Establishing the 1st Chinese National Standard for inactivated hepatitis A vaccine.

A reference standard calibrated in the International Units is needed for the quality control of hepatitis A vaccine. Thus, National Institutes for Food and Drug Control launched a project to establish a non-adsorbed inactivated hepatitis A vaccine reference as the working standard calibrated against the 1st International Standard (IS). Two national standard candidates (NSCs) were obtained from two manufacturers, and designated as NSC A (lyophilized form) and NSC B (liquid form). Six laboratories participated in the collaborative study and were asked to use their in-house validated enzyme-linked immunosorbent assay methods to detect hepatitis A vaccine antigen content. Although both exhibited good parallelism and linear relationship with IS, NSC B showed a better agreement among laboratories than NSC A. And based on suitability of the candidates, NSC B was selected. The accelerated degradation study showed that NSC B was stable at the storage temperature (≤-70 °C). Therefore NSC B was approved as the first Chinese national antigen standard for inactivated hepatitis A vaccine, with an assigned antigen content of 70 IU/ml.

Using immunoglobulin Y as an alternative antibody for the detection of hepatitis A virus in frozen liver sections.

An increasing amount of research has been conducted on immunoglobulin Y (IgY) because the use of IgY offers several advantages with respect to diagnostic testing, including its easy accessibility, low cost and translatability to large-scale production, in addition to the fact that it can be ethically produced. In a previous work, immunoglobulin was produced and purified from egg yolks (IgY) reactive to hepatitis A virus (HAV) antigens. In the present work, this anti-HAV-specific IgY was used in an indirect immunofluorescence assay to detect viral antigens in liver biopsies that were obtained from experimentally infected cynomolgus monkeys. Fields that were positive for HAV antigen were detected in liver sections using confocal microscopy. In conclusion, egg yolks from immunised hens may be a reliable source for antibody production, which can be employed for immunological studies.

Development of recombinant single-chain variable fragment against hepatitis A virus and its use in quantification of hepatitis A antigen.

Phage display technology has been utilized for identification of specific binding molecules to an antigenic target thereby enabling the rapid generation and selection of high affinity, fully human antibodies directed towards disease target appropriate for antibody therapy. In the present study, single chain Fv antibody fragment (scFv) to hepatitis A virus (HAV) was selected from phage displayed antibody library constructed from peripheral blood lymphocytes (PBLs) of a vaccinated donor. The variable heavy (V(H)) and light chains (V(L)) were amplified using cDNA as template, assembled into scFv using splicing by overlap extension PCR (SOE PCR) and cloned into phagemid vector as a fusion for display of scFv on bacteriophage. The phage displaying antibody fragments were subjected to three rounds of panning with HAV antigen on solid phase. High affinity antibodies reactive to hepatitis A virus were identified by phage ELISA and cloned into a bacterial expression vector pET20b. The scFv was purified by immobilized metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The binding activity and specificity of the scFv was established by its non-reactivity towards other human viral antigens as determined by ELISA and immunoblot analysis. The scFv was further used in the development of an in-house IC-ELISA format in combination with a commercially available mouse monoclonal antibody for the quantification of hepatitis A virus antigen in human vaccine preparations. The adjusted r² values obtained by subjecting the values obtained by quantification of the NIBSC standards using the commercial and the in-house ELISA kits by regression analysis were 0.99 and 0.95. 39 vaccine samples were subjected to quantification using both the kits. Regressional statistical analysis through the origin of the samples indicated International Unit (IU) values of 0.0416x and 0.0419x, respectively for the commercial and in-house kit respectively.

[Features of antigen-antibody interaction during use of linear synthetic peptides and multipeptide antigen modeling antigenic determinants of hepatitis A virus].

