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Mousepox: Phenotypical characterization of immune cells in conjunctiva and epidermis
300
24 June 1997 - Poster presentations
Zmmunity to viruses
P.4.04.19
Mousepox: Phenotypical characterization of immune cells in conjunctlva and epidermis
M. Gierynska, I. Spohr, A. Popis, M.G. Niemiaftowski. Division of /mmuno/ogx Depadmentof Mcmbiology & Immunology. Faculty of Veterinary Medicine, WarsawAgricultural University, Gmchowska 272, W-849 Warsaw, Poland Introduction: Mousepox, an infectfous disease caused by ectromelia virus (EV), is characterized by at least one of the following symptoms: skin lesions in form of vesicles and yellowish crusts on the back, tail base, ear lobes, paws, swelling of paws and snout, and conjunctivitis. In the present study we examined the expression of us and ya CD4+ and CD8+ T cells. and CDllb positive cells in conjunctiva and epidermis durfng mousepox. Materials and Methods: Six-to-eight-week-old BALB/c (H-2d) mice were inoculated in hind footpads with Moscow strain of EV and observed daily for clinical signs. Uninfected littermates served as control. At day 18 post infection (p.i.), mice were sacrificed and epidermis and conjuctiva were separated. Next, single cell suspension and frozen sections were prepared for flow cvtometw and cvtospin slides (single cells) and immunofluomscence analysis (frozen eectfons). Cells were were stained against ~rflTCR: FITC, ysTCR: R-PE (PHARMINGEN), CD4+: FITC, CD8 R-PE, and CD11b: FITC (SEROTEC). Frozen sections were also stained with anti-EV: FITC conjugate, and respective mAb for dendrftic cells (DC) and macrophaaes (Mg). Results: At day 76 p.t. we found in (a) conjunctiva: 11.41% of T cells (4.61% of CD4+, and 6.6% of CD8) 8.18% a,3 and yS T cells (7.05% and 1.14% respectively), and 6.37% of f&, and (b).epide&is 10.91% of ~rp and 4.18% of yS Tcells, and 0.11% of CD11b positive cells. We also confirmed by microscopic analysis in the infected eyes the presence of EV antigen, and DC and M@ Conclusions: The principal conclusion arising from this study is that CD8+ a,3 T cells dominate in peripheral (epidermal and conjunctiva) tissues at peak of mousepox. Identification the regulatory signals required for activation and selective migration of EV-specific T cells should provide an important insight into mechanisms controlling of EV infection.
P.4.04.20
Protection against lethal vesicular stomatitis virus
’ (VW) infection by hypermutated versus germilne antibodies Ulrfch Kalinke, Annette Oxenius, Constantino Lopez-Macias, Rolf M. Zlnkemagel. Hans Hengartner. lnstifute of Experimental Immunology, Schmelzbegstr 12, CH-8091 ZOrich, Switzedand Introduction: Infection of mice with vesicular stomatitisvirus (VSV), acytopathic virus closely related to rabies virus, induces a rapid onset of a virus neutralizing antibody response which protects against lethal disease. Monoclonal IgGs isolated 4 and 5 days after VSV infection were devoid of somatic mutations, bound VSV with high avidity (- log M-r) and neutralized the virus in an in vitro assay. Most secondary and all hyperimmune response IgGs analyzed were somatically mutated, however, they showed only marginally enhanced binding acidities (5 x IO9 M-l). Therefore we tested whether particular amino acid substitutions influenced the binding of hypermutated antibodies and whether germline and hypenutated antibodies differed in their in vivo protective capacity. MaterlaIr and Msthods: VH sequence comparison revealed some mutations which encoded the same Ser31 to Asn and Ser55 to Arg substitutions in diirent clones. The influence of these changes on VSV-specific binding was analyzed in an ELISA assay using single-chain Fv-CK (scFv-CK) reagents displaying either the authentic or the germline configuration, or only one, or both of the repeatedly found substitutions. The protective capacity of the antibodies was tested by treating SCID mice with graded concentrations of hypermutated or oermline IaGs and subsequent i.v. infection wfth 10s pfu VSV. Mice showino no&s in the brain 4 days after the treatment were scored as being protected: Results: The VSV-specific ELISA with the different scFv-CK reagents indicated that hypennutation was able to improve binding of VSV-specificantibodies by >I00 fold. The analysis of in vivo protection by gennline and hypemrutated IgGs revealed that most hypermutated antibodies were protective at doses of approximately 20 Kg IgG/mouse and that all germline IgGs prolonged survival of the mice. However, many germline IgGs protected only at unphysiologically high concentrations (>I00 @mouse). Conctuslons: Despite hypermutation and although IgGs may have increased binding properties by >I00 fold, they showed only 5 fold increased binding when compared to structurally related IgGs devoid of somatic mutations. This indited a certain avidity threshold for VSV-specific antibodies which was not much improved by hypennutation. Nevertheless, the minor avidity differences of germline and hypermutated IgGs seem to be crucial to reach protective IgG tiers in the serum at physiological antibody concentrations. Thus, early germline IgGs may prolong survival of infected mice until hypermutated IgGs with only marginally enhanced binding avidity appear in the serum to eventually eliminate the virus.
