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CD8+CD57+ lymphocytes in blood of children after measles virus infection
Immunity to viruses
24 June 1997 - Poster presentations
Concluskm: Our study has shown that all&c polymorphism at the HLA Class I locus is an important determinant for susceptibility to DHF. These findings suggest that HLA Class I restricted cellular immune responses may be involved in viral clearance, but may also result in severe disease due to immunopathology. To clarify this, we are now characterising cellular immune responses in naturally immune people, with particular interest in people with HlAA33 and HIA-A24.
1P.4.04.07 1 Fine-mapping of human and mouse&at antibody binding to Epstein-Barr virus nuclear antigen-l (EBNA-1): Linear and conformational epitopes J.M. Middeldorp I,*, M.J. Vetvoorl’,*, W. Puijk3, R.H. Meloen 3, P. Grootenhuis 4. ’ Bioscience Research Unit, Oganon Teknika, Boxtel, The Netherlands, 2Dept. Pathology, Free University Hospifal, Amstenlam, The Netherlands, 3Dept. Molecular lmmunologx ID-DLO, Lelystad, The Netherlands, 4Compuiational Medicinal Chemistw Oganon International, Oss, The Netherlands EBNA-1 is essential for the maintenance of EBV-genomes in latently infected (tumor) cells and is capable of eluding MHC-I mediated presentation. However anti-EBNA-1 antibodies are detected in virtually all EBV carrying individuals. To studv humoral immune recoonition of EBNA-1, we applied hiph resolution epitope mapping with all possible overlapping IP-mer peptides (PEPSCAN) to reveal the fine-specificity of human polyclonal and mouse/rat monoclonal antibody (MoAb) binding to full length EBNA-1, using 20 human sera and 3 different MoAbs (OTlX, lH4 and 2B4). MoAbs bound to the same region (AA430-450), recognising a confonnational epitope (AA430-440) with high affinity (OTIX, Kd < 1.4 nhn) or small linear epitopes (1H4 and 2H4 binding to AA440443 and AA446448 respectively). Very similar but rather restricted antibody binding patterns were found for most individuals, which included the Gly-Ala repeat region (AAQO-325) and a cluster of epitopes, located in the unique sequence of EBNA-1 (AA362-452). A 60AA synthetic combi-peptide of this region had immunoreactivity similar to full length EBNA-1 or crystal grade C-terminal DNA-binding domain of EBNA-1 (AA649-607). The latter domain was nearly negative in PEPSCAN. Regions of EBNA-1 known to be involved in DNA-looping (AA331-361 and AA372-391) or DNA-binding and dimerization (AA459-607) appeared to be virtually excluded from human antibody recognition. However, statistical analysis of the PEPSCAN data revealed weak human antibody binding within the N-terminus (AAl-90) and at 5 discrete sites within the C-terminus [AA489-500(l), AA525-535(2), AA545-555(3), AA577-595(4) and AA625-641(5)]. Alignment of these data with the crystal structure of the Off-P bindingldimerization domain of EBNA-1 showed that site 1 + 2 are localized on the EBNA-1 surface forming a confonational epitope, whereas sites 3 and 4 form independently recognised loop structures extending from protein surface. The B-barrel inner core, the DNA-binding helix (AA614-527) and -loop (AA461-469) and the flanking helix (AA472-489) are all devoid antibody recognition. (crystal coordinates were kindly provided by L. Frappier and A. Bochkarev, &Master University, Canada). PEPSCAN analysis of other EBV DNA-binding proteins (e.g. BALFP or BZLFl) revealed antibody binding sites scattered over the protein sequence, not cle&y separated intospecificdomains. These data indicate that EBNA-1 -in contrast to other EBV encoded DNAbinding proteins- is presented to the immune system in vivo as an aggregated multi-molecular protein-DNA complex, such that particular sites involved in protein-protein and protein-DNA interaction are not accessible for immune recognition.
