Viral excretion and antibody titers in children infected with hepatitis A virus from an orphanage in western India.

BACKGROUND: Hepatitis A is endemic in India and mainly causes sporadic infections. However, children in childcare centers, schools and orphanages are vulnerable to common-source outbreaks as they have naive hosts. OBJECTIVES: To investigate hepatitis A outbreak in an orphanage from Pune, India. STUDY DESIGN: Monitoring of virus excretion and anti-HAV antibody levels in hepatitis A virus (HAV) infected children. RESULTS: The orphanage housed 93 children of the age 1 month-6.5 years. Analysis of the collected serum (n=78) and stool samples (n=63) revealed 20 children to be either positive for anti-HAV IgM antibodies or excreting HAV, 14 being symptomatic and 6 asymptomatic, while 32 were already anti-HAV IgG positive either due to past HAV exposure (n=7, mean log antibody titers: 2.96) or maternal antibodies (n=25, mean log antibody titers: 1.13). Serum samples, taken 4 weeks apart, did not show any significant difference in the IgM and IgG antibody levels either. However, virus excretion decreased significantly after 15 days in symptomatic children (mean log HAV RNA copies/ml 1.03+0.30), while asymptomatic children continued to excrete higher viral loads, at constant levels (mean log HAV RNA copies/ml 2.33+0.33), for up to 90 days. CONCLUSIONS: Though virus excretion continued up to 90 days in all HAV infected children, asymptomatic children excreted higher viral loads for longer period and hence can contribute significantly in person-to-person virus transmission. All children should be vaccinated in such set ups.

Non-carrier nanoparticles adjuvant modular protein vaccine in a particle-dependent manner.

Nanoparticles are increasingly used to adjuvant vaccine formulations due to their biocompatibility, ease of manufacture and the opportunity to tailor their size, shape, and physicochemical properties. The efficacy of similarly-sized silica (Si-OH), poly (D,L-lactic-co-glycolic acid) (PLGA) and poly caprolactone (PCL) nanoparticles (nps) to adjuvant recombinant capsomere presenting antigenic M2e modular peptide from Influenza A virus (CapM2e) was investigated in vivo. Formulation of CapM2e with Si-OH or PLGA nps significantly boosted the immunogenicity of modular capsomeres, even though CapM2e was not actively attached to the nanoparticles prior to injection (i.e., formulation was by simple mixing). In contrast, PCL nps showed no significant adjuvant effect using this simple-mixing approach. The immune response induced by CapM2e alone or formulated with nps was antibody-biased with very high antigen-specific antibody titer and less than 20 cells per million splenocytes secreting interferon gamma. Modification of silica nanoparticle surface properties through amine functionalization and pegylation did not lead to significant changes in immune response. This study confirms that simple mixing-based formulation can lead to effective adjuvanting of antigenic protein, though with antibody titer dependent on nanoparticle physicochemical properties.

Bovine FcRn-mediated human immunoglobulin G transfer across the milk-blood barrier in transgenic mice.

Maternal-fetal IgGs transport occurs either prenatally or postnatally, which confers the newborns with passive immunity before their own immune system has matured. However, little is known about the mechanisms of postnatal IgGs passage in the mammary gland. To investigate how FcRn mediates the IgGs transport in the mammary gland, we first generated bFcRn and anti-HAV mAb transgenic mice, and then obtained HF transgenic mice expressing both transgenes by mating the above two strains. Transgene expression of bFcRn in the four lines was determined by qRT-PCR and western blot. We then localized the expression of bFcRn to the acinar epithelial cells in the mammary gland, and anti-HAV mAb was mainly detected in the acini with weak staining in the acinar epithelial cells. Human IgGs could be detected in both milk and serum of HF transgenic mice by western blot and ELISA. A significantly lower milk to serum ratio of human IgGs in HF mice compared with that of anti-HAV mAb mice, indicating that bFcRn could transport human IgGs across the milk-blood barrier from milk to serum during lactation in HF mice. While, there were no transport of murine IgGs, IgAs, or IgMs. These results provide understandings about the mechanisms of maternal-fetal immunity transfer in the mammary gland.

The efficacy of simulated solar disinfection (SODIS) against coxsackievirus, poliovirus and hepatitis A virus.

