Microbiology and Tumor Biology Center, Karolinska Institutet, S-171 77 Stockholm, Sweden1
Division of Clinical Virology, F68, Oral Microbiology, F88, and Basic Oral Sciences, F59, Huddinge University Hospital, S-141 86 Huddinge, Sweden2
Swedish Institute for Infectious Disease Control, S-171 82 Stockholm, Sweden3
Author for correspondence: Cristina de Carvalho Nicacio. Fax +46 8 33 07 44. e-mail cristina.de.carvalho{at}mtc.ki.se
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Abstract |
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Introduction |
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Hantaviruses are enveloped and have a three-segment negative-stranded RNA genome packed in helical nucleocapsids. The genome encodes four structural proteins: the L-segment encodes the RNA polymerase, the M-segment the two envelope glycoproteins (G1 and G2) and the S-segment encodes the nucleocapsid (N) protein (Plyusnin et al., 1996 ; Schmaljohn, 1996
).
The role of the immune response in protection, as well as in the pathogenesis of hantavirus infection, is not clear. Hantaviruses causing both HFRS and HPS infect human endothelial cells without any apparent cytopathic effect (Pensiero et al., 1992 ; Temonen et al., 1993
; Zaki et al., 1995
). The pathological manifestations seen during HFRS have therefore been suggested to be the result of virus-specific cytotoxic T-lymphocyte (CTL) responses (Ennis et al., 1997
; Van Epps et al., 1999
). Studies supporting that hypothesis have found accumulations of CD8+ CTL in kidney biopsies (Temonen et al., 1996
), and an increase in the number of activated circulating CD8+ CTL in peripheral blood mononuclear cells (PBMC) during the acute phase of HFRS (Chen & Yang, 1990
; Huang et al., 1994
). Furthermore, several inflammatory cytokines, e.g. gamma interferon (IFN-
), tumour necrosis factor-alpha (TNF-
), TNF-beta (TNF-
) and interleukin (IL)-6 have also been found at elevated levels in both kidney biopsies and sera from HFRS patients (Huang et al., 1994
; Temonen et al., 1996
; Linderholm et al., 1996
; Krakauer et al., 1994
). However, the role of the CD4+ T-helper (Th) lymphocytes is not clear. Do they support a predominantly cellular or humoral immune response, and does their cytokine production exacerbate the cytotoxic immune response giving rise to the pathology of the disease? The CD4+ Th-lymphocytes have been shown to decrease in PBMC during the acute phase of HFRS and rise to normal levels in convalescents (Chen & Yang, 1990
; Huang et al., 1994
), but the importance and consequences of this is not known.
Humoral immune responses to hantavirus antigens have been studied extensively in both animals and humans and the envelope glycoproteins are presumed to be the major elements involved in induction of protective humoral immunity to hantaviruses (Dantas et al., 1986 ; Arikawa et al., 1989
; Lundkvist & Niklasson, 1992
). The N protein of PUUV has previously been shown to be highly immunogenic in both laboratory animals and humans, and to efficiently induce protective immunity in animals (Lundkvist et al., 1993
, 1996
). Both N-specific IgM and IgG antibodies are detected in serum at the onset of symptoms in HFRS and high titres are reached and can be detected in sera drawn 1020 years post- infection (Lundkvist et al., 1993
; Settergren et al., 1991
). Since the humoral response to PUUV N is not neutralizing in vitro, the induced protection could be interpreted as mainly cell-mediated. Three CTL epitopes have been identified in the N protein of SNV (Ennis et al., 1997
), and recently, analysis of human memory CTL responses against HTNV infection identified both virus-specific and cross-reactive CTL epitopes in the N protein (Van Epps et al., 1999
). These data suggest that the N protein plays an important role in the cell-mediated immune response.
A better understanding of the mechanisms of the immune responses to hantavirus infections should help to improve diagnostics, treatment of patients and to serve as a basis for development of potential approaches for prevention by immunization. To further investigate the mechanisms for N protein-induced immunity, we have analysed both humoral and Th-lymphocyte responses to PUUV N in mice using recombinant N (rN) protein and synthetic peptides.
