Microbiology and Tumor Biology Center, Karolinska Institutet, S-171 77 Stockholm, Sweden1
Swedish Institute for Infectious Disease Control, S-171 82 Stockholm, Sweden2
Author for correspondence: ke Lundkvist (at the Swedish Institute for Infectious Disease Control). Fax +46 8 31 47 44. e-mail akelun{at}mbox.ki.se
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Abstract |
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Introduction |
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HFRS caused by PUUV is generally milder than HFRS caused by Dobrava or Hantaan hantaviruses and rarely results in haemorrhages. Although the mortality of PUUV infections is low (<0·2%), the virus causes significant morbidity in northern and eastern Europe; Russia reports around 5000, Finland 1000, Sweden 300 and Norway 50 PUUV cases each year. Sporadic outbreaks are observed in central Europe and we have recently reported major outbreaks in Bosnia and Belgium with hundreds of cases (Lundkvist et al., 1997 ; Heyman et al., 1999
).
The PUU virion consists of four structural proteins: the RNA polymerase, two envelope glycoproteins (G1 and G2) and a nucleocapsid protein (N) (Schmaljohn, 1996 ). Both glycoproteins have been shown to express epitopes that are recognized as targets for neutralizing antibodies (Lundkvist & Niklasson, 1992
).
The significance of a specific immunoglobulin M (IgM) response during acute NE and its outstanding value for serodiagnosis have been described by several authors (Zöller et al., 1993 ; Elgh et al., 1996
; Brus-Sjölander et al., 1997
). We have previously investigated the kinetics of the IgM, total IgG and IgG subclass responses to the different structural components of PUUV (Lundkvist et al., 1993a
, b, 1995
). In addition to the highly virus-neutralizing IgG response in NE-convalescent serum, IgM has been suggested to have a significant neutralizing activity during the acute course of NE (Hörling et al., 1992
). In contrast, knowledge of the IgA responses to hantavirus infections is still limited and only a few studies have been reported (Elgh et al., 1996
, 1998
; Groen et al., 1994
; Patnaik et al., 1999
).
At a production rate of 66 mg/kg/day, secretory and systemic IgA is quantitatively the most important immunoglobulin in the development of mucosal immunity and plays a major role in protecting mucosal surfaces. Neutralizing IgAs have been reported against several viruses, e.g. influenza, polio and Sendai viruses (Wang, 1986 ; Page et al., 1988
; Mazanec et al., 1987
). It has also been reported that monoclonal IgA alone was capable of protecting mice against influenza by passive transfer (Renegar & Small, 1991
). Antigen-specific IgA responses may therefore provide an important reduction in levels of the infectious pathogen at the first line of defence, the mucosal surface. Furthermore, IgA may play a role in the pathology of hantavirus infection, e.g. by immune complex formation. This study describes the characterization of the IgA response in NE patients and an evaluation of the diagnostic value of PUUV IgA-specific assays.
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Methods |
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Radial immuno-diffusion (RID) assay.
Total serum IgA was determined by RID assay as described by the manufacturer (The Binding Site). Briefly, 10 µl of the serum samples or calibrators was applied to the gel plate. The plate was incubated at room temperature for 18 h before measurement of radial diffusion. The IgA concentrations of the samples were estimated by comparison of the diffusion diameters with those of the calibrators containing known amounts of IgA. Statistical analyses were performed with Students unpaired t-test and the MannWhitney U-test.
Reagents for ELISA.
Antigen preparations (detergent-treated cell lysates of Vero E6 cells infected with PUUV strain Sotkamo) containing the structural proteins of PUUV, N, G1 and G2, were prepared as described elsewhere (Lundkvist & Niklasson, 1992 ). Bank vole (Clethrionomys glareolus) monoclonal antibodies (MAbs) 1C12, 5A2 and 5B7, specific for N, G1 and G2, respectively, were prepared and purified on protein G columns as described elsewhere (Lundkvist et al., 1991
; Lundkvist & Niklasson, 1992
). Biotin-labelled mouse MAbs (Southern Biotechnology Associates) were used for detection of human IgA1 and IgA2, followed by streptavidin-conjugated peroxidase (Sigma).
ELISAs for detection of IgA subclasses against the structural proteins of PUUV.
