Workshop C Immunity and Infection I

I Institut fur Immunologie, Universitat, 2300 Kiel, 2 Gesellschaft fur Biotechnologische Forschung, 3300 Braunschweig, 3Institut fur Klinische u. Molekular-Virologie, Universitat Erlangen-Nurnberg, 8520 Erlangen, Germany

C.l Humoral and cellular immune response to the major viral matrix protein (pp 65) of human cytomegalovirus

H. ALEXANDER!, J. STEINMANN!, W. LINDENMAIER2, B. PLACHTER3, and W. MOLLER­RUCHHOLTZ

I

In immunocompromised patients, e.g., allograft recIpIents and patients with acquired immunodeficiency syndrome, the human cytomegalovirus (HCMV)-infection is a major cause of morbidity and mortality. Strategies for prevention and treatment of HCMV require more knowledge about virus proteins which serve as dominant targets for the immune system. To characterize the human humoral response to HCMV we generated human monoclonal antibodies (HMAb) from hybridomas of the splenocytes from a 53 year-old organ donor (HCMV IgG titer 1 :400, no detectable IgM). We obtained four stable clones producing HCMV-specific IgGI HMAb. Three of them detect the abundant viral matrix phosphoprotein of pp65 in transfected cells, as shown by immunofluorescence. Additionally we measured the cellular immune response of seropositive donors by T-cell proliferation against whole HCMV and dense-body preparation. This preparation consists of > 80 % pp65 and was capable of eliciting in vitro immune reaction as the whole virus does. Furthermore, KHATTAB et al. (1991) localized three hexadecapeptides within the carboxy half (aa 303-aa 389) of pp65, which are strongly reactive in T cell proliferation and made them available to us. However, in dot blot and sandwich ELISA we could not, as yet, demonstrate reactivity of the pp65-specific HMAb to them. We are now under way to establish cell lines and clones against these peptides. Taken together, the abundant viral matrix protein (pp65) of HCMV is immunogenic for B- and T-cell response in humans. HMAb and T-cell clones to pp65 will allow us to specify the functional relevance of those virus epitopes which are important for vaccine development and passive immune therapy.

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Institute of Clinical Microbiology, University of Erlangen-Niirnberg, 8520 Erlangen, Ger­many

C.2 Production of interleukin-l during infection of mice with Yersinia enterocolitica 08

H. ULRICH BEUSCHER, ULF-PETER RAUSCH, and MARTIN ROLLINGHOFF

Here we examined the expression of IL-1 (i.e. IL-1a and IL-1B) in response to infection of mice with the enteropathogenic bacteria, Y. enterocolitica 08. Orogastrical application of the bacteria caused abscess formation restricted to the Peyer's Patches (PP). Immunohistological analysis of inflamed PP revealed an infiltration of phagocytic cells (Mac 1 pos.), beginning at day 1 after infection. The relative amount of phagocytes increased over time (day 1-6) and correlated with the presence and proliferation of the bacteria within the tissue. Between day 1 and 2, staining of the tissue sections with antisera specific for either IL-1a or IL-1B showed only IL-1B expressing cells. However, by day 3-6 both, IL-1a and IL-1B producing phago­cytes could be detected. No IL-1 positive cells were detected in other tissues such as lymph nodes, spleen, liver and kidney. This lack of expression correlated with the absence of IL-1 activity in organ extracts as well as in the plasma. These data indicate a temporal dissociation of IL-1a and IL-1B expression during an acute inflammatory response and further suggest a tissue specific production of IL-1 at the local site of inflammation.

Forschungsinstitut Borstel, 2061 Borstel, Germany, and 1 Universitat fiir Bodenkultur, 1180 Vienna, Austria

C.3 Immunogenicity and antigenicity of synthetic glycoconjugates representing the genus-specific epitope of chlamydial lipopolysaccharide (LPS)