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Methods |
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The sequence of the cloned S gene was confirmed by nucleotide sequence analysis using sequencing primers provided by the vector supplier (Qiagen). Cycle sequencing was carried out on plasmid DNA as described previously (de Carvalho Nicacio et al., 2000 a).
Synthetic peptides.
Eighty-four overlapping peptides (17 aa with 12 aa overlaps) spanning the N protein sequence of PUUV (strain Kazan-E6, aa 1433) were synthesized by multiple peptide synthesis (Syro MultiSyn Tech; Syntex) Using Fmoc-protected amino acids as previously described (Sällberg et al., 1991 ). The peptides were cleaved and deprotected according to standard protocols for Fmoc peptide synthesis.
Mice.
Inbred mice of three different haplotypes, H-2b (C57/BL6), H-2d (BALB/c) and H-2k (CBA), were used for analyses of rN immunogenicity. All mice were immunized at 48 weeks of age and were obtained from BK Universal (Sollentuna, Sweden).
Immunizations.
Determination of antibody responses was carried out on sera from groups of five to six mice immunized intraperitoneally with 20 µg of rN protein emulsified in Freunds complete adjuvant (FCA). The mice were boosted 4 weeks later subcutaneously with 50 µg of rN protein emulsified in Freunds incomplete adjuvant and sera were collected by retroorbital bleedings at 2, 4 and 6 weeks.
For proliferation assays and cytokine detection, groups of mice were immunized subcutaneously in the base of the tail with 50 µg of rN protein or control protein emulsified in FCA. For peptide immunizations, mice were injected with 100 µg of peptide emulsified in FCA.
ELISA.
Total IgG responses to PUUV N were measured by ELISA essentially as described previously (Hörling et al., 1996 ). Briefly, rabbit anti-PUUV serum diluted 1:400 in 0·05 M bicarbonate buffer, pH 9·6, was adsorbed to microtitre plates overnight at 4 °C. After blocking of non-saturated binding sites with 3% BSA, native PUUV antigen diluted in dilution buffer (0·5% BSA and 0·05% Tween 20 in PBS) was incubated for 1 h at 37 °C. Serum dilutions were incubated for 1 h at 37 °C, and specific antibody binding was detected with alkaline phosphatase (ALP)-conjugated donkey anti-mouse IgG antibodies diluted 1:5000 (Jackson Immunoresearch), followed by incubation with p-nitrophenyl phosphate substrate (Sigma). IgG subclass responses were detected with goat anti-mouse IgG1, IgG2a, IgG2b or IgG3 antibodies diluted 1:5000, followed by incubation with an ALP-conjugated rabbit anti-goat IgG diluted 1:5000 (Sigma) and substrate as above.
Identification of B-cell recognition sites.
Linear B cell epitopes were mapped by PEPSCAN (Geysen et al., 1987 ). In total, 86 peptides (10 aa with 5 aa overlaps) spanning the N protein sequence of PUUV (strain Sotkamo, aa 1433, aa identity with PUUV Kazan-E6 96·7%), synthesized on polypropylene pins, were used to locate antibody-reactive peptides. Peptide synthesis and analysis of antibody reactivity have been described previously (Lundkvist et al., 1995
). Briefly, PEPSCAN antibody reactivities were measured in sera, diluted 1:200, from rN protein immunized or non-immunized mice. Bound antibodies were detected with ALP-conjugated donkey anti-mouse IgG diluted 1:1000 (Jackson) and NPP substrate (Sigma).
Proliferation and cytokine assays.
Mice were sacrificed 911 days after immunization and draining lymph nodes (LNs) were removed. Single-cell suspensions were prepared in Clicks medium (Sigma) and plated in microtitre plates at 6x105 cells per well. Recombinant N protein, control protein or peptides were added at serial dilutions. Medium alone was used as negative control and 1 µg/ml of phytohaemagglutinin (PHA) was used as a positive control.
For measurement of T-lymphocyte proliferation after in vitro restimulation, the cells were incubated for 72 h with the addition of 1 µCi [3H]thymidine (TdR; Amersham) for the last 16 h. The labelled cells were harvested onto cellulose filters, quenched and the level of [3H]TdR incorporation was determined by a liquid scintillation -counter.