Three series of ELISAs were employed, one for each structural protein of the PUU virion. For each series, a bank vole MAb specific for the appropriate viral protein (N, G1 or G2) was used for antigen binding. Affinity-purified MAbs were diluted to 10 µg/ml in 0·05 M sodium carbonate, pH 9·6, and adsorbed to 96-well microtitre plates (Costar) overnight at 4 °C. Non-saturated binding sites were blocked overnight at 4 °C with 3% BSA in PBS.
The following reagents were diluted in ELISA buffer (PBS, 0·05% Tween 20, 0·5% BSA), added to wells and incubated for 1 h at 37 °C and the plates were washed five times with 0·9% NaCl with 0·05% Tween 20 between each step. After incubation with viral antigen and negative-control antigen (ELISA buffer), serum samples diluted 1:100 were added in duplicate to wells with antigen and to wells with negative-control antigen, followed by biotin-labelled MAbs specific for the two subclasses of human IgA. Streptavidinperoxidase was added and wells were incubated for 45 min at 37 °C followed by addition of tetramethylbenzidine (TMB) substrate (Sigma).
On all plates, one acute-phase serum was used as an internal standard. The mean absorbance of the standard duplicate was recalculated to 1·000 on each plate and the mean value of the sample duplicates was then adjusted correspondingly. The results were calculated as follows: the mean absorbance of the serum duplicates with virus antigen was reduced by the background mean absorbance obtained with negative-control antigen.
Epitope mapping (PEPSCAN).
The PEPSCAN method (Geysen et al., 1987 ), designed for identification of linear B cell antigenic sites, was used to locate antibody-reactive peptides contained within the sequence of N protein of PUUV strain Sotkamo (Vapalahti et al., 1992
). In total, 86 peptides (10-mer overlapping peptides covering the complete N protein by shifts of 5 amino acids; Lundkvist et al., 1995
) were examined. Antibody reactivities with PEPSCAN peptides were measured by ELISA, as described previously (Geysen et al., 1987
), using sera diluted 1:100. Bound antibodies were detected with peroxidase-conjugated goat anti-human IgA (DAKO) and TMB substrate according to the manufacturers instructions (Sigma).
Purification of IgA.
IgA1 was purified from acute-phase NE patient serum on Jacalin (Artocarpus integrifolia) (Sigma), as described previously (Johansen et al., 1994 ). IgG was affinity-purified on protein ASepharose as described by the manufacturer (Pharmacia).
SDSPAGE and immunoblotting.
The purity of the affinity-purified IgA1 and IgG fractions was examined by SDSPAGE and immunoblotting. Serum, IgA1 and IgG fraction samples were mixed with SDS sample buffer and applied to 420% gradient SDSpolyacrylamide gels. Gels were stained with Coomassie blue or transferred to a nitrocellulose sheet for immunoblotting analysis, which was performed essentially as described previously (Johansen et al., 1994 ). The nitrocellulose membrane was cut into strips, which were blocked with 5% milk powder in PBS (assay buffer) for 2 h at room temperature. Alkaline phosphatase-conjugated rabbit anti-human IgA and IgM (DAKO) or goat anti-human IgG (Sigma), diluted 1:1000 in assay buffer, were added and the strips were incubated for 4 h at room temperature. After four washes with PBS with 0·05% Tween 20, the reactions were developed with BCIP/NBT tablets (Sigma) dissolved in distilled water.
Neutralization assay.
Neutralizing activity of serum or purified fractions of serum against PUUV (strain Sotkamo; Vapalahti et al., 1992 ) was analysed by focus-reduction neutralization test (FRNT) (Lundkvist et al., 1997
). Briefly, samples were serially diluted and mixed with an equal volume containing 3070 focus-forming units of virus per 100 µl. The mixtures were incubated for 1 h and inoculated into wells of 6-well plates containing confluent Vero E6 cell monolayers. After adsorption for 1 h, the wells were overlaid with agarose and incubated for 12 days. PUUV-specific polyclonal rabbit antisera followed by peroxidase-labelled goat antibodies and TMB substrate were used for detection of virus-infected cells. An 80% reduction in the number of foci was used as the criterion for virus neutralization titres.
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Results |
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NE-negative sera
The mean absorbances of 27 negative sera were calculated separately for each viral protein and IgA subclass. The mean absorbance plus 3 SD was used as the cut-off value. The cut-off values of the six assays varied between 0·035 and 0·216.