) P.4.04.2i 1 Lactate dehydrogenase+levating virus (LDV): Lifelong coexistence of virus and LDV-specific immunity Manes F. van den Broek, Roman SpOrrf,Chen Even3,*, Peter G.W. Plagemann3, Edgar Hanseler’,Hans Hengartner, Rolf M. Zinkemagel. Institute of Experimental lmmunol~ Department of Patholog)! University of Zurich, Switzedand, ’ Department of C/inica/ Chemistry, University of Zurich, Switzerland, 2Dept. of Neurology, University of California, Irvine, USA, 3Deparfment of Microbiof~ Univetsiifyof Minnesota, Minneapolis, USA Viruses have developed various strategies to coexist with vertebrate hosts. Whereas viruses that destroy vital host cells must be efficiently controlled by immunity, noncytopathic viruses can escape immune surveillance by various means, including via T cell exhaustion or deletion. Lactate dehydrogenase-elevating virus (LDV) is a highly cytopathic virus exhibiting an extraordinary rate of replication from a few vlrions up to 101c-ll per ml blood within 24 h; LDV nevertheless establishes a persistent infection without harming the host. The cytotoxic and helper T cell responses to LDV were monitored in C57BU6 (H-2b), BALB/c (H-2d), C3H (H-2k) and Bl0.G (H-2a) mice. Cvtotoxic T cells spec& for the viral proteins VP-2.(orB) were found in H-2b mice, for VP9M (or6?) in H-2d, H-2k and H-2q mice, and for VPdP (06) in H-2d mice. LDVspecific CTL and helper T cell responses persisted for at least up to 250 days despite high levels (IO5 to IO7 ID5O/ml) of LDV in the blood. Thus, the cytopathic LDV which actively infects very few cells that are not essential for host survival (a subpopulation of macrophages), induces and maintains an inefficient immune response that is not exhausted. Thus, LDV infection in mice reveals a novel type of host-virus equilibdum where LDV quickly establishes persistence despite continuously induced T and B cells responses, which apparently are too slow to control the highly cytopathic and very fast replicating virus.
