Ulrich H. Koszinowski

Detection of antibodies to sendai virus by enzyme-linked immunosorbent assay (elisa).

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Sendai virus, a paramyxovirus, is described. The assay was found to be about 20-fold more sensitive than the hemagglutination inhibition assay. Differentiation between virus specific IgG and IgM is possible. The test appears to be especially useful in the study of early events in antibody formation in vivo as well as in vitro.

Involvement of Mhc Loci in immune responses that are not Ir-gene-controlled

Twenty-nine randomly chosen, soluble antigens, many of them highly complex, were used to immunize mice of two strains, C3H and B10.RIII. Lymphnode cells from the immunized mice were restimulated in vitro with the priming antigens and the proliferative response of the cells was determined. Both strains were responders to 28 of 29 antigens. Eight antigens were then used to immunize 11 congenic strains carrying different H-2 haplotypes, and the T-cell proliferative responses of these strains were determined. Again, all the strains responded to seven of the eight antigens. These experiments were then repeated, but this time -antibodies specific for the A (AαAβ) or E (EαEβ) molecules were added to the culture to block the in vitro responsiveness. In all but one of the responses, inhibition with both A-specific and E-specific antibodies was observed. The response to one antigen (Blastoinyces) was exceptional in that some strains were nonresponders to this antigen. Furthermore, the response in the responder strains was blocked with A-specific, but not with E-specific, antibodies. The study demonstrates that responses to antigens not controlled by Irr genes nevertheless require participation of class II Mhc molecules. In contrast to Ir gene-controlled responses involving either the A- or the E-molecule controlling loci (but never both), the responses not Ir-controlled involve participation of both A- and E-controlling loci. The lack of Ir-gene control is probably the result of complexity of the responses to multiple determinants. There is thus no principal difference between responses controlled and those not controlled by Ir genes: both types involve the recognition of the antigen, in the context of Mhc molecules.

Virus-specific T-cell sensitization

Syngeneic, semiallogeneic, or allogeneic spleen lymphocytes were transferred intonu/nu BALB/c mice, which were infected with vaccinia virus. Specific Sensitization of transferred thymus-derived cells was determined in vivo by mean survival time and virus titer in the spleen six days after infection, and in vitro by cell-mediated cytolysis of vaccinia virus-infected syngeneic target cells. Virus-specific Sensitization took place only after transfer of syngeneic or semiallogeneic spleen lymphocytes; allogeneic lymphocytes had no influence on mean survival time or virus titer and showed no virus-specific cytolytic activity in vitro. Infection of mice with vaccinia virus-strain WR, Elstree, DIs, or DIs-infected syngeneic fibroblasts resulted in the generation of virus-specific effector cells, while injection of a high amount of inactivated virus particles caused no Sensitization. These results suggest H-2 homology for production of virus-specific effector cells. Propagation of virus is not necessary, since early surface antigens, combined with syngeneic H-2 antigens, suffice for Sensitization of cytolytic T lymphocytes.

Interactions between vaccinia virus and sensitized macrophagesin vitro

SummaryThe action of peritoneal exudate cells (PEC) from normal and vaccinia virus infected mice on infectious vaccinia virus particles was investigatedin vitro. PEC from immune mice showed a significantly higher infectivity titre reduction (virus clearance, VC) than normal cells. This effect could be clearly attributed to the macrophage. Vaccinia virus multiplied in PEC from normal animals while there was no virus propagation in cells from immunized mice. The release of adsorbed or engulfed virus was reduced significantly in PEC from immunized animals. Anti-vaccinia-antibodies seem to activate normal macrophages to increased virus clearance. This stimulating effect was demonstrable only in the IgG fraction of the antiserum.The activity of macrophages from mice injected three times over a period of 14 days with vaccinia virus could be entirely blocked with anti-mouse-IgG, while PEC from mice injected one time six days previously were not inhibited.

Cell-mediated cytotoxicity against sendai-virus-infected cells

After injection of Sendai virus, a parainfluenza virus type I, mice generate cytotoxic lymphocytes which lyse specifically Sendai-virus-infected target cells in vitro. Their action is not inhibited by specific antibody in vitro. Killer cell activity appears 4 days after infection, reaches a maximum on the 7th day and disappears on the 14th to 16th day. Decrease of cytotoxic cell activity is correlated with an increase of haemagglutinating antibodies. The cytotoxic effector cell could be characterized as a thymus-derived cell, there is no specific activity in antibody-dependent cell-mediated cytolysis (ADCC). The degree of cytotoxic effector cell activity is only slightly influenced by the dose of injected infective virus. Using different syngeneic Sendai-virus-infected cells as targets for cell-mediated cytotoxicity, a tumor line was not lysed by cytotoxic lymphocytes in spite of viral surface antigens. Preliminary experiments were performed to demonstrate the H-2 gene restriction of the cytotoxic interaction. Using macrophages and tumor cells as targets only syngeneic infected target cells were lysed.

Increased cellular immunity against host cell antigens induced by vacciniavirus

Guinea pigs show an increased cell-mediated immunity against host cell antigens after active immunization with vaccinia virus infected tissue culture cells. This immunologic adjuvant effect was observed in a heterologous system comprising primary rabbit kidney cells, primary fibroblasts from the mouse, permanent monkey kidney cells andin vivo infected mouse brain cells. The quantitative determination of the cellular immune response was carried out both in macrophage migration inhibition and in lymphocyte transformation tests. Cell-mediated immunity appears on the third day after immunization. A prerequisite for the adjuvant effect is the reproduction of the virus in the cell against which the increased cell-mediated immune response is directed.

