and DEAE chromatography. After evaluation of antigenicity by western blotting, PD was covalently conjugated to yeast-derived recombinant HBsAg by cross-linking with glutaraldehyde. Intramuscular immunization with the conjugate induced higher level of HBsAg-specific antibody than did HBsAg alone (p < 0.05), and was comparable to commercial Hepatitis B vaccine. During the surveillance period (days 35-105), anti-HBs titers were hold high. Moreover, the conjugated vaccine enhanced Th1 immune responses, while Th2 responses were also activated and induced an antibody response, as determined by IFN-γ ELISPOT and IgG1/IgG2a ratio assays. CONCLUSIONS:Recombinant truncated PD covalently conjugated to HBsAg antigen enhanced the immunogenicity of the antigen in mice simultaneously by humoral and cellular immune response, which would facilitate therapeutic hepatitis B vaccines

ELISPOT detection of <i>in vitro</i> IFN-γ production by splenocytes of vaccinated mice.

<p> Splenocytes from two spleens were pooled for a total of three samples per immunization group and analyzed by ELISPOT. (A) Numbers of spleen cells targeting HEV and (B) numbers of spleen cells targeting HAV were detected in the mouse groups immunized as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123400#pone.0123400.g002" target="_blank">Fig 2</a>. Error bars represent the SEM. *, <i>P</i><0.05, **, <i>P</i><0.01, ***, <i>P</i><0.001; ns, no significant difference.</p

Proportions of CD4⁺ and CD8⁺ T cell subsets in splenotypes of vaccinated mice.

<p>(A) CD4⁺ T cell subsets and CD8+ T cell subsets of all groups. (B) CD4⁺/CD8⁺ T cell ratios of experimental groups and PBS control group. Mouse groups were immunized as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123400#pone.0123400.g002" target="_blank">Fig 2</a>. Error bars represent the SEM. *, <i>P</i><0.05, **, <i>P</i><0.01, ***, <i>P</i><0.001; ns, no significant difference.</p

Profiles of HEV-specific IgA production in mucosal immune response to vaccination.

<p>HEV IgA was detected by indirect ELISA in (A) vaginal secretions, (B) intestine, (C) respiratory tract and (D) stool homogenates of the mouse groups immunized as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123400#pone.0123400.g002" target="_blank">Fig 2</a>. Each serum effective dose (presented as the GMT) is the reciprocal value of the maximum positive dilution. Error bars represent the SEM. *, <i>P</i><0.05, **, <i>P</i><0.01, ***, <i>P</i><0.001; ns, no significant difference.</p

Epidemiology of hepatitis E virus in China: results from the Third National Viral Hepatitis Prevalence Survey, 2005-2006.

In China, hepatitis E virus (HEV) is prevalent and causes disease, but its epidemiological profile is not well understood. We used a commercial enzyme-linked immunosorbent assay to detect total antibodies to hepatitis E virus in 15,862 serum samples collected during the Third National Viral Hepatitis Prevalence Survey. The results were analyzed to calculate estimates of HEV seroprevalence and to examine the effects of some putative risk factors. The seroprevalence of HEV in the general Chinese population during the period from 2005 through 2006 was 23.46% (95% confidence interval [CI], 18.41%-28.50%). The farming population, the age group of 15-60 year olds, and those living in the Midwest or Mideast region and in Xinjiang province had the highest seroprevalence estimates. The prevalence of HEV is high in China. The seroprevalence rate of HEV shows an unbalanced distribution among areas with different geographic location and economic development levels. The characteristics of the distribution associated may be due to the route of HEV transmission (via contaminated water or animal reservoirs). Within the same region, the seroprevalence of HEV is generally increased with age

In vitro IL-4 and IFN-γ production by splenocytes of vaccinated mice stimulated by HBsAg or PD.

<p>(A) IL-4 responses determined by ELISPOT assay. 1–2, HBsAg and PD-specific IL-4 responses of splenocytes from the conjugated vaccine group, respectively; 3–4, HBsAg-specific IL-4 responses of splenocytes isolated respectively from the commercial vaccine and HBsAg alone groups; 5, PBS group. (B) IFN-γ responses determined by ELISPOT assay. 1–2, HBsAg and PD-specific IFN-γ responses of splenocytes from the conjugated vaccine group, respectively; 3–4, HBsAg-specific IFN-γ responses of splenocytes isolated respectively from the commercial vaccine and HBsAg alone groups; 5, PBS group. The symbol “*” represents statistical significant difference between the groups in the segment ends.</p

Western blotting analysis of PD and schematic diagram of the dose-response relationship.

<p>Lane 1, 10 μl of sonicate from induced bacteria; lane 2, 10 μl of sonicate from non-induced bacteria; lanes 3–5, 1:10, 1:20, and 1:40 dilutions of eluate in 100 mM NaCl in Buffer A.</p