L. BRADE, Y. Fu, M. BAUMANN, P. KOSMA\ and H. BRADE

Chlamydiae are pathogenic, obligatory intracellular parasites which carryon their surface a genus-specific LPS antigen which has been cloned and expressed in E. coli. The tetrasaccharide aKdo(2-8)-aKdo(2-4)-aKdo(2-6)-BGlcNAc, a partial structure of chlamydial LPS represent­ing the genus-specific epitope, was synthesized and covalently linked to bovine serum albumine (BSA) resulting in an artificial glycoconjugate antigen. The glycoconjugate was used to immunize mice to prepare chlamydia-specific monoclonal antibodies which were selected using chlamydia-specific LPS antigens and the structurally and antigenically related Re-type LPS of a Salmonella minnesota rough mutant. Characterization of the selected antibodies was performed by i) hemagglutination using recombinant chlamydia-specific LPS, ii) inhibition assays with synthetic polyacrylamide derivatives containing the genus-specific epitope or partial structures thereof, iii) enzyme immuno assay using recombinant LPS and synthetic BSA-glycoconjugates and iv) by immunofluorescence using L929 mono layers infected with Chlamydia psittaci or C. trachomatis. Chlamydia-specific antibodies were obtained which recognized as the minimal structure for binding the 2.8-linked Kdo disaccharide or the Kdo trisaccharide and which were hundred times higher in affinity than those obtained after immunization with chlamydial elementary bodies.

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Dept. of Immunology, University, 7900 Ulm, Germany

C.4 T cell responses to antigens of intracellular bacteria

SABINE DAUGELAT, HEINZ GULLE, BERND SCHOEL, and STEFAN H. E. KAUFMANN

We are interested in characterizing major T cell antigens of intracellular bacteria. A new method which was developed in our laboratory (H. GULLE et a!., J. Imm. Meth. 133, 253, 1990) allows direct T cell screening of two-dimensionally separated proteins. A mixture of bacterial proteins is first separated by 2D-PAGE and then transferred into 480 distinct soluble fractions. We have used this technique to screen secreted antigens of actively growing Mycobacterium tuberculosis H37Rv organisms with peripheral blood mononuclear leuko­cytes of tuberculosis patients and healthy contacts. Tuberculosis patients and PPD+ contacts preferentially recognized a cluster of acidic proteins in the molecular weight range of 20-100 kDa whereas PPD- contacts did not respond to these antigens. Lysates of Listeria monocy­togenes EGD organisms were separated and transferred by the same method. Screening these fractions with T lymphocytes purified from spleens of infected C57BLl6 mice resulted in a specific proliferation pattern. We are currently investigating the kinetics of the proliferative response as well as cytokine production in order to correlate T cell specificity with the biological function of these cells. This procedure could ultimately lead to the identification of potential vaccine antigens.

Heinrich-Pette-Institut, 2000 Hamburg 20, Germany

C.S Antiviral immune responses of fully allogeneic irradiation bone marrow mouse chimeras

L. FANG, A. GESSNER, K. KUHLCKE, J. GOSSMANN, and F. LEHMANN-GRUBE

In lymphocytic choriomeningitis (LCM) virus-infected chimeras of the type [BIO.BR -> BID] higher infectious titers were attained in spleens and livers than in the organs of the mice used for their construction and the subsequent elimination was retarded, but eventually the virus was cleared. Virus-specific cytotoxic T lymphocytes (CTL) and antiviral effector cells were determined by chromium-release assay and adoptive immunization, respec­tively. Their numbers were lower in chimeras than in BI0.BR or BID mice, and fewer were restricted for the donor's haplotype than for the recipient's haplotype. Comparable results were obtained when CTL and their precursors were enumerated by limiting dilution assay. Transfusion of immune spleen cells from either BI0.BR or BID mice resulted in incomplete virus elimination from the spleens of infected chimeras, while transfusion of a mixture of the two types of immune cells led to rapid clearance. We conclude that in the chimeras cells of both donor and recipient haelotypes participate in the infection with LCM virus, the control of which is mediated by H-2 - and H-2k-restricted CTL. The numbers of spleen cells releasing antiviral antibodies of both major classes (IgM and IgG) were virtually identical in infected chimeras and in BIO and BIO.BR mice, and the same was true with regard to single cells producing interferon-yo

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Heinrich-Pette-Institut, 2000 Hamburg 20, Germany

