Characterization of human serotype 1 astrovirus-neutralizing epitopes

Astroviruses are important agents of pediatric gastroenteritis. To better understand astrovirus antigenic structure and the basis of protective immunity, monoclonal antibodies (MAbs) were produced against serotype 1 human astrovirus. Four MAbs were generated. One MAb (8G4) was nonneutralizing but reacted to all seven serotypes of astrovirus by enzyme-linked immunosorbentassay (ELISA) and immunoperoxidase staining of infected cells. Three MAbs were found to have potent neutralizing activity against astrovirus. The first (5B7) was serotype 1 specific, another (7C2) neutralized all seven human astrovirus serotypes, while the third (3B2) neutralized serotypes 1 and 7. Immunoprecipitation of radiolabeled astrovirus proteins from supernatants of astrovirus-infected cells showed that all three neutralizing antibodies reacted with VP29. MAb 5B7 also reacted strongly with VP26. A competition ELISA showed that all three neutralizing antibodies competed with each other for binding to purified astrovirus virions, suggesting that their epitopes were topographically in close proximity. None of the neutralizing MAbs competed with nonneutralizing MAb 8G4. The neutralizing MAbs were used to select antigenic variant astroviruses, which were then studied in neutralization assays. These assays also suggested a close relationship between the respective epitopes. All three neutralizing MAbs were able to prevent attachment of radiolabeled astrovirus particles to human Caco 2 intestinal cell monolayers. Taken together, these data suggest that the astrovirus capsid protein VP29 may be important in viral neutralization, heterotypic immunity, and virus attachment to target cells.     

Generation of neutralizing human monoclonal antibodies against parvovirus B19 proteins

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 microgram/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 micrograms/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.     

Preferential selection of receptor-binding variants of influenza virus hemagglutinin by the neutralizing antibody repertoire of transgenic mice expressing a human immunoglobulin mu minigene

An analysis was made of the neutralizing antibody repertoire, for influenza virus hemagglutinin (HA) of transgenic mice expressing a human immunoglobulin mu (IgH) minigene, by monoclonal antibody (MAb) selection and sequencing of the HA genes of X31 (H3N2 subtype) laboratory variants. Whereas previously reported laboratory variants, selected in ovo with high-affinity murine MAbs of the IgG class, differed from wild-type virus by a single amino acid residue change in one of the major antigenic sites, neutralizing MAbs from transgenic donors selected novel variant viruses with altered receptor-binding specificity and contained residue changes in both the receptor-binding pocket (HA1 225 or HA1 226) and an antigenic site (HA1 135, HA1 145, or HA1 158). Changes in receptor-binding specificities of the variant viruses were confirmed by their resistance to inhibition by horse serum glycoproteins and altered binding to neoglycoproteins. The residue changes in variant virus V-21.2 (HA1 135 G-->R, 225 G-->D) abrogated neutralization by each of the MAbs; nevertheless V-21.2 was recognized by its own selecting MAb in enzyme-linked immunosorbent assay and therefore qualified as an adsorptive mutant rather than an antigenic variant. We consider that a low-affinity neutralizing antibody response may preferentially select for receptor-binding variants of influenza virus HA.     

Two amino acid residues confer type specificity to a neutralizing, conformationally dependent epitope on human papillomavirus type 11

Characterization of virus binding by neutralizing antibodies is important both in understanding early events in viral infectivity and in development of vaccines. Neutralizing monoclonal antibodies (MAbs) to human papillomavirus type 11 (HPV11) have been described, but mapping the binding site has been difficult because of the conformational nature of key type-specific neutralization epitopes on the L1 coat protein. We have determined those residues of the L1 protein of HPV11 which confer type specificity to the binding of HPV11-neutralizing MAbs. Binding of three HPV11-specific neutralizing MAbs could be redirected to HPV6 L1 virus-like particles in which as few as two substitutions of corresponding amino acid residues from HPV11 L1 have been made, thus demonstrating the importance of these residues to MAb binding through the transfer of a conformationally dependent epitope. In addition, a fourth neutralizing MAb could be distinguished from the other neutralizing MAbs in terms of the amino acid residues which affect binding, suggesting the possibility that it neutralizes HPV11 through a different mechanism.     