AIM: To study features of antigen-antibody interaction during use of linear synthetic peptides and multipeptide antigen modelling antigenic determinants of hepatitis A virus (HAV) and to evaluate perspectives for use of heterogeneous tetrameric multipeptide antigens for detection of HAV serological markers. MATERIALS AND METHODS: Linear peptides VP1 and VP3 were synthesized by fluorenylmethyloxycarbonyl (Fmoc)-polyamide solid phase method. MAP4 (VP1+VP3) was synthesized according to 9-Fmoc strategy. Interaction of these peptides with anti-HAV IgM positive sera from patients with HA was studied by noncompetitive and competitive methods of immunoenzyme assay. RESULTS. Using immunoenzyme assay, high heterogeneity of immune response in patients with HA (62 and 67% in two groups) was shown. MAP4 (VP1+VP3), unlike the combination of linear peptides VP1 and VP3, interacted with anti-HAV IgM in 41 - 45% of sera and, at the same time, did not lead to false positive results. CONCLUSION: Population of HAV is not so uniform which is usually assumed. It could be reasonable to use heterogenous multipeptide antigens, including those containing VP1 (11 - 25 a.r.) and VP3 (110 - 121 a.r.), for the development of new assays for HA diagnostics.

Identification of hepatitis A virus mimotopes by phage display, antigenicity and immunogenicity.

A phage-displayed peptide approach was used to identify ligands mimicking antigenic determinants of hepatitis A virus (HAV) for the first time. Bacteriophages displaying HAV mimotopes were isolated from a phage-display peptide library by affinity selection on serum antibodies from hepatitis A patients. Selected phage-peptides were screened for reactivity with sera from HAV infected patients and healthy controls. Four cloned peptides with different sequences were identified as mimotopes of HAV; three of them showed similarity in their amino acid sequences with at least one of the VP3 and VP1 antigenic proteins of HAV. One clone was recognised by 92% of the positive sera. The phagotopes competed effectively with HAV for absorption of anti-HAV-specific antibodies in human sera, as determined by ELISA. The four phage clones induced neutralising anti-HAV antibodies in immunised mice. These results demonstrate the potential of this method to elucidate the disease related epitopes of HAV and to use these mimotopes in diagnostic applications or in the development of a mimotope-based hepatitis A vaccine without the necessity of manipulation of the virus.

Epitope-based recombinant diagnostic antigen to distinguish natural infection from vaccination with hepatitis A virus vaccines.

Hepatitis A virus (HAV) infection can stimulate the production of antibodies to structural and non-structural proteins of the virus. However, vaccination with an inactivated or attenuated HAV vaccine produces antibodies mainly against structural proteins, whereas no or very limited antibodies are produced against the non-structural proteins. Current diagnostic assays to determine exposure to HAV, such as the Abbott HAV AB test, detect antibodies only to the structural proteins and so are not able to distinguish a natural infection from vaccination with an inactivated or attenuated virus. Here, we constructed a recombinant tandem multi-epitope diagnostic antigen (designated 'H1') based on the immune-dominant epitopes of the non-structural proteins of HAV to distinguish the two situations. H1 protein expressed in Escherichia coli and purified by affinity and anion exchange chromatography was applied in a double-antigen sandwich ELISA for the detection of anti-non-structural HAV proteins, which was confirmed to distinguish a natural infection from vaccination with an inactivated or attenuated HAV vaccine.

Differential effect of interleukin-2 treatment on primary and secondary immunizations in HIV infected individuals.

OBJECTIVES: As interleukin (IL)-2 therapy increases CD4 cell counts in HIV infected subjects, it emerged as a candidate for the partial restoration of immune competence in this disease. METHODS: We studied the frequencies of antigen-specific T cells using single cell resolution cytokine ELISPOT assays and titers of specific antibodies before and after immunization of HIV infected subjects who were treated with HAART or HAART plus IL-2. RESULTS: Subjects seronegative to hepatitis A were vaccinated with hepatitis A antigen. In the non-IL-2 treated group, hepatitis A-specific T cells producing IL-2 and IL-4 along with specific antibodies were induced, showing that these subjects are immune competent and capable of mounting a primary immune response. Additional IL-2 treatment had no significant effect on this primary T cell response; however, booster immunizations with tetanus toxoid or the gp120 depleted HIV vaccine Remune induced higher frequencies of specific interferon (IFN)-gamma producing T cells in IL-2 treated subjects. No impact of IL-2 treatment on these secondary B cell responses was seen. CONCLUSION: Overall, our study showed that IL-2 therapy had no immune enhancing effect on the induction of a primary response, but increased the frequency of IFN-gamma producing memory cells after booster immunization.