C.6 Control of a virus infection of the mouse by cytotoxic T lymphocytes (CTL) after loss of the major histocompatibility complex-encoded class I molecule that presents the immunodominant viral CTL-epitope

C. GEGIN and F. LEHMANN-GRUBE

For controlling infection of the mouse with the lymphocytic choriomeningitis (LCM) virus, CTL are essential. In the infected BALB!c mouse the arising LCM virus-specific CTL are exclusively restricted by the class I major histocompatibility complex-encoded molecule «L,,; K- or D-restricted anti-viral CTL cannot be detected. Thus, the infected L-deficient BALB/c mutant, C_H_2dm2 should not eliminate the virus but rather become a carrier. The experimental evidence proves that the C_H_2dm2 mouse is perfectly capable of getting rid of the virus, which is explained by the generation of K- and D-restricted CTL. Why such cells remain undetecta­ble in BALB/c mice is currently unexplained, because there is no lack of the corresponding precursors and the corresponding virus antigen is presented. Despite the sole presence of L­restricted CTL, transfusion of day-8-immune spleen cells from BALBI c into infected (L­deficient) mutant mice results in accelerated virus elimination from the organs of the latter. We interpret this as further evidence for our opinion that CTL-mediated lysis of virus-infected target cells is not the (only) mechanism by which this infection is controlled in vivo.

Institut fur Klinische Mikrobiologie, Universitiit Erlangen-Nurnberg, 8520 Erlangen, Ger­many

C.7 Expression of large amounts of murine IL-10 in E. coli and its modulating effect on the cellular immune response to L. major

ANDRE GESSNER and MARTIN ROLLlNGHOFF

The pattern of Iymphokine expression is important for the clinical outcome of an infection of mice with the protozoan Leishmania major. In resistant C57BLl6 mice predominantly CD4+ T cells of the Th1 type are activated to produce IFN-y and IL-2, whereas in BALB/c mice, which succumb the infection, large numbers of antigen-specific Th2 type T cells, producing IL-4, can be found. Clones of the latter type are known to produce IL-10, formerly known as cytokine synthesis inhibitory factor (CSIF) (MOORE et aI., 1990). This Iymphokine was initially characterized as a factor which suppresses IFN-y production by Th1 cells (FIORENTINO, BOND, and MOSMANN, 1989).

To study the function of this cytokine with regard to its effect on the modulation of the CD4+ cell driven cellular immune response to L. major large amounts of the protein were expressed in E. coli as glutathion S-transferase-fusion protein, purified and digested with thrombin to remove the N-terminal part, yielding functional active murine IL-10. Results concerning in vivo and in vitro effects of this recombinant product during experimentally induced leishmaniasis will be presented.

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IInstitut fur Genetik u. Mikrobiologie, Universitat, 8700 Wurzburg, 2Institut fur Med. Mikrobiologie, Universitat, 2400 Lubeck, Germany

e.S Studies on the interaction of virulent and avirulent mutants of Legionella pneumophila with macrophages, lung fibroblasts and Acanthamoeba cells

J. HACKER I, R. MARRE2, L. BENDERI, M. OTTI, and M. STEINERTI

Legionella pneumophila (Lp), the causative agent of Legionnaires' diseases is a Gram­negative bacterial organism. Lp is able to invade professional and non-professional phagocytic cells and is also able to penetrate free living amoebae. To study the mechanisms of interaction of Lp and eukaryotic cells non-virulent bacterial mutant strains were isolated. Compared to their virulent counterparts these mutants did not show any genomic alterations indicated by the use of pulsed field gel electrophoresis but two-dimensional SDS-PAGE. While the virulent strains were able to multiply intracellularly in macrophage-like cell lines and Acanthamoebae, a property which increased following passage of bacteriae through lung fibroblast cells the non virulent mutants were unable to survive in the eukaryots. Our studies further indicate that a membrane-associated protein of Lp, termed Mip «<macrophage infectivity potentiator») is necessary for intracellular multiplication. The amino acid-sequence of Mip shows similarities to the sequence of protein kinase C inhibitor II. Studies are underway to determine the exact role of this protein in the infectious process.