P.4.04.08
CDEWDW lymphocytes in blood of children after measles virus infection
B. Aronsson I,*, L. Smedman 1.2,M. Troye-Blomberg *. ‘Dept.of Immunology, Wennergren InstiUte, Stockholm Universe Sweden, * Kamiinska Institure. Dept. of PediaMzs, S:t G&an’s Childrsn’s Hospital, Stockholm, Sweden A general suppression of cell mediated immunity follows measles infection. This immunosuppression is reflected in viw as suppressed DTH responses to recall antigens, such as tuberculin and in vitm as abnormal lymphokine production and suppressed lymphoproliferative responses to mitogen. This general immunosuppression probably contributes to a more severe and complicated course of the disease. It is of great importance to achieve more knowledge about the post-measles immunosuppression. One way could be to identify an in vitro parameter of clinical relevance. It is well established that immunity to measles is mediated by CD8+ cytotoxic T cells. CD6+ T cells w-expressing CD57 (Leu-7) comprise functional suppressor T cells. CD8+CD57+ lymphocytes are able to suppress both T cell proliferation and B cell differentiation in vitro. This subpopulation of T cells is expanded in the petipheral of patients with congenital common vatfable immunodeficiency
291
(CVID) and also in some chmnical virus infections, e.g. cytomegalovirus infection (CMV) and human immunodeficiency virus (HIV). The aim of our study was to investigate if this marker is enhanced during measles infection and whether it is involved in the immunosuppression. During 1994 and 1995 there were small outbreaks of measles among children in Stockholm. Twentylwo of these children were examined twice, during and three to four months after infection. As controls age matched children were examined in the same way. The blood samples taken from the children were examined for the percentage of CD8+CD57+ T cells. Other markers as CD4, CD14, CD3. CD56 and CD20 were also included. In the measles patients, the difference in proportion of CD8+CD57+ T lymphocytes, shows a statistically significant regression on the timing of the first examination (p -Z 0.05, single-sided test). This fits in with a raised proportion immediately after the disease which waned back to normal during the following months. Thus, our data suggest that CDSICD57 double-positive lymphocytes expand during the acute infection. However, whether these cells are involved in the general immunosuppression seen under measles infection still remains to be elucidated.
P.4.04.09
Influence of revaccination with a single addltlonal dose of recombinant hepatitis B vaccine in Iranian healthy nonresponder neonates
Faze1Shokri ‘, Abdollah Amani * . ’ Dept. of Immunology School of Public Health, Tehran Univ of Medical Sciences, Tehran, IR Iran, 2B/cod Tmnsfusion Center of Sanandaj, Sanandaj, IR Iran Effective control of HBV transmission in areas of high and intermediate endemicity would not be possible without mass vaccination of neonates and children; not all vaccinees, however, respond to vaccination. Revaccination of nonresponders has been proposed to induce sercconversion. In this study 49 Iranian neonates who failed to develop a protective anti-HBs response following primary vaccination with triple doses of recombinant hepatitis B vaccine, were classified as hype-responders (anti-HBs ~1 ~10 lU/l) or non-responders (anti-HBs
1P.4.04.10 1 Effect of ageing and cells fractionation on the antiviral conetitutive immunity of murine resident peritoneal cells B. Orzechowska. B. Domaraczenko, 2. Biach-Olszewska. Lab. Viro/., L. Hirszfe/d Inst. Immunol. Exp. Ther., hl. Acad. Sci.. Wrcdaw, Bland Introduction: It was shown previously that resident peritoneal cells (RPC) of BALB/c mice. cultured in vitro at 26°C release spontaneously IFN-B which seems to play an important role in host defence against viral infections. On the contrary, incubation of the cells at 3pC inhibits the IFN-p synthesis at the level of transcription. In spite of this, the freshly isolated RPC, incubated at 3PC express the constitutive antiviral activity. After several days in culture, however, the RPC lose the immunity and acquire the sensitivity to vesicular stomatitis virus (VSV) infection. This study was undertaken to show the effect of RPC fractionation on their constiive immunity. Materials and Methods: Resident peritoneal ceils (RPC) were washed out from peritoneal cavfly of 8 weeks old BALB/c female mice. The cells were suspended in Eagle minimum essential medium (EMEM) supplemented with 10% of calf seturn, at concentration of 1-2 x 106cells/ml. RPC were separated into two fractions by 2 h adhesion to glass. The constitutive immunity was detected by the incapability of the cells to VSV replication. VSV was titrated in mouse cell line Lsn. ReeulB: RPC freshly isolated from individual mice were resistant to VSV infection. Separation of the cells into two fractions: adhering and non adhering, resulted in lost of the constitutive immunity by the cells attached to glass. VSV multiplied in the cells to high titers (IO’ TCID 50/i@ cells). In contrast, the absence of detectable virus replication rate was noticed in non adhering cells. Mixture of both cell fractions restored the resistance of the cells to viral infection. Con&&n: Our results show that the ceils non adhering to glass are responsible for the constitutlve antiviral defence of murine RPC.