( P.4.04.22 1 Induction of Tcell dependent VSV neutralizing antibodies by a fusion protein comprlslng VW-G and the human IgGl Fc portion C. L6pez-Macfas, U. Kalinke, T. Fehr, E. Bucher, R.M. Zinkemagel, H. Hengartner. institute of Experimental /mmuno/o~ University of ZOrfch, Zririch, Switzerland Introduction: Antigens which crosslink receptors on B cells poorly need T-cell help, whereas B cells can be activated by strongly crosslinklng antigen in the absence of T-cell help. One example of a strongly crosslinking antfgens is vesicular stomathis virus (VSV). The glycoprotefn of VSV (VSV-G), the only protein in the viral envelope, is densely packed and highly organized with a spacing of 5-10 nm. Neutralizing antibodies bind one major antigenic site of VSV-G. This feature allows VSV-G to behave as a T independent antigen Type 1 (Tl-1). Nonorganized forms of VSV-G, such as irregular aggregated recombinant VSV-G behave as TI-2 antigens. To analyze the correlation between antigen organization and the efficiency of B cell activation. we have produced a recombinant fusion protein comprising VSV-G and the Fc portion of human IgGl (Fc-0). the fusion protein has high solublfii. Matewlals& Methods: The VSV-G gene was cloned between the EcoRl and Hindlll sites of the expression vector pCD4-HglCEl(encoding for the Fc portion of human IgGl). The construct was then transfected into the hybrfdoma J558L. Protein was purified from culture supematant and used to immunlze C57BU8 mice. Neutralizing serum antibodies against VSV were used as the read-out for B-cell activation. Resuftru Intravenous immunization with graded concentrations of Fc-G (10, 3, 1,0.3 pg) failed to induce B cell responses. However, intraperftoneal administration of the Fc-G producing hybrfdoma induced neutralizing IgG antibodies which appeared at day 12 after immunization. No IgM antibodies were detected. This result suggested the requirement of T-cell help for B-ceil activation by the soluble form of VSV-G. The induction of neutralizing antibodies depended on a continuous antigen supply and/or induction of an allogenic response against the hybridoma cells (H-p) providing presumably an inflamatory reaction and T-cell help. The T cell dependence (TO) of neutralizing antibodies induced by Fc-G is being currently investigated by Immunization of athymic mice and mice depleted of CD4+ T cells. Conclusion: This study indicated that expression of VSV-G in a soluble Fc-G behaves as TD antigen, compared to highly repetitive organization of VSV-G in viral particles (R-1) and to the poorly organized recombinant VSV-G (R-2) forming micells.
IP.4.04.23
Cytokine diversity of Influenza specific memory TH clones from a single murine progenitor
C.M. Graham, C.A. Smith, D.B. Thomas. National lnstifufe rbr Medicat Research, London, UK Introductfon: CD4+ memory T cell clones from a C57Bl/lO (H-F) single donor previously infected with influenza virus (H3N2) and which recognise haemag-
24 June 1997 - Poster presentations
Zmmunity to viruses
P.4.04.19
Mousepox: Phenotypical characterization of immune cells in conjunctlva and epidermis
M. Gierynska, I. Spohr, A. Popis, M.G. Niemiaftowski. Division of /mmuno/ogx Depadmentof Mcmbiology & Immunology. Faculty of Veterinary Medicine, WarsawAgricultural University, Gmchowska 272, W-849 Warsaw, Poland Introduction: Mousepox, an infectfous disease caused by ectromelia virus (EV), is characterized by at least one of the following symptoms: skin lesions in form of vesicles and yellowish crusts on the back, tail base, ear lobes, paws, swelling of paws and snout, and conjunctivitis. In the present study we examined the expression of us and ya CD4+ and CD8+ T cells. and CDllb positive cells in conjunctiva and epidermis durfng mousepox. Materials and Methods: Six-to-eight-week-old BALB/c (H-2d) mice were inoculated in hind footpads with Moscow strain of EV and observed daily for clinical signs. Uninfected littermates served as control. At day 18 post infection (p.i.), mice were sacrificed and epidermis and conjuctiva were separated. Next, single cell suspension and frozen sections were prepared for flow cvtometw and cvtospin slides (single cells) and immunofluomscence analysis (frozen eectfons). Cells were were stained against ~rflTCR: FITC, ysTCR: R-PE (PHARMINGEN), CD4+: FITC, CD8 R-PE, and CD11b: FITC (SEROTEC). Frozen sections were also stained with anti-EV: FITC conjugate, and respective mAb for dendrftic cells (DC) and macrophaaes (Mg). Results: At day 76 p.t. we found in (a) conjunctiva: 11.41% of T cells (4.61% of CD4+, and 6.6% of CD8) 8.18% a,3 and yS T cells (7.05% and 1.14% respectively), and 6.37% of f&, and (b).epide&is 10.91% of ~rp and 4.18% of yS Tcells, and 0.11% of CD11b positive cells. We also confirmed by microscopic analysis in the infected eyes the presence of EV antigen, and DC and M@ Conclusions: The principal conclusion arising from this study is that CD8+ a,3 T cells dominate in peripheral (epidermal and conjunctiva) tissues at peak of mousepox. Identification the regulatory signals required for activation and selective migration of EV-specific T cells should provide an important insight into mechanisms controlling of EV infection.