I Max-Planck-Institut fUr Immunbiologie, 7800 Freiburg, 2 Institut fur Immunologie, Univer­sitat, 6900 Heidelberg, 3 Abt. fur angewandte Immunologie, Deutsches Krebsforschungszen­trum, 6900 Heidelberg, Germany

e. 9 Qualitative and quantitative analysis of T cell responses during B. burgdorferi infection in mice

N. HONARVARI, M. KRAMER2, u. E. SCHAIBLE I, R. WALLICH3, and M. M. SIMONI

We have shown in previous studies that immunocompetent mice of various inbred strains but not immunodeficiency (SCID) mice are able to control B. burgdorferi infection and disease. In addition we have found that SCID mice which develop chronic arthritis, carditis and hepatitis upon experimental inoculation with B. burgdorferi are protected against the disease by passive transfer of either presensitized spleen cells or by polyclonal B. burgdorferi specific immune sera and monoclonal antibodies (mAb) to the outer surface protein A or B (OspA, OspB). These data suggest an important role for T cells in the generation of protective immune responses in mice. Previous experiments have shown that CD4+ and CD8+ T cells from various inbred strains are primed in vivo to B. burgdorferi and that they can be specifically restimulated in vitro by either intact spirochetes or by total celllysates. In order to get more information on the quality and quantity of T cells activated during a spirochetal infection in mice we are presently analyzing their reactivities to individual B. burgdorferi antigens, their frequencies and their functional capacities. For this purpose a method has been developed which allows the purification of individual B. burgdorferi structures from complex protein preparations. It is hoped that these studies will help to understand the molecular mechanisms underlying the induction of protective immunity against B. burgdorferi infection. In addition they may also provide information on the role of T cells in the pathogenesis of Lyme disease.

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1 Institut fur Med. Virologie u. Immunologie, 4300 Essen, Germany, 2 The Wistar Institute for Anatomy and Biology, Philadelphia, PA, USA

C.lO Alterations of the rhesus monkeys immune system following rabies virus infection and monoclonal antibody treatment

E. KREUZFELDERt, O. THRAENHARTt, 1. MARCUSt, and B. DIETZSCHOLD2

To establish efficacy of monoclonal antibody (mAbs) treatment for rabies virus infection eighteen 3.5 to 4.5 kg in weight rhesus monkeys were administered one intramuscular dose of 0.5 ml (= 105

.2 MICLD5o) Thai dog sc 2960 virus strain. 12 monkeys were treated with 0.5 ml

of a cocktail of 5 murine mAbs. Whereas 6 monkeys received a dose of 40 IU mAbs/ml given in the previously infected area (group 1), further 6 monkeys were treated with 200 IU mAbsl ml (group 2). Animals were bled for antibody, IL-2 receptor(R) measurement and T cell enumeration before infection and 8, 16 and 37 days after infection. All 3 monkeys with a survival time of longer than 90 days showed a marked reduction of CD2 cells below 10 %. Moreover long survivors showed the highest anti-rabies virus antibody concentration measured by ELISA. These antibodies had no neutralizing capacity measured by rapid fluorescent inhibition test. Decrease of percentages of CD2 cells from virus-infected monkeys without mAbs treatment (group 3) seems to be related to increased IL-2R concentrations, therefore activation of T cells, and increased survival time. CD2 percentages of group 1 monkeys also seem related to increased survival time. In contrast, decreased IL-2R receptor concentrations seem to characterize the two long survivors of group 1 monkeys. In summary, influence of rabies infection on the immune system of monkeys is shown. Whereas mAbs untreated animals showed signs of activation of the immune system in relation to survival, the mAbs treatment showed a complex pattern of immune reactivity. This reactivity is likely an expression of anti-murine and anti-rabies response.

! Institut fur angewandte Immunologie u. Biotechnologie Hamburg GmbH, 2000 Hamburg 65, 2Institut fur Hygiene u. Mikrobiologie, Universitat, 8700 Wurzburg, 3 Hygienisches Institut, 2000 Hamburg, Germany

C.ll Evaluation of monoclonal antibodies and polyclonal antisera against Borrelia burgdorferi antigens for the diagnosis of lyme disease

T. MEYER!, J. BERGER!, H. KARCH2, M. MOSKOPHIDIS3

, D. KEESER!, and R. ARNDT!