24 June 1997 - Poster presentations
Concluskm: Our study has shown that all&c polymorphism at the HLA Class I locus is an important determinant for susceptibility to DHF. These findings suggest that HLA Class I restricted cellular immune responses may be involved in viral clearance, but may also result in severe disease due to immunopathology. To clarify this, we are now characterising cellular immune responses in naturally immune people, with particular interest in people with HlAA33 and HIA-A24.
1P.4.04.07 1 Fine-mapping of human and mouse&at antibody binding to Epstein-Barr virus nuclear antigen-l (EBNA-1): Linear and conformational epitopes J.M. Middeldorp I,*, M.J. Vetvoorl’,*, W. Puijk3, R.H. Meloen 3, P. Grootenhuis 4. ’ Bioscience Research Unit, Oganon Teknika, Boxtel, The Netherlands, 2Dept. Pathology, Free University Hospifal, Amstenlam, The Netherlands, 3Dept. Molecular lmmunologx ID-DLO, Lelystad, The Netherlands, 4Compuiational Medicinal Chemistw Oganon International, Oss, The Netherlands EBNA-1 is essential for the maintenance of EBV-genomes in latently infected (tumor) cells and is capable of eluding MHC-I mediated presentation. However anti-EBNA-1 antibodies are detected in virtually all EBV carrying individuals. To studv humoral immune recoonition of EBNA-1, we applied hiph resolution epitope mapping with all possible overlapping IP-mer peptides (PEPSCAN) to reveal the fine-specificity of human polyclonal and mouse/rat monoclonal antibody (MoAb) binding to full length EBNA-1, using 20 human sera and 3 different MoAbs (OTlX, lH4 and 2B4). MoAbs bound to the same region (AA430-450), recognising a confonnational epitope (AA430-440) with high affinity (OTIX, Kd < 1.4 nhn) or small linear epitopes (1H4 and 2H4 binding to AA440443 and AA446448 respectively). Very similar but rather restricted antibody binding patterns were found for most individuals, which included the Gly-Ala repeat region (AAQO-325) and a cluster of epitopes, located in the unique sequence of EBNA-1 (AA362-452). A 60AA synthetic combi-peptide of this region had immunoreactivity similar to full length EBNA-1 or crystal grade C-terminal DNA-binding domain of EBNA-1 (AA649-607). The latter domain was nearly negative in PEPSCAN. Regions of EBNA-1 known to be involved in DNA-looping (AA331-361 and AA372-391) or DNA-binding and dimerization (AA459-607) appeared to be virtually excluded from human antibody recognition. However, statistical analysis of the PEPSCAN data revealed weak human antibody binding within the N-terminus (AAl-90) and at 5 discrete sites within the C-terminus [AA489-500(l), AA525-535(2), AA545-555(3), AA577-595(4) and AA625-641(5)]. Alignment of these data with the crystal structure of the Off-P bindingldimerization domain of EBNA-1 showed that site 1 + 2 are localized on the EBNA-1 surface forming a confonational epitope, whereas sites 3 and 4 form independently recognised loop structures extending from protein surface. The B-barrel inner core, the DNA-binding helix (AA614-527) and -loop (AA461-469) and the flanking helix (AA472-489) are all devoid antibody recognition. (crystal coordinates were kindly provided by L. Frappier and A. Bochkarev, &Master University, Canada). PEPSCAN analysis of other EBV DNA-binding proteins (e.g. BALFP or BZLFl) revealed antibody binding sites scattered over the protein sequence, not cle&y separated intospecificdomains. These data indicate that EBNA-1 -in contrast to other EBV encoded DNAbinding proteins- is presented to the immune system in vivo as an aggregated multi-molecular protein-DNA complex, such that particular sites involved in protein-protein and protein-DNA interaction are not accessible for immune recognition.
