Immunogenicity and antigenicity of chimeric picornaviruses which express hepatitis A virus (HAV) peptide sequences: evidence for a neutralization domain near the amino terminus of VP1 of HAV.

We evaluated the antigenic characteristics of chimeric picornaviruses created by inserting peptide sequences from hepatitis A virus (HAV) capsid proteins into the B-C loop of VP1 of Sabin strain type 1 poliovirus (PV-1). Fifteen viable chimeras were generated. Each retained the ability to be neutralized by polyclonal PV-1 antisera. Two chimeras (H15 and H2) stimulated production of low levels of HAV neutralizing antibodies in immunized rabbits or mice, although in both cases only a small fraction of immunized animals produced this response. The H15 chimera, which contains residues 13-24 of HAV VP1, elicited HAV neutralizing antibodies in three of nine rabbits and at least one of seven immunized mice. These results indicate that a neutralization domain exists in this region of VP1. However, human sera with high titers of antibodies to HAV failed to neutralize or immunoprecipitate this chimera, suggesting the absence of a significant antibody response to this neutralization domain following natural infection. Sera from rabbits immunized with H15 that did not develop HAV neutralizing antibodies contained antibodies reactive with the HAV peptide segment expressed by the H15 virus, indicating substantial differences in the specificities of antibodies elicited by this peptide segment among individual immunized rabbits. The H15 peptide insert was an effective antigen, as indicated by a high level of sensitivity of the H15 chimera to neutralization by a related anti-peptide antibody which was itself devoid of HAV neutralizing activity. One of 16 rabbits immunized with the H2 chimera (residues 101-108 of HAV VP1) developed HAV neutralizing antibodies, confirming both the presence and the highly conformational nature of a neutralization antigenic site involving these residues of HAV.     

Use of linear and multiple antigenic peptides in the immunodiagnosis of acute hepatitis A virus infection

The reactivities of two panels of anti-HAV human sera from geographically distinct areas (Chile and Spain) to synthetic peptides from the VP1, VP2 and VP3 hepatitis A virus capsid proteins were examined by an enzyme-linked immunosorbent assay (ELISA) procedure. Two and four branched multiple antigenic peptides (MAPs) and palmitoylated peptides were compared with free synthetic sequences for the detection of IgM anti-HAV antibodies in the two panels of human sera. Our results showed that acute hepatitis A patient sera recognized preferentially homogeneous two branched MAPs and palmitic acid conjugated peptides. The palmitoyl-derived VP3(110-121) peptide and the corresponding dimeric MAP were the most sensitive and appropriate for serological studies of HAV-infected patients by ELISA, sensitivity and specificity being higher than 90% and 95%, respectively. These peptide-based tests open up new avenues in the development of peptide-based immunosorbent assays for the detection of acute HAV disease.     

A new continuous epitope of hepatitis A virus

A new continuous epitope of hepatitis A virus (HAV) was defined in the VP3 protein. Convalescent sera recognised the synthetic peptide 3110–3121 (FWRGDLVFDFQV). The replacement of the arginine, glycine, or aspartic acid at positions 112, 113, or 114, respectively by other aminoacids induced the loss of synthetic peptide recognition by human convalescent sera, thereby confirming the presence of an epitope in the original VP3(110-121) sequence. Shorter VP3 peptides such as VP3(110-119), VP3(110-117), and VP3(110-116) and a tandem repeat of VP3(111-116) failed to react with convalescent sera, indicating the importance of the entire peptide in the epitope structure. The maximum inhibition of human convalescent binding to HAV by the VP3(110-121) peptide was around 60%, and 50% inhibition was achieved at a peptide concentration of 2.3–2.4 μg/ml. Antibodies generated by this peptide bound to intact HAV and neutralised its infectivity. Antipeptide antibodies inhibited convalescent serum binding to HAV. Monoclonal antibodies H7C27 and MAK-4E7 inhibited completely binding of the antipeptide antibodies to HAV. J. Med. Virol. 54:95–102, 1998. © 1998 Wiley-Liss,Inc.     