The antimicrobial activity of simulated solar disinfection (SODIS) against enteric waterborne viruses including coxsackievirus-B5, poliovirus-2 and hepatitis A virus was investigated in this study. Assays were conducted in transparent 12-well polystyrene microtitre plates containing the appropriate viral test suspension. Plates were exposed to simulated sunlight at an optical irradiance of 550 Wm(-2) (watts per square metre) delivered from a SUNTEST™ CPS+ solar simulator for 6 hours. Aliquots of the viral test suspensions were taken at set time points and the level of inactivation of the viruses was determined by either culture on a HeLa cell monolayer for coxsackievirus-B5 and poliovirus-2 or by utilising a chromogenic antibody-based approach for hepatitis A virus. With coxsackievirus-B5, poliovirus-2 and hepatitis A virus, exposure to SODIS at an optical irradiance of 550 Wm(-2) for 1-2 hours resulted in complete inactivation of each virus. The findings from this study suggest that under appropriate conditions SODIS may be an effective technique for the inactivation of enteric viruses in drinking water. However, further verification studies need to be performed using natural sunlight in the region where the SODIS technology is to be employed to validate our results.

Breastmilk hepatitis A virus RNA in nursing mothers with acute hepatitis A virus infection.

Breastmilk specimens from three women with acute hepatitis A virus (HAV) infection were studied. Anti-HAV immunoglobulin M and immunoglobulin G antibodies were detected in serum and breastmilk specimens of the three women. The three women also had serum HAV RNA. However, HAV RNA was detected only in two of the three breastmilk specimens. It is interesting that none of the three infants contracted clinical HAV infection. Furthermore, mothers with HAV infection should not be encouraged to discontinue breastfeeding.

Production of monoclonal antibodies with a controlled N-glycosylation pattern in seeds of Arabidopsis thaliana.

Seed-specific expression is an appealing alternative technology for the production of recombinant proteins in transgenic plants. Whereas attractive yields of recombinant proteins have been achieved by this method, little attention has been paid to the intracellular deposition and the quality of such products. Here, we demonstrate a comparative study of two antiviral monoclonal antibodies (mAbs) (HA78 against Hepatitis A virus; 2G12 against HIV) expressed in seeds of Arabidopsis wild-type (wt) plants and glycosylation mutants lacking plant specific N-glycan residues. We demonstrate that 2G12 is produced with complex N-glycans at great uniformity in the wt as well as in the glycosylation mutant, carrying a single dominant glycosylation species, GnGnXF and GnGn, respectively. HA78 in contrast, contains additionally to complex N-glycans significant amounts of oligo-mannosidic structures, which are typical for endoplasmic reticulum (ER)-retained proteins. A detailed subcellular localization study demonstrated the deposition of both antibodies virtually exclusively in the extracellular space, illustrating their efficient secretion. In addition, although a KDEL-tagged version of 2G12 exhibited an ER-typical N-glycosylation pattern, it was surprisingly detected in protein storage vacuoles. The different antibody variants showed different levels of degradation with hardly any degradation products detectable for HA78 carrying GnGnXF glycans. Finally, we demonstrate functional integrity of the HA78 and 2G12 glycoforms using viral inhibition assays. Our data therefore demonstrate the usability of transgenic seeds for the generation of mAbs with a controlled N-glycosylation pattern, thus expanding the possibilities for the production of optimally glycosylated proteins with enhanced biological activities for the use as human therapeutics.

[Clinical features of patients with fulminant hepatitis A requiring emergency liver transplantation: comparison with acute liver failure due to other causes].

BACKGROUND/AIMS: According to recent prevalence of hepatitis A virus (HAV) infection, acute liver failure (ALF) due to HAV infection is observed frequently in parallel. The aim of this study was to elucidate the clinical, laboratory, and pathologic features of patients who have undergone emergency liver transplantation (LT) due to fulminant HAV infection. METHODS: Clinical, laboratory, and pathologic data of 11 transplant recipients with anti-HAV IgM-positive ALF between December 2007 and May 2009 were analyzed, and compared with data of 10 recipients who underwent LT for the management of ALF due to other causes. RESULTS: The median age of the patients with HAV-related ALF was 34 years (range: 15-43 years). The levels of hemoglobin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine were higher and the level of bilirubin was lower in the HAV-related ALF group than in the other group (P=0.005, 0.001, 0.001, 0.010, and 0.003, respectively). The time from the onset of initial symptoms to the development of encephalopathy was shorter in the HAV-related ALF group than in the other group (median 5 days, range: 4-13 days; P<0.001). In patients with HAV-related ALF, laboratory findings and clinical prognostic parameters including the Acute Liver Failure Study Group prognostic index, King's College criteria, and model for endstage liver disease (MELD) and Child-Pugh scores were not associated with the grade of hepatic encephalopathy or time of progression to encephalopathy. CONCLUSIONS: The results of this study indicate that the clinical condition of patients with HAV-related ALF requiring emergency LT aggravates rapidly. Prognostic parameters are not sufficient for discriminating transplant candidates in patients with fulminant hepatitis A.