P.4.04.08
CDEWDW lymphocytes in blood of children after measles virus infection
B. Aronsson I,*, L. Smedman 1.2,M. Troye-Blomberg *. ‘Dept.of Immunology, Wennergren InstiUte, Stockholm Universe Sweden, * Kamiinska Institure. Dept. of PediaMzs, S:t G&an’s Childrsn’s Hospital, Stockholm, Sweden A general suppression of cell mediated immunity follows measles infection. This immunosuppression is reflected in viw as suppressed DTH responses to recall antigens, such as tuberculin and in vitm as abnormal lymphokine production and suppressed lymphoproliferative responses to mitogen. This general immunosuppression probably contributes to a more severe and complicated course of the disease. It is of great importance to achieve more knowledge about the post-measles immunosuppression. One way could be to identify an in vitro parameter of clinical relevance. It is well established that immunity to measles is mediated by CD8+ cytotoxic T cells. CD6+ T cells w-expressing CD57 (Leu-7) comprise functional suppressor T cells. CD8+CD57+ lymphocytes are able to suppress both T cell proliferation and B cell differentiation in vitro. This subpopulation of T cells is expanded in the petipheral of patients with congenital common vatfable immunodeficiency
291
(CVID) and also in some chmnical virus infections, e.g. cytomegalovirus infection (CMV) and human immunodeficiency virus (HIV). The aim of our study was to investigate if this marker is enhanced during measles infection and whether it is involved in the immunosuppression. During 1994 and 1995 there were small outbreaks of measles among children in Stockholm. Twentylwo of these children were examined twice, during and three to four months after infection. As controls age matched children were examined in the same way. The blood samples taken from the children were examined for the percentage of CD8+CD57+ T cells. Other markers as CD4, CD14, CD3. CD56 and CD20 were also included. In the measles patients, the difference in proportion of CD8+CD57+ T lymphocytes, shows a statistically significant regression on the timing of the first examination (p -Z 0.05, single-sided test). This fits in with a raised proportion immediately after the disease which waned back to normal during the following months. Thus, our data suggest that CDSICD57 double-positive lymphocytes expand during the acute infection. However, whether these cells are involved in the general immunosuppression seen under measles infection still remains to be elucidated.
P.4.04.09
Influence of revaccination with a single addltlonal dose of recombinant hepatitis B vaccine in Iranian healthy nonresponder neonates
Faze1Shokri ‘, Abdollah Amani * . ’ Dept. of Immunology School of Public Health, Tehran Univ of Medical Sciences, Tehran, IR Iran, 2B/cod Tmnsfusion Center of Sanandaj, Sanandaj, IR Iran Effective control of HBV transmission in areas of high and intermediate endemicity would not be possible without mass vaccination of neonates and children; not all vaccinees, however, respond to vaccination. Revaccination of nonresponders has been proposed to induce sercconversion. In this study 49 Iranian neonates who failed to develop a protective anti-HBs response following primary vaccination with triple doses of recombinant hepatitis B vaccine, were classified as hype-responders (anti-HBs ~1 ~10 lU/l) or non-responders (anti-HBs
1P.4.04.10 1 Effect of ageing and cells fractionation on the antiviral conetitutive immunity of murine resident peritoneal cells B. Orzechowska. B. Domaraczenko, 2. Biach-Olszewska. Lab. Viro/., L. Hirszfe/d Inst. Immunol. Exp. Ther., hl. Acad. Sci.. Wrcdaw, Bland Introduction: It was shown previously that resident peritoneal cells (RPC) of BALB/c mice. cultured in vitro at 26°C release spontaneously IFN-B which seems to play an important role in host defence against viral infections. On the contrary, incubation of the cells at 3pC inhibits the IFN-p synthesis at the level of transcription. In spite of this, the freshly isolated RPC, incubated at 3PC express the constitutive antiviral activity. After several days in culture, however, the RPC lose the immunity and acquire the sensitivity to vesicular stomatitis virus (VSV) infection. This study was undertaken to show the effect of RPC fractionation on their constiive immunity. Materials and Methods: Resident peritoneal ceils (RPC) were washed out from peritoneal cavfly of 8 weeks old BALB/c female mice. The cells were suspended in Eagle minimum essential medium (EMEM) supplemented with 10% of calf seturn, at concentration of 1-2 x 106cells/ml. RPC were separated into two fractions by 2 h adhesion to glass. The constitutive immunity was detected by the incapability of the cells to VSV replication. VSV was titrated in mouse cell line Lsn. ReeulB: RPC freshly isolated from individual mice were resistant to VSV infection. Separation of the cells into two fractions: adhering and non adhering, resulted in lost of the constitutive immunity by the cells attached to glass. VSV multiplied in the cells to high titers (IO’ TCID 50/i@ cells). In contrast, the absence of detectable virus replication rate was noticed in non adhering cells. Mixture of both cell fractions restored the resistance of the cells to viral infection. Con&&n: Our results show that the ceils non adhering to glass are responsible for the constitutlve antiviral defence of murine RPC.