Epitope mapping
In total, 86 overlapping 10-mer peptides of PUUV (strain Sotkamo), covering the whole N protein in 5-amino-acid shifts, were used for epitope mapping by the PEPSCAN method. Several linear B cell antigenic sites were identified over the entire N protein when a pool of NE sera, drawn from 10 acute-phase patients, was analysed. The results are shown in Fig. 3 as absorbances, with the reactivities of a pool from 10 non-exposed donors subtracted. The majority of the antigenic regions detected were located in the C-terminal part of the protein, represented by major peaks at amino acids 256265, 351360 and 411420.
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Eight sera that contained low or undetectable levels of PUUV-specific IgM but were from patients with clinical symptoms of acute-phase NE were analysed for N-specific IgA1 (Table 3). All eight sera reacted as positive; four sera with high absorbances (0·7771·540) and four sera with reactivities slightly above the cut-off value (0·1000·157; cut-off=0·079). Since two of these sera were negative for PUUV-specific IgM, the results clearly revealed the value of IgA1 detection as a complement to the IgM assay.
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Discussion |
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The patterns of the IgA1 responses against the different viral components were reminiscent of the findings for IgM in a previous study (Lundkvist et al., 1993a ). Like the measurement of PUUV-specific serum IgM, as well as IgG3 (Lundkvist et al., 1993b
; Groen et al., 1994
), detection of specific IgA proved to be an adequate method for determination of the stage of infection: a significant decrease was seen already in the early convalescent samples (drawn 1641 days after onset of disease). Similar observations have been made for other virus infections that primarily infect mucosal surfaces, e.g. measles, rota- and enteroviruses (Friedman et al., 1989
; Coulson et al., 1990
; Pozzetto et al., 1990
).
Our finding of the presence of IgA1 in several of the 2-year and 10-year NE convalescent samples is noteworthy, because of the suggested role of persistent IgA as a marker for prolonged antigenic stimulation (Friedman & Eichler, 1991 ; Nilsen et al., 1991
). Hantaviruses persist, usually for life, in their natural rodent carriers and PUUV has been detected 1 year after experimental infection (Bogdanov et al., 1987
). In a previous study, we noted elevated levels of PUUV-specific IgG4, especially directed to G2, in 2-year and 1020-year NE-convalescent sera. Since virus-specific IgG4 has previously been demonstrated in virus infections that tend to persist in the host, e.g. hepatitis B, herpes simplex and varicella-zoster viruses (Asano et al., 1987
, 1988
; McBride & Ward, 1987
; Sällberg et al., 1990
), we thereby speculated on prolonged/persistent antigenic stimulation during/after PUUV infection (Lundkvist et al., 1993b
). In line with this, PUUV N and G2 proteins and RNA have recently been reported to persist in experimentally infected cynomolgus macaques for 7, 10 and at least 30 weeks, respectively (Groen et al., 1995
). Although the kinetics of the human IgA response to PUUV might support the presence of a prolonged or repeated antigenic stimulation, the question of whether viral RNA and/or viral antigens actually remain in the human body for an extended period after the initial PUUV infection is still unclear. In several attempts, using highly sensitive RTPCR, PUUV RNA has been found in only a minority of patient samples and only within the first days after onset of disease (Hörling et al., 1995
; Plyusnin et al., 1997
, 1999
). Several other explanations for long-term memory preservation and antibody synthesis have been proposed, e.g. that viral antigens persist in the form of antigenantibody complexes on the surface of follicular dendritic cells (Tew et al., 1980
), that continuing antibody synthesis is stimulated by idiotypeanti-idiotype interactions (Morris et al., 1985
) and that B and/or T cell memory may be maintained by cross-reactive stimulation (Beverley, 1990
). However, the detection of virus-specific IgA1 in 2-year and 1020-year convalescent samples may not be unique to PUUV infection; it may also be explained, at least partially, by the comparatively higher sensitivities of the PUUV assays. Extended studies, e.g. by direct analysis of the presence of viral RNA and antigens in biopsy material from convalescents or from experimentally infected monkeys, are needed for a better understanding of this issue.
Mapping of epitopes in the PUUV N protein revealed several antigenic regions recognized by the human IgA response. At least six regions were detected by a pool of 10 acute-phase NE sera, with pronounced activity against three regions in the C-terminal part of the protein. This pattern does not match completely the pattern of the total IgG response, which has been shown previously to be more equally distributed over the whole protein (Lundkvist et al., 1995 ; Vapalahti et al., 1995
).