Diagnosis of Lyme disease is often impeded by unsuccessful cultivation of Borrelia burgdorferi (Bb), lack of anamnestic information and difficulties in interpreting results of serologic investigations. In order to improve diagnosis of Lyme borreliosis we employed monoclonal antibodies (mAbs) and polyclonal antisera against Bb-antigens (OspA, Flagellin) to immunologically detect these antigens in the urine of patients with presumed Lyme disease. A comparison of the antigen detection assay with serological methods (Immunoblot, ELISA) revealed that of 49 patients 24 were positive by both assays. Ten patients had Bb-antigens in the urine while no serological response was detectable. In the urine of two patients with IgM­antibodies against Bb no antigen could be identified. We further investigated 5 patients that were positive with respect to the presence of Bb-antigen in the urine and subsequently were

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treated with antibiotics. Urine samples of these patients were analyzed with the Bb-antibodies during this treatment period. In all 5 cases no Bb-antigens were immunologically detectable after 2-6 weeks of antibiotic treatment. The antigen detection assay seems to be a useful complement to Lyme disease diagnostics and furthermore provides a convenient method for monitoring the efficiency of antibiotic treatment of Lyme disease.

Institut fur Klinische Mikrobiologie, Universitiit Erlangen-Nurnberg, 8520 Erlangen, Ger­many

C.12 Langerhans cells can serve as host cells for Leishmania major, the cause of cutaneous leishmaniasis, and are potent stimulators of an antigen-specific T-cell immune response

HEIDRUN MOLL, ANTJE WILL, CHRISTINE BLANK, and MARTIN ROLLINGHOFF

Cutaneous leishmaniasis is initiated by the bite of an infected sandfly and inoculation of Leishmania major parasites into the mammalian skin. Macrophages are known to playa central role in the course of infection because they are the prime host cells supporting the replication of parasites and function as antigen-presenting cells modulating the specific immune activity. However, in addition to macrophages in the dermis, the skin contains epidermal Langerhans cells (LC) which can present antigen to T cells. Therefore, we examined murine LC for their ability to serve as host cells and to induce aT-cell response to L. major. Using double-labelling immunohistological techniques, we could clearly show that LC can be infected by L. major both in vitro and in vivo. Furthermore, LC can present L. major antigen to T cells from antigen-primed mice in vitro. The T cell-stimulatory effect of epidermal cells was much greater than that of spleen cells, a standard population of antigen-presenting cells. It was antigen­specific and could be induced by lysates of parasites and by live organisms. In addition, epidermal LC were able to induce a T-cell response to purified L. major lipophosphoglycan in vitro. The data suggest that LC take up parasites in the skin and are important antigen­presenting cells in cutaneous leishmaniasis. They may perform a critical function by transport­ing antigen into lymphoid tissues for initiation of the immune response and by stimulating T cells that infiltrate the cutaneous lesion.

W.H.O.-IRTC, Institute of Biochemistry, University of Lausanne, 1055 Epalinges, Switzer­land

C.13 Contribution of CD8+ T cells to resistance in Leishmania major infections

1. MOLLER, P. KROPF, G. MILON, and J. LOUIS

In the murine model of experimentally induced cutaneous leishmaniasis the importance of CD4+ T cells in resolution of cutaneous lesions is firmly established. The contribution of CDS+ T cells is still controversial. The extreme susceptibility of normal BALB/c mice to

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infection with L. major can be overcome by various immune interventions. We enabled BALB/c mice to resolve their infections by a) administration of a-CD4 mAb at the beginning of infection and b) treatment with a-IL4 mAb and investigated whether the state of resistance to infection seen in BALB/c mice as a result of these two manipulations depends upon CDS+ T cells. Our results indicate that CDS+ T cells are involved in the resolution of lesions and the reduction of parasite burden which occurs in BALB/c mice, rendered resistant by treatment with a-IL4 mAb. A protective effect of parasite specific CDS+ T cells could also be revealed in susceptible mice, rendered resistant by injection of a single dose of a-CD4 mAb at the beginning of infection. Moreover, the immunity to reinfection, which is seen in BALB/c mice, cured by a single injection of a-CD4 mAb, mainly depended upon the activity of CDS+ T cells. The delayed type hypersensitivity (DTH) response of BALB/c mice, cured as a result of immune intervention with a-CD4 mAb, depended at least in part upon CDS+ T cells. Furthermore, an expansion of specific CDS+ T cells, able to transfer specific DTH reactions, was observed in the spleens of cured BALB/c mice. CDS+ T cells contributed significantly to IFN-y production observed after specific stimulation of lymphoid cells from BALB/C mice, cured as a result of administration of a-CD4 mAb at the beginning of infection.