P.4.04.20
Protection against lethal vesicular stomatitis virus
’ (VW) infection by hypermutated versus germilne antibodies Ulrfch Kalinke, Annette Oxenius, Constantino Lopez-Macias, Rolf M. Zlnkemagel. Hans Hengartner. lnstifute of Experimental Immunology, Schmelzbegstr 12, CH-8091 ZOrich, Switzedand Introduction: Infection of mice with vesicular stomatitisvirus (VSV), acytopathic virus closely related to rabies virus, induces a rapid onset of a virus neutralizing antibody response which protects against lethal disease. Monoclonal IgGs isolated 4 and 5 days after VSV infection were devoid of somatic mutations, bound VSV with high avidity (- log M-r) and neutralized the virus in an in vitro assay. Most secondary and all hyperimmune response IgGs analyzed were somatically mutated, however, they showed only marginally enhanced binding acidities (5 x IO9 M-l). Therefore we tested whether particular amino acid substitutions influenced the binding of hypermutated antibodies and whether germline and hypenutated antibodies differed in their in vivo protective capacity. MaterlaIr and Msthods: VH sequence comparison revealed some mutations which encoded the same Ser31 to Asn and Ser55 to Arg substitutions in diirent clones. The influence of these changes on VSV-specific binding was analyzed in an ELISA assay using single-chain Fv-CK (scFv-CK) reagents displaying either the authentic or the germline configuration, or only one, or both of the repeatedly found substitutions. The protective capacity of the antibodies was tested by treating SCID mice with graded concentrations of hypermutated or oermline IaGs and subsequent i.v. infection wfth 10s pfu VSV. Mice showino no&s in the brain 4 days after the treatment were scored as being protected: Results: The VSV-specific ELISA with the different scFv-CK reagents indicated that hypennutation was able to improve binding of VSV-specificantibodies by >I00 fold. The analysis of in vivo protection by gennline and hypemrutated IgGs revealed that most hypermutated antibodies were protective at doses of approximately 20 Kg IgG/mouse and that all germline IgGs prolonged survival of the mice. However, many germline IgGs protected only at unphysiologically high concentrations (>I00 @mouse). Conctuslons: Despite hypermutation and although IgGs may have increased binding properties by >I00 fold, they showed only 5 fold increased binding when compared to structurally related IgGs devoid of somatic mutations. This indited a certain avidity threshold for VSV-specific antibodies which was not much improved by hypennutation. Nevertheless, the minor avidity differences of germline and hypermutated IgGs seem to be crucial to reach protective IgG tiers in the serum at physiological antibody concentrations. Thus, early germline IgGs may prolong survival of infected mice until hypermutated IgGs with only marginally enhanced binding avidity appear in the serum to eventually eliminate the virus.