Expression of hepatitis A virus capsid sequences in insect cells

A cDNA coding for hepatitis A virus (HAV) VP1 and portions of the flanking VP3 and P2 sequences was inserted into the genome of Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter and translational start codon. Cells infected with recombinant virus produced high levels of a 55 kDa protein, identified as containing HAV VP1 by reactivity with anti-VP1 serum. The expressed protein also reacted on immunoblots with human HAV convalescent sera as well as sera from rabbits immunized with intact HAV. This protein was found predominantly in the cytoplasm of infected insect cells, probably as an insoluble aggregate.     

Immune response to hepatitis A virus capsid proteins after infection.

This study was undertaken to determine the immune response of humans to viral capsid polypeptides of hepatitis A virus (HAV) after natural infection, which is very important for vaccine development. Antiviral capsids in 73 serum samples from patients with acute and chronic HAV infections were analyzed by immunoblotting against individual HAV capsid polypeptides (VP1, VP2, VP3, and VP4) by using a cell culture-based HAV antigen. For reference, total anti-HAV immunoglobulin G (IgG) and anti-HAV IgM were also determined by radioimmunoassay. As a result, a dominant immune response against VP1 (98% IgG, 94% IgM) was found in the acute phase. However, many other sera also reacted with VP0 (88% IgG; 35% IgM) and VP3 (81% IgG and 29% IgM). In contrast to the acute phase, anti-VP1, anti-VP0, and anti-VP3, IgG antibodies against all three viral proteins (29, 29, and 73% respectively), especially those against VP3, were found years after onset of HAV disease and over long periods in the sera of hepatitis patients. These results suggest that antibodies for capsid polypeptides are present over an extended period in the sera of HAV-infected patients. They are likely of importance in maintaining long-term immunity.     

Hepatitis in Albanian children: molecular analysis of hepatitis A virus isolates

Hepatitis A is a common disease in developing countries and Albania has a high prevalence of this disease associated to young age. In spite of the occurrence of a unique serotype there are different genotypes classified from I to VII. Genotype characterisation of HAV isolates circulating in Albania has been undertaken, as well as the study of the occurrence of antigenic variants in the proteins VP3 and VP1. To evaluate the genetic variability of the Albanian hepatitis A virus (HAV) isolates, samples were collected from 12 different cities, and the VP1/2A junction amplified and sequenced. These sequences were aligned and a phylogenetic analysis performed. Additionally, the amino half sequence of the protein VP3 and the complete sequence of the VP1 was determined. Anti-HAV IgM were present in 66.2% of all the sera. Fifty HAV isolates were amplified and the analysis revealed that all the isolates were sub-genotype IA with only limited mutations. When the deduced amino acid sequences were obtained, the alignment showed only two amino acids substitutions at positions 22 and 34 of the 2A protein. A higher genomic stability of the VP1/2A region, in contrast with what occurs in other parts of the world could be observed, indicating high endemicity of HAV in Albania. In addition, two potential antigenic variants were detected. The first at position 46 of VP3 in seven isolates and the second at position 23 of VP1 in six isolates.     

Immunoreactivity of human and rabbit antisera to hepatitis A virus

Rabbit antibodies produced by immunization with complete hepatitis A virions (HAV) recognized all the viral structural proteins and neutralized HAV infectivity in cell culture. Rabbit antibodies to chromatographically purified individual viral proteins and to synthetic peptides representing epitopes on the structural viral protein VP1 neither recognized whole virus nor neutralized infectivity, indicating that native epitopes on the virus surface are necessary for virus recognition and neutralization. Human anti-HAV-positive sera of the acute and convalescent phase of disease recognized and neutralized viral particles. Analysis of the immunoreactivity of these human sera in immunoblot showed that the IgM antibody preferentially recognizes the structural viral proteins VP0 and VP3 of HAV, whereas IgA and IgG antibodies reacted more strongly with VP1.     