Our previous work has suggested the presence of high levels of neutralizing serum IgM during the acute phase of NE (Lundkvist et al., 1993a ). In that study, five acute-phase sera, all with substantial levels of IgM directed to both envelope proteins, maintained their neutralizing activity after IgG depletion. However, no attention was paid to virus-specific serum IgA. In the present study, we purified IgA1 and IgG and studied the respective neutralizing activities separately. Interestingly, we found significant levels of neutralizing IgA1 in three of four PUUV IgA-positive sera, which also shows the importance of the IgA response in terms of virus inhibition. When the neutralizing activities of the IgA and IgG fractions were compared with the neutralizing activity of the original serum, the results suggested that the major neutralizing activity was not caused by these two Ig classes. Thus, the results are in line with our previous data, indicating that the major antibody-mediated virus neutralization during the acute stage of NE is caused by PUUV-specific IgM, supported in some cases by IgA and/or IgG. In line with this, maternal IgA and IgG have been shown to protect infant rats from lethal doses of Seoul virus, after transfer either in utero or by breastfeeding (Dohmae et al., 1993
; Dohmae & Nishimune, 1998
). Since hantaviruses are mainly spread via aerosol and primarily infect via the respiratory tract, the presence of neutralizing IgA is interesting in terms of the recovery from the acute infection and also for long-term immunity in convalescents.
Secretory IgA (S-IgA) responses are unique to mucosal surfaces and secretions are mainly induced by infection or immunization (e.g. intranasal, oral, rectal or vaginal) via the mucosal route. In humans, serum IgA is predominantly detected as a monomer, whereas dimeric or polymeric S-IgA is found in mucosal secretions. Transport of S-IgA across epithelial surfaces to external secretions, where antigen-specific S-IgA interacts with potential pathogens and inhibits their interaction with the host, may be the most important mechanism provided by S-IgA. S-IgA antibodies are particularly effective in virus neutralization, probably mainly because of the presence of multiple antigen-binding sites. Mazanec et al. (1992) have hypothesized that IgA may interfere with virus replication by binding to newly synthesized viral proteins within infected cells and may thereby neutralize microbial pathogens intracellularly. Antigen-specific S-IgA responses may provide an important reduction in levels of the infectious pathogen at the first line of defence, the mucosal surface. Further studies on S-IgA during clinical disease and after recovery will be needed to understand better the mechanisms and importance of PUUV-specific IgA.
Two previous studies have evaluated the diagnostic value of PUUV-specific serum IgA (Groen et al., 1994 ; Elgh et al., 1996
). In the first study, all acute-phase patient samples examined were found to be positive for PUUV-specific IgA, although a comparatively small number of sera (18) were analysed. In contrast, in the latter study, where a larger number (108) of patient sera were analysed, the detection of IgA by recombinant N ELISA was regarded as being of minor clinical relevance, mainly because of the low sensitivity (63·0%) of the assay. The discrepancies were suggested to be caused by differences in the size of the serum panels or by the use of recombinant instead of native viral antigen (Elgh et al., 1996
). In a more recent study, 13/17 (76%) sera drawn 28 days post-onset of disease were found to be positive by an IgA ELISA based on truncated recombinant PUUV N, while 17/17 (100%) of the sera drawn at days 515 were positive for PUUV-specific IgA (Elgh et al., 1998
). In line with our data, one IgA-positive serum was completely negative for virus-specific IgM and IgG (Elgh et al., 1998
).
In the present study, we found initially that the sensitivity of different IgA ELISA protocols varied to a large extent. By the selected combination of MAb-captured antigen and biotinstreptavidin amplification, we obtained highly specific and sensitive assays. The highest sensitivity (93%, n=100) was shown for the anti-N IgA1 ELISA, probably indicating that the majority of the PUUV-specific IgA1 response is directed to N. When eight sera from NE patients with undetectable or borderline levels of IgM were analysed, all sera were found to be positive for PUUV-specific IgA1. Thus, our study clearly indicates a diagnostic value for IgA1 as a complement to IgM and IgG detection.
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Acknowledgments |
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References |
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Received 10 November 1999;
accepted 10 February 2000.