) P.4.04.2i 1 Lactate dehydrogenase+levating virus (LDV): Lifelong coexistence of virus and LDV-specific immunity Manes F. van den Broek, Roman SpOrrf,Chen Even3,*, Peter G.W. Plagemann3, Edgar Hanseler’,Hans Hengartner, Rolf M. Zinkemagel. Institute of Experimental lmmunol~ Department of Patholog)! University of Zurich, Switzedand, ’ Department of C/inica/ Chemistry, University of Zurich, Switzerland, 2Dept. of Neurology, University of California, Irvine, USA, 3Deparfment of Microbiof~ Univetsiifyof Minnesota, Minneapolis, USA Viruses have developed various strategies to coexist with vertebrate hosts. Whereas viruses that destroy vital host cells must be efficiently controlled by immunity, noncytopathic viruses can escape immune surveillance by various means, including via T cell exhaustion or deletion. Lactate dehydrogenase-elevating virus (LDV) is a highly cytopathic virus exhibiting an extraordinary rate of replication from a few vlrions up to 101c-ll per ml blood within 24 h; LDV nevertheless establishes a persistent infection without harming the host. The cytotoxic and helper T cell responses to LDV were monitored in C57BU6 (H-2b), BALB/c (H-2d), C3H (H-2k) and Bl0.G (H-2a) mice. Cvtotoxic T cells spec& for the viral proteins VP-2.(orB) were found in H-2b mice, for VP9M (or6?) in H-2d, H-2k and H-2q mice, and for VPdP (06) in H-2d mice. LDVspecific CTL and helper T cell responses persisted for at least up to 250 days despite high levels (IO5 to IO7 ID5O/ml) of LDV in the blood. Thus, the cytopathic LDV which actively infects very few cells that are not essential for host survival (a subpopulation of macrophages), induces and maintains an inefficient immune response that is not exhausted. Thus, LDV infection in mice reveals a novel type of host-virus equilibdum where LDV quickly establishes persistence despite continuously induced T and B cells responses, which apparently are too slow to control the highly cytopathic and very fast replicating virus.
( P.4.04.22 1 Induction of Tcell dependent VSV neutralizing antibodies by a fusion protein comprlslng VW-G and the human IgGl Fc portion C. L6pez-Macfas, U. Kalinke, T. Fehr, E. Bucher, R.M. Zinkemagel, H. Hengartner. institute of Experimental /mmuno/o~ University of ZOrfch, Zririch, Switzerland Introduction: Antigens which crosslink receptors on B cells poorly need T-cell help, whereas B cells can be activated by strongly crosslinklng antigen in the absence of T-cell help. One example of a strongly crosslinking antfgens is vesicular stomathis virus (VSV). The glycoprotefn of VSV (VSV-G), the only protein in the viral envelope, is densely packed and highly organized with a spacing of 5-10 nm. Neutralizing antibodies bind one major antigenic site of VSV-G. This feature allows VSV-G to behave as a T independent antigen Type 1 (Tl-1). Nonorganized forms of VSV-G, such as irregular aggregated recombinant VSV-G behave as TI-2 antigens. To analyze the correlation between antigen organization and the efficiency of B cell activation. we have produced a recombinant fusion protein comprising VSV-G and the Fc portion of human IgGl (Fc-0). the fusion protein has high solublfii. Matewlals& Methods: The VSV-G gene was cloned between the EcoRl and Hindlll sites of the expression vector pCD4-HglCEl(encoding for the Fc portion of human IgGl). The construct was then transfected into the hybrfdoma J558L. Protein was purified from culture supematant and used to immunlze C57BU8 mice. Neutralizing serum antibodies against VSV were used as the read-out for B-cell activation. Resuftru Intravenous immunization with graded concentrations of Fc-G (10, 3, 1,0.3 pg) failed to induce B cell responses. However, intraperftoneal administration of the Fc-G producing hybrfdoma induced neutralizing IgG antibodies which appeared at day 12 after immunization. No IgM antibodies were detected. This result suggested the requirement of T-cell help for B-ceil activation by the soluble form of VSV-G. The induction of neutralizing antibodies depended on a continuous antigen supply and/or induction of an allogenic response against the hybridoma cells (H-p) providing presumably an inflamatory reaction and T-cell help. The T cell dependence (TO) of neutralizing antibodies induced by Fc-G is being currently investigated by Immunization of athymic mice and mice depleted of CD4+ T cells. Conclusion: This study indicated that expression of VSV-G in a soluble Fc-G behaves as TD antigen, compared to highly repetitive organization of VSV-G in viral particles (R-1) and to the poorly organized recombinant VSV-G (R-2) forming micells.
IP.4.04.23
Cytokine diversity of Influenza specific memory TH clones from a single murine progenitor
C.M. Graham, C.A. Smith, D.B. Thomas. National lnstifufe rbr Medicat Research, London, UK Introductfon: CD4+ memory T cell clones from a C57Bl/lO (H-F) single donor previously infected with influenza virus (H3N2) and which recognise haemag-