Molecular epidemiology of hepatitis A virus in patients in the Ahwaz region of Iran

Hepatitis A virus (HAV) is one of the etiologic agents of acute viral hepatitis, an important public health problem worldwide. The aim of this study was to investigate the genetic diversity of HAV in Southwest Iran (Ahwaz). A total of 59 sera were collected from acutely ill patients with anti-HAV IgM antibodies during 2009 and 2010 were tested also by RT-PCR targeting the 5' NCR for molecular diagnosis and examined in the VP1-2A and VP3-VP1 regions for genotyping. Twelve (20%) patients were detected VP1-2A by RT-PCR and 10 patients had VP3-VP1. The resulting amplicons were sequenced for genotype identification. All HAV strains were identified as subgenotype IB. Phylogenetic analysis revealed an extensive genetic heterogeneity among the strains. Seven hundred sixty-five S→F and 788 K→R amino acid substitutions in IRI49 isolate were found. It is concluded that subgenotype 1b is the sole genotype HAV in this region.     

Genetic analysis of HAV strains in Tunisia reveals two new antigenic variants

To evaluate the genetic variability of hepatitis A virus (HAV) isolates in Tunisia, serum samples were collected from 99 patients in different Tunisian areas in 2003 containing 92 cases with acute hepatitis, five with severe acute hepatitis and two with fulminant hepatitis. The entire VP1 gene was amplified and sequenced. Sequences were then aligned and a phylogenetic analysis was performed. Additionally, the amino acid (aa) sequence of the VP1 was determined. The analysis of Tunisian HAV isolates revealed that all the isolates were sub-genotype IA with 96.4%–99.8% of identity and showed the emergence of two novel antigenic variants. The Tun31-03 antigenic variant, with a 38 aa deletion containing Met156, Val171, Leu174 and Ala176 and located between 150 and 187 aa of the VP1 protein where neutralization escape mutations, was found. The second antigenic variant, Tun36-03, was isolated from a patient with fulminant hepatitis and presented a substitution of Thr by Pro at position 10 of the VP1 protein. This amino acid is located in a peptide presenting an antigenically reactive epitope of the VP1 protein. This substitution has never been described previously.     

Recombinant proteins VP1 and VP3 of hepatitis A virus prime for neutralizing response

Six overlapping genomic regions of capsid proteins VP1 and VP3 of hepatitis A virus (HAV) inserted into the expression vectors pBD or pUR respectively expressed beta-galactosidase-HAV fusion proteins. The recombinant proteins were poorly soluble so they were difficult to detect by human anti-HAV sera in radioimmunoassay, but the fusion proteins dissolved in sodium dodecyl sulfate reacted with human and rabbit anti-HAV-positive sera in immunoblots. Antisera against VP1 and VP3 recombinant proteins reacted with the respective structural proteins of HAV in immunoblots. Two recombinant proteins, one including the first 120 amino acids of the N-terminus of VP1 and the other containing all of VP1 except for the first 60 N-terminal amino acids, induced a transient neutralizing antibody response in rabbits. Antisera directed against other regions of VP1 and VP3 neither neutralized viral infectivity nor recognized native virus in a competitive radioimmunoassay. However, when immunized animals were challenged with a sub-immunogenic dose of HAV, all animals responded with stable virus-neutralizing antibodies.     

Identification of amino acids located in the antibody binding sites of human hepatitis A virus.

Antigenic mutants of human hepatitis A virus (human-HAV) were isolated by their resistance to neutralizing monoclonal antibodies raised to human-HAV. The nucleotide sequence determined for the capsid regions of 12 mutants identified amino acid changes that clustered in three non-overlapping sites; one in VP3 and two in VP1. All mutants had a change at amino acid residue 70 in VP3, indicating its primary importance for antibody binding. Ten mutants had two amino acid changes occurring in the VP3 site as well as one in one of the two VP1 sites. These data suggest that both sites in VP1 interact with the single VP3 site to form the immunodominant epitope of HAV. The amino acid changes found in the antigenic mutants of human-HAV selected in this study were located in the same positions as changes found in strains of HAV isolated from Old World monkeys. These simian strains of HAV are not recognized by most monoclonal antibodies raised to human-HAV, suggesting that the observed amino acid changes are part of the antibody binding site.     