Characterization of anti-idiotypic antibodies mimicking antibody- and receptor-binding sites on hepatitis A virus.

Two anti-idiotypic monoclonal antibodies (mAb2s; named 94-2 and 94-7), were generated from a BALB/c mouse immunized with human monoclonal anti-hepatitis A virus (HAV) neutralizing antibody KF94. We characterized the properties of the mAb2s and determined interactions between mAb2s, KF94 and HAV using enzyme-linked immunosorbent assay, immunofluorescence assay and HAV infectivity assay. Inactivated HAV inhibited mAb2 binding to KF94, indicating that the mAb2s mimicked the HAV neutralization site that was complementary to the paratope of KF94. MAb2 94-7 competed with an anti-HAV cellular receptor antibody for binding to HAV-susceptible cells and partially blocked virus infection. We speculated that mAb2 94-7 mimicked a portion of the HAV receptor-binding site. The ability to generate mAb2 implies that HAV receptor-binding sites are exposed on the surface of HAV, permitting antibody access.

[Clinical features of acute hepatitis A in recent two years].

BACKGROUND/AIMS: The purpose of this study was to characterize the clinical features of acute hepatitis A in Seoul and Gyeonggi province during the recent 2 years. METHODS: We reviewed the medical records of 222 patients who were diagnosed as acute hepatitis A between August 2005 and March 2007 at the Konkuk University Hospital and Korea University, Ansan Hospital. The clinical manifestation, serological tests, and image findings were analyzed. RESULTS: Median age of the patients was 28.1 years and the age groups of highest incidence were the second and third decade. The frequent symptoms were anorexia (66.4%), fatigue (49.2%), fever (47.7%), and abdominal discomfort (42.5%). Fourteen cases (6.3%) showed renal insufficiency, and hemodialysis was performed in one. Cholestatic hepatitis in 2 cases, relapsing hepatitis in 4 cases and prolonged hepatitis in 13 cases were observed. However, there was no case of fulminant hepatitis or death. The underlying diseases including chronic hepatitis B, diabetes mellitus and thyroid disorder did not affect the disease severity of hepatitis A. IgM anti-HAV was not detected initially in 6.7% of the patients. Anti-HEV (IgM) was detected simultaneously in 3 of 150 patients. CONCLUSIONS: The age of patients with acute hepatitis A has been increased in the recent years. Most patients recovered uneventfully. However, unusual patterns of severe hepatitis and renal insufficiency occurred in considerable number of cases. Follow-up serologic test for IgM anti-HAV is needed in seronegative cases with hepatitis A.

Immunogenicity and safety of a virosomal hepatitis A vaccine (Epaxal) in the elderly.

BACKGROUND: Protection against hepatitis A virus (HAV) in the elderly is becoming more important as more senior travelers visit areas of high HAV endemicity, and less have protective antibodies acquired after natural infection during childhood. This study assessed the immunogenicity and safety of hepatitis A vaccine in elderly compared to young adults. METHODS: In this open, uncontrolled study, subjects of 18 to 45 years or < or = 50 years of age received two doses of aluminum-free, virosomal HAV vaccine, Epaxal (Berna Biotech Ltd, formerly Swiss Serum and Vaccine Institute, Bern, Switzerland) 12 months apart. RESULTS: After both the basic and the booster doses, geometric mean titers (GMT) for anti-HAV antibodies were 1.7-fold higher in subjects younger than 45 years compared with those < or = 50 years of age. The proportional increase in GMT after the booster dose, however, was similar in younger and older subjects. Seroprotection (< or = 20 mIU/mL) rates in the younger and older subjects were 100 and 65%, respectively, after the first vaccination and 100 and 97%, respectively, after the booster dose. Systemic and local adverse events were mainly mild and short-lived. CONCLUSION: These data show that HAV virosomal vaccine (Epaxal) is well tolerated and immunogenic in elderly subjects. The clinical relevance of lower seroconversion rates after the primary dose is unknown in this population of travelers.