Gesteigerte Immunogenität von Plasmamembranen vaccinia virusinfizierter BHK-Zellen

Vacciniavirusinduzierte Zelloberflächenveränderungen können durch den zytoziden Effekt virusspezifischer Antikörper auf infizierte Zellen sowie durch die Agglutination virusinfizierter Zellen durch Concanavalin A demonstriert werden. Diese Veränderungen treten zeitlich korreliert mit einer gesteigerten Immunogenität der Wirtszelle 2–3 Stunden nach Infektionsbeginn bereits vor der Produktion reifer infektiöser Viruspartikeln auf. Eine gesteigerte Antikörperproduktion gegen Wirtszellantigene wird auch nach Immunisierung mit isolierten Plasmamembranen vacciniavirusinfizierter Zellen erreicht, während Zellkernfraktionen diesen Effekt nicht auslösen. Bei den zytotoxischen Antikörpern, die zu den 7 S-Immunglobulinen gehören, handelt es sich — wie durch Absorption mit BHK-Zellmembranen gezeigt werden kann — um spezifisch gegen BHK-Zellen gerichtete Antikörper. Cell surface alterations induced by vaccinia virus can be demonstrated either by the cytocidal effect of specific antibodies against vaccinia virus on the infected cells or agglutination of the infected cells by Concanavalin A. This change occurs 2–3 hours after infection, that is before any mature particles of vaccinia virus were produced. It coincides with an increased immunogenicity of the host cell. The production of cytotoxic antibodies against host cell antigens was also increased after active immunization with plasma membranes isolated from vaccinia virus infected BHK-cells, whereas the fraction of cell nuclei did not show up this effect. The cytotoxic antibodies which proved to be 7 S immunoglobulines, are specifically directed against BHK-cells as could be shown by absorption tests with membranes of BHK-cells.

Cytotoxic interactions of virus specific effector cells with virus infected targets of different cell type.

The action of Sendai virus specific cytolytic T lymphocytes (CTL) against Sendai virus infected macrophages was found to be H‐2 restricted while Sendai virus infected cell lines, including fibroblasts and tumour cells, were lysed across the H‐2 barrier to some extent. The properties of the Meth‐A tumour cell, which was resistant to lysis by allogenic killer cells was investigated. Sendai virus specific CTL failed to kill Sendai virus infected Meth‐A cells but after vaccinia virus infection these target cells were susceptible to lysis by vaccinia virus specific CTL.

Cellular immunity to HB antigen (Australia antigen)-in vitro reactions in guineapigs and human beings.

Guinea pigs were inoculated with purified HB-Antigen emulsified in Freunds complete adjuvant. After 16 days all animals had developed antibodies and cell-mediated immunity as revealed by macrophage-migration-test and lymphocyte transformation.12 healthy normal persons, 11 persistent HB-antigen-carriers and 7 HB-antibody-carriers were tested for cellular immunity against HB-antigen using the leucocyte-migration-test. Specific reactions were found only in those persons, who had HB-antibody in their serum by means of counterimmunoelectrophoresis or radioimmunassay.ZusammenfassungMeerschweinchen wurden mit gereinigtem HB-Antigen in Verbindung mit komplettem Freundschen Adjuvans immunisiert. Nach 16 Tagen entwickelten alle Tiere Antikörper und eine spezifische cellulÄre ImmunitÄt gegen HB-Antigen, nachweisbar durch Makrophagenmigrationshemmungstest und Lymphocyten-Transformation.12 gesunde Kontrollpersonen, 11 Patienten mit HB-Antigen-Persistenz und 7 HB-AntikörpertrÄger wurden im Leukocytenmigrationstest auf cellulÄre ImmunitÄt gegen HB-Antigen untersucht. Spezifische Reaktionen wurden nur bei den Personen gefunden, bei denen sich durch überwanderungselektrophorese oder Radioimmunassay HB-Antikörper nachweisen lieen.

CellulÄre ImmunitÄt gegen HB-Antigen (Australia-Antigen) —In-vitro-reaktionen bei Meerschweinchen und Menschen

Guinea pigs were inoculated with purified HB-Antigen emulsified in Freunds complete adjuvant. After 16 days all animals had developed antibodies and cell-mediated immunity as revealed by macrophage-migration-test and lymphocyte transformation.12 healthy normal persons, 11 persistent HB-antigen-carriers and 7 HB-antibody-carriers were tested for cellular immunity against HB-antigen using the leucocyte-migration-test. Specific reactions were found only in those persons, who had HB-antibody in their serum by means of counterimmunoelectrophoresis or radioimmunassay.ZusammenfassungMeerschweinchen wurden mit gereinigtem HB-Antigen in Verbindung mit komplettem Freundschen Adjuvans immunisiert. Nach 16 Tagen entwickelten alle Tiere Antikörper und eine spezifische cellulÄre ImmunitÄt gegen HB-Antigen, nachweisbar durch Makrophagenmigrationshemmungstest und Lymphocyten-Transformation.12 gesunde Kontrollpersonen, 11 Patienten mit HB-Antigen-Persistenz und 7 HB-AntikörpertrÄger wurden im Leukocytenmigrationstest auf cellulÄre ImmunitÄt gegen HB-Antigen untersucht. Spezifische Reaktionen wurden nur bei den Personen gefunden, bei denen sich durch überwanderungselektrophorese oder Radioimmunassay HB-Antikörper nachweisen lieen.