For determination of cytokine concentrations in in vitro restimulated cell cultures, supernatants were harvested at 24 h for measurement of IL-2 and at 48 h for IL-4, IL-6 and IFN-. Cytokine concentrations were measured by ELISA according to the manufacturers instructions (Endogen, Cambridge, MC, USA).
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Results |
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Fine specificity of the humoral responses
Three pools of sera from groups (n=6) of H-2b, H-2d and H-2k mice, drawn 2 weeks after the second immunization, were analysed for reactivities against antigenic regions within the PUUV N protein. Mapping was performed with PEPSCAN against 86 overlapping decapeptides corresponding to the whole N sequence. The three serum pools all displayed similar reactivity patterns against the peptides (Fig. 3ac
), and several antigenic regions were detected. The highest reactivity was seen against aa 1120, showing that the major antigenic region of the protein is located within the amino-terminal part. Some reactivity could also be seen against the central part of the protein, namely aa 166180, 221245, 281290 and 296305. Only the H-2k serum pool reacted against peptides within the carboxy-terminal part of the protein, representing aa 396405 and 411420.
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Discussion |
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Based on the in vitro neutralizing activity of G1- and G2-specific MAbs (Dantas et al., 1986 ; Arikawa et al., 1989
, 1992
; Lundkvist & Niklasson, 1992
), and on passive transfer experiments (Zhang et al., 1989
; Schmaljohn et al., 1990
;
. Lundkvist and others, unpublished) the envelope glycoproteins have been assumed to be the main inducers of protective humoral responses. The role of cell-mediated immunity in hantavirus infection has not been studied in detail. However, it has been reported that the cellular response is important both in protection from and in the pathology of hantavirus infections. Virus-specific T-lymphocytes in passive transfer studies in vivo, and both human and mice CTLs in in vitro studies, have been demonstrated to have protective and antiviral activity against hantaviruses (Asada et al., 1987
, 1988
, 1989
; Yoshimatsu et al., 1993
; Ennis et al., 1997
; van Epps et al., 1999
).
The importance of the N protein in induction of immunity to hantaviruses is still not clear. N-specific MAbs have been shown to partially protect mice from HTNV infection and bank voles from PUUV infection (Yoshimatsu et al., 1993 , 1996
;
. Lundkvist and others, unpublished). Accordingly, the humoral response to the N protein may, in addition to the glycoprotein-specific response, be of importance for immunity, e.g. via antibody-dependent cell-mediated cytotoxicity and/or complement-mediated cytolysis, since the N protein has not been shown to induce neutralizing antibodies. However, the N protein has also been implicated in cellular immunity in hantavirus infection. In challenge experiments, baculovirus-expressed N protein has been shown to confer complete protection against HTNV in hamsters, without inducing any neutralizing antibodies (Schmaljohn et al., 1990
). Also, the amino-terminal aa 1118 of N protein were shown to be sufficient to give complete protection against PUUV challenge (Lundkvist et al., 1996
).
Mapping of the IgG responses by PEPSCAN detected epitopes throughout the N protein; however, the majority of the detected reactivities were found in the amino-terminal part of the protein. This agrees with earlier studies, in which sera from PUUV-infected bank voles showed the highest activity against the amino-terminal 1120 aa (Lundkvist et al., 1996 ). In contrast, the human IgG responses in NE patient sera have been shown to be broader, with reactivity against peptides spanning the whole N protein (Vapalahti et al., 1995
; Lundkvist et al., 1995
), while the human IgA responses in NE patient sera were shown to be mainly directed to the carboxy-terminal part of the protein (de Carvalho Nicacio et al., 2000
b). It should be noted that conformational antibody epitopes, several of which have been located at the amino-terminal part of the PUUV N protein by MAbs (Lundkvist et al., 1991
, 1995
, 1996
), could not be investigated by the methods used in the present study.