Dept. of Pathology, Free University Hospital, De Boelelaan 1117, 10Sl HV Amsterdam, The Netherlands

C.14 Hexapeptides related to retroviral TM-proteins suppress fMLP­induced responses in human monocytes and granulocytes

R. A. J. OOSTENDORP and R. J. SCHEPER

Retroviral transmembrane envelope (TM-)proteins playa major role in retrovirus-associated immunosuppression. CKS-17, a synthetic peptide derived from the MuLV TM-protein plSE, inhibits various immune-effector functions. In previous studies we have shown that the CKS-17-derived hexapeptide LDLLFL inhibits IL-2 induced lymphoproliferation, NK-activity and fMLP-induced monocyte and granulocyte polarization. Here, we investigated the mechanism of action of LDLLFL. Inhibition of polarization correlates with decreased [Ca2+1i-influx in response to fMLP: at 10-8 M fMLP almost complete inhibition; at 10-6 M fMLP no suppression. In contrast, [Ca2+1i-influx in response to CSa or LTB4 is not inhibited by LDLLFL. These results suggest that LDLLFL interferes with binding of fMLP to its receptor. This was confirmed in fMLP-receptor binding studies in which we used both fluorescent labelled and radiolabelled fMLP. Subsequently we studied whether other immune-effector functions, shown previously to be inhibited by LDLLFL, were modulated by fMLP. We found that neither proliferation of the IL-2 dependent cell line HT-2 nor the inhibition of the proliferation of this cell line by LDLLFL were modulated by fMLP. Our results indicate that one mechanism of retroviral TM-proteins to inhibit monocyte and granulocyte function is to interfere with inflammatory responses to bacterial cell-wall fragments. Modulation of these responses is, however, not the only mechanism by which retroviral TM-proteins interfere with immune-effector mechanisms.

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Dept. of Immunology, University, 7900 Ulm, and 1 Institute for Genetics and Microbiology, University, 8700 Wurzburg, Germany

C.t5 Differential recognition of Hly+ and Hly- Listeria monocytogenes by CD8+ cytolytic T cells

GUDRUN SZALAY, WERNER GOEBEL \ and STEFAN H. E. KAUFMANN

The intracellular pathogen Listeria monocytogenes (LM) is frequently used as a model of T cell-mediated immunity in the murine system and the hemolytic toxin listeriolysin 0 is a major virulence factor. Inactivation of the listeriolysin gene by transposon mutagenesis or by selection of spontaneous deletion mutants led to the loss of hemolytic activity and to a Hly­phenotype (S. KA THARIOU et aI., J. Bact. 169, 1291, 1987). We investigated the capacity of Hly+ and Hly- LM to grow in mouse bone marrow derived macrophages (BMM) and the cytolytic potential of CD8+ lines and clones derived from Hly+ LM-infected C57BLl6 mice. Hly+ LM strain EGD and WTD could grow intracellularly whereas Hly- Tn-mutants M3 and M20 as well as the deletion mutant SLCC 53 failed to do so. A positively sorted CD8+ line and clone not only killed HLy+ LM infected BMM but also HLy- LM infected BMM though often to a smaller degree. Interestingly, SLCC 53-infected BMM were more effectively lysed than BMM treated with EGD. These results show a dissociation between listerial virulence and the recognition of infected BMM by CD8+ T cells.