Sequential changes in hepatitis A virus genotype distribution in Estonia during 1994 to 2001

Hepatitis A virus (HAV) isolates from a large outbreak and from non-outbreak cases in Estonia were characterized by sequencing the aminoterminal VP1 region. From January 1998 to December 1999, a total of 1084 cases of hepatitis A were reported to the Harjumaa-Tallinn and Ida-Virumaa Health Protection Services in Estonia. The attack rate was highest among males aged 15-29. Initial cases were noted to be associated with injecting drug use. IgM anti-HAV positive sera were available from 107 hospitalized outbreak cases and from 68 patients sampled during 1994 to 2001. HAV RNA was detected in 42% of sera from 1994-1996 and in 88% of sera from 1998-2001. It was possible to obtain HAV sequences from 83 outbreak and 29 background cases. The outbreak strain was represented by five different sequences, all belonging to subtype IIIA. During the outbreak, this IIIA strain also spread into the general population. All available non-outbreak isolates from 1994 to 2001 but one belonged to genotype IA and formed distinct clusters as compared to isolates from other parts of the world. One subtype IIIA isolate from 1995 was unrelated to the outbreak strain. Subtype IA had been dominating in Estonia during 1994-2001, but the outbreak strain from 1998 to 1999 was IIIA. This subtype was encountered previously in addicts in Sweden during the 1980s and in Norway at the end of the 1990s. This study supports the use of limited sequencing within the aminoterminal VP1 region for studying the molecular epidemiology of hepatitis A.     

Characterization of mimotopes mimicking an immunodominant conformational epitope on the hepatitis C virus NS3 helicase

The hepatitis C virus (HCV) nonstructural 3 (NS3) protein is composed of an amino terminal protease and a carboxyl terminal RNA helicase. NS3 contains major antigenic epitopes. The antibody response to NS3 appears early in the course of infection and is focused on the helicase region. However, this response cannot be defined by short synthetic peptides indicating the recognition of conformation-dependent epitopes. In this study, we have screened a dodecapeptide library displayed on phage with anti-NS3 mouse monoclonal antibodies (mAbs) that compete with each other and human anti-HCV NS3 positive sera. Two peptides (mimotopes) were selected that appeared to mimic an immunodominant epitope since they were recognized specifically by the different anti-NS3 mAbs of the study and by human sera from HCV infected patients. Homology search between the two mimotopes and the NS3 sequence showed that one of the two peptides shared amino acid similarities with NS3 at residues 1396-1398 on a very accessible loop as visualized on the three-dimensional structure of the helicase domain whereas the other one had two amino acids similar to nearby residues 1376 and 1378. Reproduced as synthetic dodecapeptides, the two mimotopes were recognized specifically by 19 and 22, respectively, out of 49 sera from HCV infected patients. These mimotopes allowed also the detection of anti-NS3 antibodies in sera of HCV patients at the seroconversion stage. These results suggest that the two NS3 mimotopes are potential tools for the diagnosis of HCV infection.     

Antigenic Epitopes of the Hepatitis A Virus Polyprotein

Forty-two antigenic domains were identified across the hepatitis A virus (HAV) polyprotein by using a set of 237 overlapping 20-mer synthetic peptides spanning the entire HAV polyprotein and a panel of serum samples from acutely HAV-infected patients. The term "antigenic domain" is used in this study to define a protein region spanned with consecutive overlapping immunoreactive peptides. Nineteen antigenic domains were found within the structural proteins, and 22 were found within the nonstructural proteins, with 1 domain spanning the junction of VP1 and P2A proteins. Five of these domains were considered immunodominant, as judged by both the breadth and the strength of their immunoreactivity. One domain is located within the VP2 protein at position 57-90 aa. A second domain, located at position 767-842 aa, contains the C-terminal part of the VP1 protein and the entire P2A protein. A third domain, located at position 1403-1456 aa, comprises the C-terminal part of the P2C protein and the N-terminal half of the P3A protein. The fourth domain, located at position 1500-1519 aa, includes almost the entire P3B, and the last domain, located at position 1719-1764 aa, contains the C-terminal region of the P3C protein and the N-terminal region of the P3D protein. It is interesting to note that four of the five most immunoreactive domains are derived from small HAV proteins and/or encompass protein cleavage sites separating different HAV proteins. The HAV-specific immunoreactivity of each antigenically reactive peptide was confirmed by using seven HAV seroconversion panels. Collectively, these data demonstrate that HAV structural and nonstructural proteins contain antigenic epitopes that can be efficiently modeled with short synthetic peptides.     