Immune responses after rN immunization varied with mouse haplotype. The highest responder was the H-2k haplotype (CBA), while the H-2d haplotype (BALB/c) was intermediate and the H-2b haplotype (C57/Bl6) was the lowest responder. Earlier studies have shown that certain HLA restriction elements can be associated with course of disease during hantavirus infection. Mustonen et al. (1998) reported an increased frequency of a more severe course of NE in patients with HLA B8 and DRB1*0301 alleles, while HLA B27 on the other hand was associated with mild disease. The differences in responder status observed in the present study were of course not indicators of disease severity as the mouse model used is not an infection or disease model. But the responder status in these mice varied when both humoral and cellular responses were studied, indicating that haplotype plays an important role in immune responses against PUUV N protein.
Inflammatory cytokines have been suggested to play important roles in the pathogenesis of hantavirus infection. Several studies have detected elevated levels of IFN-, TNF-
and IL-6 on lymphocytes and in sera from HFRS and HPS patients (Huang et al., 1994
; Ennis et al., 1997
; Krakauer et al., 1994
; Linderholm et al., 1996
; Mori et al., 1999
). Increased expression of TNF-
, TNF-
and platelet-derived growth factor has also been detected in kidney biopsies from NE patients (Temonen et al., 1996
). In the present study we analysed the cytokine profile of in vitro rN-restimulated lymphocytes. These cells produced high concentrations of IFN-
and IL-2, and lower concentrations of IL-6 and IL-4, indicating that the PUUV N protein predominately induced Th1 type cytokines, but also Th2 cytokines in this system.
In mice the IgG subclass distribution is known to correlate with cytokine profile. IgG1 production is mainly promoted by Th2 cytokines, and in contrast, IgG2a production is promoted by Th1 cytokines (Stevens et al., 1988 ). The subclass distribution after rN immunization seen in this study did not show a clear predominance of either IgG1 or IgG2a after boost injection, suggesting a mixed Th1/Th2 response, concordant with the cytokine response.
Four T-cell recognition sites were mapped within the N protein by using peptides. Two of the regions were located in the highly immunogenic amino-terminal part of the protein, aa 627 and 96117 (Lundkvist et al., 1996 ). The other two regions were located in the highly variable central part of N, aa 211232 and 256277. The authenticity of these regions was confirmed by recall of peptide-primed lymphocytes with rN protein in vitro. This is the first study in which proliferative T-lymphocyte responses to PUU hantavirus have been mapped. Two earlier studies have identified CD8+ and CD4+ CTL epitopes on the N protein of SNV and HTNV (Ennis et al., 1997
; van Epps et al., 1999
). In the study by van Epps et al., a human CD8+ CTL epitope in the HTNV N protein was identified, aa 1220, corresponding to the first region detected in the present study (aa 627). This poses the question of cross-reactivity and whether this specific epitope/region is an important T-cell epitope among all hantaviruses. When van Epps et al. compared the cross-reactivity of CTLs against target cells pulsed with peptides representing sequences from different hantaviruses, only the HTNV and the SEOV peptides, which differ only at aa 12, resulted in cross-reactive lysis of target cells. These results may seem discouraging, but as the same epitope could be important for both CTL and Th-lymphocytes, the restricted activation of CTLs may not be applicable for Th-lymphocytes, which possibly have a broader activation pattern leading to T-cell help via cytokine excretion for antibody production and CTL activation. Furthermore, all regions detected in the present study have earlier been shown to react with antibodies in human NE sera (Vapalahti et al., 1995
; Lundkvist et al., 1995
). In addition, the two regions in the central part of N (aa 211232 and 256277) have also been shown to react with sera from experimentally and naturally infected bank voles, the natural reservoir of PUUV (Lundkvist et al., 1996
).
In conclusion, T-cell reactive regions on PUUV N protein have been identified using mouse lymph node lymphocytes. Further studies are needed to define the specific epitopes and the role of these epitopes in the immune responses against other hantaviruses and their role in protection against virus infection.
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Acknowledgments |
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This project was supported by the Swedish Medical Research Council (Projects 12177 and 12642), the Swedish Society of Medicine and by the European Community (Contracts BMH4-CT97-2499 and QLK2-CT-1999-01119).
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Received 4 July 2000;
accepted 4 October 2000.