Institute of Immunology and Transfusion Medicine, University of Lubeck Medical School, 2400 Lubeck, Germany

C.t6 Quantification of latently infected B-lymphocytes in Epstein-Barr Virus-seropositive, healthy individuals by the polymerase chain reaction

H. WAGNER, G. BEIN, A. BITSCH, and H. KIRCHNER

In order to estimate the amount of latently infected B-lymphocytes in blood products of Epstein-Barr-Virus (EBV) seropositive individuals we designed highly sensitive and specific oligonucleotide primers from DNA sequences unique to the BAM HI-W region being directly repeated up to 11 times within the EBV genome. This segment of the EBV genome was enzymatically amplified by the polymerase chain reaction. Culture supernatant from HSV-l, HSV-2, and CMV and DNA from EBV-seronegative did not give false positive results after amplification. By using the primers we detected EBV -related sequences in culture supernatant of the EBV-B type, P3HR-l, up to a dilution of 1:50000 at a background of 150000 EBV­negative cells. We also amplified up to 10 copies of a plasmid containing the BAM HI-W region of the EBV-A strain, B95-8, at a background of 1 ug EBV-negative DNA. Therefore it will be possible to detect one latently EBV -infected cell among 150000 EBV -negative cells, a situation expected to exit in vivo in EBV-seropositive individuals. If the results of amplifying the plasmid dilution are used as quantitative standard, it will be possible to estimate the amount of infected B-cells in latently EBV -infected individuals.

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! Angewandte Immunologie, Deutsches Krebsforschungszentrum, 6900 Heidelberg, 2Institut fur Immunologie, Universitiit, 6900 Heidelberg, 3 Max-Planck-Institut fur Immunbiologie, 7800 Freiburg, Germany

C.l7 Molecular analyses identify two distinct classes of Borrelia burgdorferi

R. WALLICH!, G. KOBER!, S. E. MOTER2, M. D. KRAMER2, U. E. SCHAIBLE3, and M. M. SIMON3

To investigate the genotypical and phenotypical heterogeneity among different isolates of the Lyme disease spirochete Borrelia burgdorferi we have analyzed 30 different European and North American strains. Employing (i) Southern blot hybridization and (ii) polymerase chain reaction using probes/primers that detect either the plasmid located Outer surface protein (Osp) genes or a chromosomal gene encoding the 41-kDa protein flagellin, as well as (iii) Western blot analyses all Borrelia burgdorferi strains were subdivided into two distinct groups. Almost all North American strains tested to date fall into a single reactivity group, whereas European strains fall into two groups, one of which is similar to the American type. This classification may have a clinical correlate reflected in differences between predominant spectra of Lyme disease as seen in Europe and North America. Furthermore, these results may have important implications for improvement of diagnostic test systems and development of potential vaccines.

Heinrich-Pette-Institut fUr Experimentelle Virologie u. Immunologie, 2000 Hamburg 20, Germany

C.l8 Analysis of structural domains of SV 40 T -antigen involved in mediating TSTA-activity

JENS ZERRAHN and WOLFGANG DEPPERT

In simian virus 40 (SV40)-transformed cells, the virally encoded SV40 T-antigen (T-Ag) forms the tumor-specific transplantation antigen (TSTA). Preimmunization of mice with soluble, purified T-Ag leads to rejection of syngeneic T-Ag-expressing tumor cells, regardless of whether tumor formation is T-Ag-dependent (mKSA cells), or T-Ag-independent (MethA cells mediated process, with an essential contribution of CDS+ T-cells. The requirement of CDS+ T-cells for this process was shown by in vivo depletion of the CD8+ subset. Since exogenous, soluble T -Ag induces the activation of CD8+ T -cells one has to assume that it can be presented in a MHC-I restricted manner, like an endogenous antigen. Induction of anti­TSTA activity by SV40 T-Ag is not dependent on the native conformation of the protein, as tumor protection is also observed after immunization with SDS-denatured T-Ag. This suggests that sequence determinants are responsible for introducing T-Ag into the MHC-I­presenting pathway. To identify structural domains on T-Ag responsible for its TSTA activity, various fragments of T -Ag were expressed in bacterial expression vectors, purified, SDS­denatured, and used to immunize BALB/c (H_2d

) mice. Both, N- and C-terminal fragments were able to induce rejection of syngeneic SV40 tumor cells (mKSA), demonstrating that different structural domains on T -Ag can induce its activity.