Antigenic Sites of Coxsackievirus A9

Antigenic analysis of coxsackievirus A9 (CAV9) was carried out by using a peptide scanning method. Immunogenic regions in the capsid proteins VP1, VP2, and VP3 were recognized by antibodies in the sera of virus-immunized rabbits. The peptide sequences were scanned using a 12-amino-acid window and three-residue shift. Three immunogenic regions, located in the N- and C-terminal parts of VP1 and in the N-terminus of VP3, were identified. Trypsin treatment of the virus, known to cleave off the C-terminus of VP1 containing a functional RGD motif, completely abolished the reactivity against this region but did not have any other significant effect on antigenicity. In further studies, it was found that the RGD motif itself was poorly immunogenic whereas antibody-binding sites were located at both sides of the motif. New antigenic sites emerged after heat treatment of CAV9 at 56 or 100°C prior to immunization; in particular, loop structures between β strands in VP2 exhibited increased immunogenicity. New antigenic sites in VP1 and VP3 also appeared after the treatments. In spite of the markedly altered reactivity in peptide scanning, the virus treated at 56°C elicited high titers of neutralizing antibodies. To reveal cross-reactive antigenic sites, antisera raised against coxsackievirus B3 and echovirus 11 were also tested. The cross-reactive antigenic sites were located mainly in the N-terminal parts of VP1 and VP3.     

Genetic analysis of HAV strains isolated from patients with acute hepatitis in Korea, 2005-2006

Hepatitis A virus (HAV) is a causative agent of acute viral hepatitis, which represents a significant public health problem. HAV is usually transmitted by oral-fecal route and prevalent not only in developing countries but also in developed countries worldwide. To characterize the HAV wild type strains circulating in Korea, the VP3/VP1 and VP1/P2A junction regions were detected by RT-PCR from HAV IgM positives during 2005 and 2006. Among 160 HAV IgM positive sera, 30% (n = 48) were positive for HAV RNA. Additionally, the VP3/VP1 junction regions were detected all six stools, which collected from outbreak in Gyeonggi province. Phylogenetic analysis of the sequences obtained from 54 distinct HAV isolates revealed that most of the strains (n = 45) belonged to genotype IA and the others including nine strains belonged to genotype IIIA. Interestingly, a Q --> S amino acid change was dominantly observed at position 810 of the VP1/P2A junction region in 14 isolates. The molecular epidemiology of HAV infection in Korea has changed with the co-circulation of at least two genotypes and 810Q --> S amino acid substitutions were found to be prevalent. These results strongly suggest that various HAV strains, including genotype IIIA, might be imported from high-endemic countries into Korea.     

Increased circulation of hepatitis A virus genotype IIIA over the last decade in St Petersburg, Russia

The current study, covering the period 2004–2009, is a part of long‐term monitoring for hepatitis A virus (HAV) strains circulating in St Petersburg, Russia. The HAV RNA was isolated directly from the sera of hepatitis A patients and RT‐PCR was carried out using primer pairs for VP1/2A and VP1 genomic regions. PCR products were sequenced and 324 nucleotides from VP1/2A and 332 from the VP1 region were used for phylogenetic analysis. The results show that the IA subtype was the most common circulating subtype during the follow‐up period, as found in the previous study: almost 90% of the isolated HAV strains belonged to the IA subtype. The large hepatitis A food‐borne outbreak in St Petersburg in 2005 was caused by HAV IA. However, the proportion of HAV isolates belonging to subtype IIIA significantly increased in the period 2001–2009 (7.9%) compared to the period 1997–2000 (none found). The subtype IIIA was first found in St Petersburg in 2001 among a group of intravenous drug users. The increase in its circulation during the decade suggests that this previously unusual genotype has been permanently introduced into the general population of St Petersburg. These results indicate the usefulness of molecular epidemiological methods for studying changes in the circulation of HAV strains. J. Med. Virol. 84:1528–1534, 2012. © 2012 Wiley Periodicals, Inc.