Antigenic and genomic diversity of human rotavirus VP4 in two consecutive epidemic seasons in Mexico

In the present investigation we characterized the antigenic diversity of the VP4 and VP7 proteins in 309 and 261 human rotavirus strains isolated during two consecutive epidemic seasons, respectively, in three different regions of Mexico. G3 was found to be the prevalent VP7 serotype during the first year, being superseded by serotype G1 strains during the second season. To antigenically characterize the VP4 protein of the strains isolated, we used five neutralizing monoclonal antibodies (MAbs) which showed specificity for VP4 serotypes P1A, P1B, and P2 in earlier studies. Eight different patterns of reactivity with these MAbs were found, and the prevalence of three of these patterns varied from one season to the next. The P genotype of a subset of 52 samples was determined by PCR. Among the strains characterized as genotype P[4] and P[8] there were three and five different VP4 MAb reactivity patterns, respectively, indicating that the diversity of neutralization epitopes in VP4 is greater than that previously appreciated by the genomic typing methods.     

Somatostatin-14, somatostatin-28, and prosomatostatin[1-10] are independently and efficiently processed from prosomatostatin in the constitutive secretory pathway in islet somatostatin tumor cells (1027B2)

Seven neutralizing monoclonal antibodies (nMAbs) produced against serotype Asia-1 foot-and-mouth disease virus (FMDV) were used to select neutralization-resistant variants. Seven single and six multiple antibody-resistant variants were selected to identify neutralization antigenic sites on FMDV Asia-1. The variants no longer reacted with nMAbs which were used to select them when tested by microneutralization test (MNT), radioimmunoassay (RIA) and agar gel immunodiffusion (AGID) assay. Based on the binding and neutralization patterns of the variants, the nMAbs could be divided into discrete groups indicating the presence of three independent antigenic sites with evidence for occurrence of possibly a fourth site on the virus surface. Site 1 was present on 140S, 12Sps and VP1 and thus was conformation-independent. Sites 2 and 3 were restricted to the intact virion (140S) and thus were more conformation-dependent. Site 4 present on 140S virions and 12S protein subunits was less conformation-dependent. The site 3 nMAbs neutralized the infectivity of all the ten different Asia-1 virus isolates tested indicating that this site is conserved in Asia-1 virus serotype. Both cross-neutralization of different Asia-1 viruses with the nMAbs and cross-inhibition assays between MAbs demonstrated that the nMAbs recognized at least six different epitopes on Asia-1 virus.     

Comparison of neutralizing epitopes among infectious bursal disease viruses using radioimmunoprecipitation

Monoclonal antibodies (MAbs) were used to analyze the relatedness of neutralizing epitopes of infectious bursal disease virus (IBDV) strains. The MAb B69 neutralized the homologous D78 virus but not the MD and Del-A variant strains of IBDV. MAbs 33E8 and R63 neutralized D78 and variant strains MD and Del-A. A cDNA clone consisting of a 1000-base-pair fragment of the VP2 gene from the Del-A IBDV strain was translated using an in vitro system. Six peptides were observed following translation, which represented a full-length product (35.5 kilodaltons) and five truncated products. The translated peptides were radioimmunoprecipitated using MAbs B69, 33E8, and R63. Only MAb R63 immunoprecipitated the in vitro translation products from Del-A. MAbs B69 and 33E8 did not immunoprecipitate the in vitro-translated VP2 peptides. The D78 and Del-A viruses were radiolabeled in vivo using 35S-methionine. Proteins from these viruses were examined by radioimmuno-precipitation with MAbs B69, 33E8, and R63. Although the background made interpretation of the results difficult for MAb B69, this MAb clearly immunoprecipitated VP2 of D78. MAb 33E8 immunoprecipitated the D78 VP3 protein, and R63 immunoprecipitated the VP2 protein. MAb R63 also immunoprecipitated the 35S-methionine-labeled VP2 protein from Del-A. It was concluded that the neutralizing epitope represented by 33E8 was located on VP3 and that the epitope represented by R63 is located on both classic and variant viruses in the region of VP2 that is generally thought to contain sequence variability among IBDV strains.     