[Characteristics of cellular immunity in children with acute viral hepatitis A]. / Kharakteristika pokazatelei kletochnogo immuniteta u detei, bol'nykh ostrym virusnym gepatitom A.

AIM: To study the populational composition of lymphocytes and the specific features of production of cytokines in children with acute viral hepatitis A (AVHA). MATERIALS AND METHODS: 128 patients aged 11 to 14 years who had AVHA, moderate AVHA being in 83.5% were examined. In 87.2% of the children, the disease was cyclic. The etiology of the disease was verified by simultaneously detecting anti-HAV IgM (enzyme immunoassay) and by the presence of HAV RNA (polymerase chain reaction) in the blood. Peripheral lymphocytes (CD) were phenotypes in the indirect immunofluorescence test using monoclonal antibodies; cytokines were determined by the enzyme immunoassay; serum beta 2-microglobulin was done by radioimmunoassay. RESULTS: The children with AVHA were found to have elevated levels of tumor necrosis beta-factor, interleukin-1 beta (IL-1 beta), and IL-4 in the icteric period, as well as a decrease in their levels at convalescence. IL-6 was detected in individual patients only in the first 3 days of the icteric period. The peak of AVHA was characterized by relative lymphocytosis, by decreases in the counts of T helper/inductor cells and natural killer cells, by increases in the count of CD25 cells, convalescence, by preserved lymphocytosis, by the increased levels of T lymphocytes that carry the markers CD3, CD4, CD8, CD16, CD25, and CD95, and by higher toxicity of beta 2-microglobulin.

[The characterization analysis of HAV recombinant antigen was expressed by vaccinia virus vector].

OBJECTIVE: To find a suitable cell line for hepatitis A antigen expressed by vaccinia virus vector and to find a way of inactivation and preservation of the HAV recombinant antigen. Methods Series of cell lines such as K4,143, HEL, Hep-2 and Vero were inoculated with vaccinia virus that can express HAV recombinant antigen. ELISA was used to determine the contents of expression antigen. The characterization of the HAV antigen expressed by vaccinia virus was then analyzed after it was treated with different methods. RESULTS: The expression of HAV recombinant antigen in K4,143 and HEL cell lines was a little more than expression in Hep-2 and Vero cell lines. The antigenicity is obviously higher when HAV recombinant antigen was inactivated by beta-propiolactone other than it was inactivated by formalin. It was best to preserve the prepared HAV recombinant antigen under -40 degrees C condition. CONCLUSIONS: The application of vaccinia virus vector in hepatitis A antigen preparation was very useful and promising.

Changing epidemiology of hepatitis A infection in Izmir, Turkey.

The seroprevalence study was conducted in order to determine the current seroepidemiology hepatitis A in Izmir, Turkey and to evaluate the epidemiological shift in HAV serostatus. Blood samples collected from 595 subjects aged 1-60 years were analyzed for anti-HAV IgG antibodies. The current study results were compared with those of a previous study conducted in 1998 involving the same location. There was a marked decrease in the prevalence of anti-HAV between 1998 and 2008. While anti-HAV seroprevalence rates in the current study were 4.6% in children aged 1-4 years, 23% in children aged 10-14 years, and 85% in young adults aged 20-29 years, the prevalence rates were 36% in the 1-4 years age group, 65% in the 10-14 years age group, and 95% in young adults in the previous study, indicating a shift in HAV seroprevalence from the younger to the higher age groups. As HAV infection in childhood is decreasing, the pool of susceptible adolescents and young adults is increasing in Izmir, Turkey. The majority of adolescent population is susceptible to HAV infection. The potential risk of HAV epidemics still exists. The situation of Turkey, suggested to need for mass immunization. Also, introduction of hepatitis A vaccination into the national immunization schedule of Turkey should be considered.