Radiotherapy in the treatment of dermatologic malignancies

A large set of monoclonal antibodies (MAbs) directed against the fusion glycoprotein complex F1F2 of bovine respiratory syncytial virus (BRSV) and several polyclonal sera from infected or vaccinated animals were tested in Pepscan to locate linear epitopes on the F-protein. The polyclonal sera mapped to antigenic sites that correspond exactly to known antigenic sites on the F protein of human RSV. Only the neutralizing MAb 3 could be mapped with Pepscan. MAb 3 reacted with three successive overlapping linear peptides that shared the amino acid sequence 173STNKAVVSLS182. The sequence of this novel neutralization site is conserved in all known BRSV- and human RSV-strains and is located on the N-terminus of F1, adjacent to the hydrophobic, putative fusion-related region. This region is probably part of a central coiled-coil stem that is structurally conserved in paramyxovirus fusion and orthomyxovirus hemagglutinin glycoproteins. This linear conserved epitope may be a potential candidate for a peptide-based vaccine which can induce neutralizing antibodies against all groups and subgroups of RSV. Furthermore, the proposed structural features of the neutralization site may aid in the design of a peptide-based vaccine.     

Improved mimicry of a foot-and-mouth disease virus antigenic site by a viral peptide displayed on beta-galactosidase surface

A major antigenic site (site A) of foot-and-mouth disease virus includes multiple overlapping epitopes located within the flexible G-H loop of capsid protein VP1. We have studied the antigenicity of several recombinant E. coli beta-galactosidases displaying the site A from a serotype C virus in different surface regions of the bacterial enzyme. In each one of the explored insertion sites, the recombinant peptide shows different specificity with a set of anti-virus monoclonal antibodies directed to site A. In some of them, the inserted stretch mimics better than free or haemocyanin-coupled peptide the antigenicity of site A in the intact virus. In particular, an insertion within an exposed loop involved in the activating interface of beta-galactosidase (amino acids 272 to 287) led to a significant improvement of the overall reactivity. Since insertions at this site renders proteins enzymatically active, the activating interface could be an adequate place for the presentation of foreign antigens in correctly assembled beta-galactosidase tetramers. These results also suggest that anti-virus antibodies directed against the major antigenic site of FMDV recognize different conformations of the G-H loop, which are better reproduced in some of the recombinant proteins because of the dissimilar restrictions imposed by each particular insertion site.     

Structure of a neutralizing antibody bound monovalently to human rhinovirus 2

The structure of a complex between human rhinovirus 2 (HRV2) and the Fab fragment of neutralizing monoclonal antibody (MAb) 3B10 has been determined to 25-A resolution by cryoelectron microscopy and three-dimensional reconstruction techniques. The footprint of 3B10 on HRV2 is very similar to that of neutralizing MAb 8F5, which binds bivalently across the icosahedral twofold axis. However, the 3B10 Fab fragment (Fab-3B10) is bound in an orientation, inclined at approximately 45 degrees to the surface of the virus capsid, which is compatible only with monovalent binding of the antibody. The canyon around the fivefold axis is not directly obstructed by the bound Fab. The X-ray structures of a closely related HRV (HRV1A) and a Fab fragment were fitted to the density maps of the HRV2-Fab-3B10 complex obtained by cryoelectron microscope techniques. The footprint of 3B10 on the viral surface is largely on VP2 but also covers the VP3 loop centered on residue 3064 and the VP1 loop centered on residue 1267. MAb 3B10 can interact directly with VP2 residue 2164, the site of an escape mutation on VP2, and with VP1 residues 1264 to 1267, the site of a deletion escape mutation. Deletion of these residues shortens the VP1 loop, moving it away from the MAb binding site. All structural and biochemical evidence indicates that MAb 3B10 binds to a conformation epitope on HRV2.     