Using immunoglobulin Y as an alternative antibody for the detection of hepatitis A virus in frozen liver sections

An increasing amount of research has been conducted on immunoglobulin Y (IgY) because the use of IgY offers several advantages with respect to diagnostic testing, including its easy accessibility, low cost and translatability to large-scale production, in addition to the fact that it can be ethically produced. In a previous work, immunoglobulin was produced and purified from egg yolks (IgY) reactive to hepatitis A virus (HAV) antigens. In the present work, this anti-HAV-specific IgY was used in an indirect immunofluorescence assay to detect viral antigens in liver biopsies that were obtained from experimentally infected cynomolgus monkeys. Fields that were positive for HAV antigen were detected in liver sections using confocal microscopy. In conclusion, egg yolks from immunised hens may be a reliable source for antibody production, which can be employed for immunological studies.

Efficient neutralizing activity of cocktailed recombinant human antibodies against hepatitis A virus infection in vitro and in vivo.

Hepatitis A virus (HAV) is the major pathogen responsible for acute infectious hepatitis A, a disease that is prevalent worldwide. Although HAV immunization effectively prevents infection, primary immunizations must be administered at least 2 weeks prior to HAV exposure. In contrast, passive immunization with pooled human immunoglobulin (Ig) can provide immediate and rapid protection from HAV infection. Because the use of human sera-derived Igs carries the risk of contamination, we sought to develop recombinant HAV-neutralizing human antibodies. We prepared a combinatorial phage display library of recombinant human anti-HAV antibodies from RNA extracted from the blood lymphocytes of a convalescent hepatitis A patient. Two recombinant human IgG antibodies, HAIgG16 and HAIgG78, were screened from the antibody library by their ability to bind with high affinity to purified, inactivated HAV virions. These antibodies recognized different epitopes of the HAV virion capsid, and competed with both patient sera and well-characterized neutralizing mouse monoclonal antibodies. A cocktailed mixture of HAIgG16 and HAIgG78 at a 3:1 ratio was prepared to compare its combined biological activity with that conferred by each antibody individually. The cocktailed antibodies displayed a stronger neutralizing activity in vitro than that observed with either HAIgG16 and HAIgG78 alone. To determine the in vivo neutralizing abilities of these antibodies, rhesus monkeys were inoculated with cocktailed antibodies and challenged with HAV. Whereas control animals developed hepatitis A and seroconverted to the HAV antibody, animals receiving cocktailed antibodies were protected either from viral infection or from developing clinical hepatitis. These results demonstrate that recombinant human antibody preparations could be used to prevent or treat early-stage HAV infection.

[Comparison of the Chinese LJ strain structural gene with HM175, MBB, LA strains and the expression of hepatitis A virus antigen by LJ/HM175 recombinant vaccinia virus].

The nucleotide and amino acid sequences of a Chinese hepatitis A virus Long-Jia (LJ) strain were compared with that of HM175, MBB and LA strains in structural genes (nt 630-3049). The most extensive nucleotide homology was identified between LJ and MBB strains. The identity rates of nucleotide were 95.4%, 96.7% and 91.4%, respectively. Variation rates of amino acid were 0.91%, 0.91% and 2.98%, respectively. A total of 23 amino acid differences located in whole capsid region between LJ and LA strain, especially in VP1. Only 7 amino acid differences located in VP2 and VP3 between LJ and HM175/MBB strain. Restriction enzyme sites increased 10, 13 and 30 sites in 56 restriction enzymes tested, and decreases 15, 1 and 27 sites, respectively. BstE II (nt 2810) and Pvu I (nt 2013) were the peculiar sites of LJ strain. Hind III, Pst I and Sac I sites were identical among the four strains. After structural gene (nt 745-2993) of HM175 strain was replaced by LJ strain, the complete hepatitis A virus cDNA open reading frame was inserted into pJSA1175 (vaccinia virus expression vector) downstream of promoter 7.5 k. Hepatitis A virus antigen expressed was 1:16 in titer by sandwich ELISA. Band-patterns of anti-VP0, anti-VP1 and anti-VP1, 2, 3, were as same as that of HM175 strain and natural hepatitis A virus antigen by Western blot analysis.

Antigenic hepatitis A virus structures may be produced in Escherichia coli.

The synthesis of 14S pentamers and 70S empty capsids of hepatitis A virus (HAV) has been accomplished by expressing the viral genome for periods of time longer than 4 h in Escherichia coli. HAV pentamers (14S) self-assembled into capsids (70S) in vitro. The antibodies induced by these structures recognized and neutralized HAV.