Two dominant neutralizing antigenic determinants of canine parvovirus are found on the threefold spike of the virus capsid

The 25-nm diameter parvovirus capsid is assembled from 60 copies of a sequence common to the overlapping VP1 and VP2 proteins. Here we examine the epitope specificity's of 28 monoclonal antibodies (MAb) prepared against canine parvovirus (CPV), feline panleukopenia virus (FPV), and raccoon-dog parvovirus or blue (Arctic) fox parvovirus. Comparing the reactivity of those MAb with various MAb-selected escape mutants, or with natural variants of CPV or mink enteritis virus (MEV) which differ at known sequences, showed that the binding of 20 of those MAb was strongly affected by variations of two regions on the threefold spike of the CPV capsid. One region was adjacent to the tip of the threefold spike, and the second was around VP2 residue 300, on the shoulder of that structure. MAb recognizing both antigenic sites efficiently neutralized the virus infectivity and inhibited hemagglutination. Mutations leading to natural antigenic variation have also been observed in both those sites in naturally variant strains of CPV or MEV, suggesting that they are important antigenic structures on these parvoviruses. The bindings of several MAb were not affected by the mutations at those antigenic sites, indicating that they recognized other, and perhaps conserved, structures.     

Characterization of three novel monoclonal antibodies against hepatitis C virus core protein

Three novel monoclonal antibodies (MAbs) were established against a recombinant hepatitis C virus (HCV) core protein derived from cloned genotype 1b HCV cDNA. MAbs C7-50 and C8-59 recognize a conserved linear epitope represented by amino acid residues 21 to 40 of the nucleocapsid protein. MAb C8-48 is directed against a strain-specific conformational epitope located within the first 82 amino acids. A sensitive two-site MAb-based immunoradiometric assay was established using antibodies directed against distinct epitopes on the nucleocapsid protein. Processed 21 kDa core protein was detected by immunoblotting in human hepatocellular carcinoma cell lines and primary adult rat hepatocytes transfected with a cytomegalovirus promoter-driven expression construct. Immunofluorescence microscopy studies revealed a granular and vesicular cytoplasmic staining pattern. MAb C7-50 was used successfully to detect HCV core antigen in chronically infected chimpanzee liver tissue. These MAbs represent important reagents for the study of HCV biology and for the development of immunodiagnostic assays.     

Identification and mapping of the gene encoding the glycoprotein complex gp82-gp105 of human herpesvirus 6 and mapping of the neutralizing epitope recognized by monoclonal antibodies

Monoclonal antibodies (MAbs) 2D4, 2D6, and 13D6 against human herpesvirus 6 (HHV-6) variant A strain GS recognized virion envelope glycoprotein complex gp82-gp105 and neutralized the infectivity of HHV-6 variant A group isolates. A 624-bp genomic fragment (82G) was identified from an HHV-6 strain GS genomic library constructed in the lambda gt11 expression system by immunoscreening with MAb 2D6. Rabbit antibodies against the fusion protein expressed from the genomic insert recognized glycoprotein complex gp82-gp105 from HHV-6-infected cells, thus confirming that the genomic fragment is a portion of the gene(s) that encodes gp82-gp105. This genomic insert hybridized specifically with viral DNAs from HHV-6 variant A strains GS and U1101 under high-stringency conditions but hybridized with HHV-6 variant B strain Z-29 DNA only under low-stringency conditions. DNA sequence analysis of the insert revealed a 167-amino-acid single open reading frame with an open 5' end and a stop codon at the 3' end. Hybridization studies with HHV-6A strain U1102 DNA localized the gp82-gp105-encoding gene to the unique long region near the direct repeat at the right end of the genome. To locate the neutralizing epitope(s) recognized by the MAbs, a series of deletions from the 3' end of the gene were constructed with exonuclease III, and fusion proteins from deletion constructs were tested for reactivity with MAbs in a Western immunoblot assay. Sequencing of deletion constructs at the reactive-nonreactive transition point localized the epitope recognized by the three neutralizing MAbs within or near a repeat amino acid sequence (NIYFNIY) of the putative protein. This repeat sequence region is surrounded on either side by two potential N-glycosylation sites and three cysteine residues.     

Identification of domains in bluetongue virus VP3 molecules essential for the assembly of virus cores

Bluetongue virus (BTV) cores consist of the viral genome and five proteins, including two major components (VP3 and VP7) and three minor components (VP1, VP4, and VP6). VP3 proteins form an inner scaffold for the deposition on the core of the surface layer of VP7. VP3 also encapsidates and interacts with the three minor proteins. The BTV VP3 protein consists of 901 amino acids and has a sequence that is a highly conserved among BTV serotypes and other orbiviruses (e.g., epizootic hemorrhagic disease virus and African horse sickness virus). To locate sites of interaction between VP3 and the other structural proteins, we have analyzed the effects of a number of VP3 deletion mutants representing conserved regions of the protein, using as an assay the formation of core-like particles (CLPs) expressed by recombinant baculoviruses. Five of the VP3 deletion mutants interacted with the coexpressed VP7 and made CLPs. These CLPs also incorporated the three minor proteins. One mutant, lacking VP3 amino acid residues 499 to 508, failed to make CLPs. Further mutational analyses have demonstrated that a methionine at residue 500 of VP3 and an arginine at residue 502 were both required for CLP formation.     

Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (II). Epitope mapping by monoclonal antibodies

PSP94 has shown potential to be a serum biomarker for evaluating prostate cancer. Studies of the epitope structure is crucial for this endeavour. In this article, we have used 15 different monoclonal antibodies (MAb) to analyse the epitope structure of PSP94 and to compare with the results obtained from our previous work using polyclonal antibody and recombinant PSP94. Firstly, we determined the relative activities of the 15 MAb population by direct and competitive ELISA. The two predominant MAbs (MAb PSP-6 and -19) in 15 MAbs were selected for further studies of the epitope structure. By comparing the binding activities of recombinant GST-PSP94 and natural PSP94 with MAbs, and by comparing their affinity with MAbs in an in vitro denaturing experiment, PSP94 was shown to have a similar, prevalently linear epitope structure as we demonstrated by polyclonal antibody. Using recombinant GST fusion protein with PSP94 and with each half of the N- and C-terminal 47 amino acids (GST-PSP-N47/C47) in E. coli cells, the different epitopes recognized by 15 monoclonal antibodies were delineated and the polar distribution of the epitope structure of PSP94 was characterized. Results of direct ELISA of recombinant N47 and C47 and their competitive binding against natural PSP94 (competitive ELISA) showed that the N- and C-termini represent the immuno-dominant and immuno-recessive area separately. A majority of the monoclonal antibodies (12/15) showed preferential binding of the N-terminal sequence of the PSP94 protein. Using GST-PSP-N47 as a standard protein, an epitope map of the 15 monoclonal antibodies was obtained. The results of this study will help to define the clinical utility of PSP94.     

Reactivities of feline calicivirus field isolates with monoclonal antibodies detected by enzyme-linked immunosorbent assay

Reactivities of feline calicivirus (FCV) field isolates with monoclonal antibodies (MAbs) were examined by enzyme-linked immunosorbent assay (ELISA). The reactivities of the viruses in ELISA were different from our previous results using the neutralization tests (NT). Many isolates were positive in ELISA with MAbs which recognized neutralizing epitope 3B and/or 4. However, most were negative in NT in our previous study. After absorption of two FCV strains with host cells, the non-infectious virus fluid still reacted with MAb, which recognized epitope 3B and/or 4 in ELISA. These results indicated the possibility that neutralizing epitopes are expressed on non-infectious virus particles or exist as proteinaceous molecules in virus fluid.     

Synergistic neutralization of a chimeric SIV/HIV type 1 virus with combinations of human anti-HIV type 1 envelope monoclonal antibodies or hyperimmune globulins

A panel of 14 human IgG monoclonal antibodies (MAbs) specific for envelope antigens of the human immunodeficiency virus type 1 (HIV-1), 2 high-titer human anti-HIV-1 immunoglobulin (HIVIG) preparations, and 15 combinations of MAbs or MAb/HIVIG were tested for their ability to neutralize infection of cultured human T cells (MT-2) with a chimeric simian immunodeficiency virus (SHIV-vpu+), which expressed HIV-1 IIIB envelope antigens. Eleven MAbs and both HIVIGs were neutralizing. When used alone, the anti-CD4-binding site MAb b12, the anti-gp41 MAb 2F5, and the anti-gp120 MAb 2G12 were the most potent. When combination regimens involving two MAbs targeting different epitopes were tested, synergy was seen in all paired MAbs, except for one combination that revealed additive effects. The lowest effective antibody concentration for 50% viral neutralization (EC50) and EC90 were achieved with combinations of MAbs b12, 2F5, 2G12, and the anti-V3 MAb 694/98D. Depending on the combination regimen, the concentration of MAbs required to reach 90% virus neutralization was reduced approximately 2- to 25-fold as compared to the dose requirement of individual MAbs to produce the same effect. Synergy of the combination regimens implies that combinations of antibodies may have a role in passive immunoprophylaxis against HIV-1. The ability of SHIV to replicate in rhesus macaques will allow us to test such approaches in vivo.     

Role of magnetic resonance in hemodialysis patients with amyloid shoulder arthropathy]

Monoclonal antibodies (MAbs) were generated by immunizing mice with a truncated recombinant protein corresponding to the immunodominant region (residues 1-120) of hepatitis C virus (HCV) nucleocapsid protein. The specific recognition by either human sera or mouse monoclonal antibodies of overlapping peptides spanning the core region 1-120 as well as the comparison with epitopes described earlier allowed the fine mapping of HCV core. Within the region 1-120, the major antigenic domain could be restricted to the first 45 amino acids. Indeed, the peptide S42G (residues 2-45) allowed the detection of an anti-HCV core response by all anticore-positive human sera examined. According to their epitope localization, three groups of mouse MABs could be evidenced that were directed against different regions of core. Group II MAbs recognized a strictly linear epitope (QDVKF, residues 20-24), whereas group I MABs were directed against a conformational epitope mainly located at the amino acid residues (QIVGG, 29-33). The epitope of group III MABs was also conformational (PRGRRQPI, residues 58-65). These three epitopes appeared close but different from the three major human epitopes RKTKRNTN, VYLLPR, and GRTWAQPGYPWPLY (residues 7-17, 34-39, and 73-86, respectively). Group II MAB 7G12A8 and group I MAB 19D9D6 were used in a sandwich ELISA for the capture and the detection, respectively, of viral core antigen in sera of patients with chronic HCV infection. After treatment of sera with triton x 100 in acidic conditions, amounts of viral antigen as low as 20 pg/ml of sera could be detected.     

Fowl adenovirus recombinant expressing VP2 of infectious bursal disease virus induces protective immunity against bursal disease

The right hand end Nde I fragment 3 (90.8-100 map units) of the fowl adenovirus serotype 10 (FAV-10) was characterised so as to allow the location of an insertion site for recombinant vector construction. Infectious bursal disease virus (IBDV) VP2 gene from the Australian classical strain 002/73, under the control of the FAV-10 major late promoter/leader sequence (MLP/LS) was inserted into a unique Not I site that was generated at 99.5 map units. This recombinant virus was produced without deletion of any portion of the FAV-10 genome. When administered to specific pathogen free (SPF) chickens intravenously, intraperitoneally, subcutaneously or intramuscularly, it was shown that the FAV-10/VP2 recombinant induced a serum VP2 antibody response and protected chickens against challenge with IBDV V877, an intermediate virulent classical strain. Birds were not protected when the recombinant was delivered via the conjunctival sac.     

IgG immune response to B19 parvovirus VP1 and VP2 linear epitopes by immunoblot assay

Human B19 parvovirus recombinant capsid proteins VP1 and VP2 were expressed in E. coli and purified. Recombinant proteins were used to detect a specific IgG immune response against VP1 and VP2 linear epitopes by immunoblot assay. A total of 222 serum samples from 218 apparently immunocompetent subjects with different clinical conditions and laboratory evaluations with regards to B19 infection were analyzed. The sera had previously been tested for B19 DNA and for specific IgM and IgG against VP2 conformational antigens by ELISA assay. The data show that, during the active or very recent phase of infection, IgG anti-VP1 linear epitopes appear in concomitance and with the same frequency as IgG anti-VP2 conformational antigens. IgG against conformational VP2 antigens and against linear VP1 epitopes seem to persist for months or years in the majority of individuals. IgG against VP2 linear epitopes are generally present during the active or very recent phase of infection and during the convalescent phase, while they are present only in about 20% of subjects with signs of a past B19 infection.