Characterization of the C5a anphylatoxin effector domain and selection of a C5a-R antagonist using C5a phage libraries

Monday, 23 June 1997 - Oral presentations

( 0.3.03.5

[ Roles of the complement receptors, CR1 (CD35) and CR3 (CD1 1b/CD1 8), in regulation of the immune complex-elicited respiratory burst of neutrophils in whole blood

C.H. Nielsen ‘, S. Antonsen 2, S.H. Matthiesen ‘, R.G.Q. Leslie ‘. ’ Dept.of Medical Microbiology. Institute of Medical Sidogy, Odsnse UniverSily,Odsnse, Denmark, 2Dept. of Clinical Chemis@ Odense University Hospital, Odense, Denmark Introduction: The complement receptor, CRI, on erythrocytes (E) is involved in the transport of freshly opsonised immune complexes (C) from the circulation for disposal in the liver, whereas the receptors, CR1 and CR3, on myeioid cells are involved in complex uptake and in providing stimuiatory signals for activities such as the release of lysosomal contents and a respiratory burst (RB). We have previously shown that the presence of E efficiently restricts IC deposition on monocytes and neutrophils whilst others have repotted a CRI-dependent protective effect of E against IC-induced increase in endothellal permeability, mediated via neutmphiis (1). In the present study we examine the role of E CR1 in regulation of neutrophil activation by stimuli involving their CR. Mat&ale and Method% The binding to neutrophils of immune complexes (IC), comprising of FITC-labelled Tetanus Toxoid (lT) and human anti-n Abs, and the consequent respiratory burst (RB) were investigated in whole blood cell preparations suspended in 75% serum, using flow cytometry. The studies were perfom7ed both in absence and presence of function-blocking mAbs against CR1 and CR3 (3DQ and Ab44, respectively). In addition, in some experiments, the levels of the iysosomai components, iactofenfn and elastase in culture supematants were measured. Rssuk Blockade of CR1 on washed blood cells, using the mAb, 3D9, resulted in a l.Q-fold increase in the IC-elicited neutrophii RB after 5 min. of incubation, rising to 3.1-fold after 40 min. This enhancement was not due to increased ICdeposition on the neutrophiis. Blockade of CR3 abrogated the 3DQ-induced rise in RB activity and inhibited the IC-binding to neutmphiis in a whole blood ceil preparation, with or without 3D9, by ca. 40% from 15-40 min., while reducing their RB over 40 min. to appmximateiy one third. Blockade of CR1 on either E or leukocytes, before mixing the populations, revealed that the potentiation of the RB by 3DQwas associated with abrogation of E-CR1 function, whereas blockade of leukocyte-CR1 had a diminishing effect. Exposure to IC in high concentrations induced release of both specific and azumphillc granule contents from neutrophiis. The latter was CRddependent, in that blockade of the receptor inhibited the iactofenin release by one third during 40 min. of incubation. Conclusion: CR3 plays a significant role in the IC-mediated generation of a RB and expulsion of specific granules by neutmphiis, while CR1 on whole blood cells, ptimatily E-CRl, has the capacity to restrict the IC-elicited RB in neutmphiis. We propose that E exert their restrictive effect in two ways: by direct competition for Cdbcoated IC and by mediating the rapid conversion of the complex-bound C3b to CBdg, thereby rendetfng the IC incapable of engaging neutmphil CR3. [I] Beynon H. L. et al. (1994) J. Immunoi. 153, 3160.

10.3.03.6

1 PoHyR&rphlsmof the factor H related proteins

B.C. Died, A. Cannich, E.H. Weiss. lnstifut firrAnthmpo/ogie und Humangenetik, Ludwig-Maximiiians-Universitit MUnchen, Germany Introduction: Human complement factor H regulating the alternative compiement activation pathway, is a serum protein composed of 20 repetitive domains (SCR) that are encoded by individual exons. The gene gave rise to an expanding family of related genes which are composed of a subset of newly recombined factor H homologous exons, in part linked to novel SCR sequences. Recent evidence suggests that some of the encoded serum glycoproteins (factor H related proteins, FHRPs) might be involved in the regulation of LPS binding. Materials and Methods: By RT-PCR from liver RNA we detenined the sequences of FHRPl, 2 and 3. The genes were isolated and characterized by YAC and Lambda clones. Transient and stable transfection systems were used to produce recombinant proteins for serological and functlonal analyses. Results: The genes encoding FHRPl to 3 are closed linked to the factor H gene. Extensive polymorphism was observed for FHRPl and novel alleles were defined for FHRPP and FHRP3. Moreover, the frequency of a FHRPP null allele was determined. in addition, a differential mRNA expression pattern of the factor H gene family was observed. Functional characterization of the FHRPl variants is in progress. Conclusion: The amplification and diversity of the factor H related proteins point to a fine tuning of their function at the gene and possibly at the allele level. Recombinant production of the different variants will allow to analyze their function.

Complement activation, function and receptors

10.3.03.7

19

1 Localisation of the collectin blndlng site within Clq-receptor (collectin receptor)

G.R. Stuart’, N. Lynch 2, W. Schwaeble2, R.B. Sim’. ’ MRC immunochemistry Unit, Dept. of Eiochemistq University of Oxford, Oxfoti UK, *Dept. of Microbiology and Immunology University of Leicester, Lsicesfec UK Introduction: The Cl q receptor (Cl qWcollectin receptor) is located on the surface of leukocytes, endothelial cells, fibroblasts, platelets and specialised epltheiia. Binding of Clq to ClqR elicits a range of immunological responses, such as phagocytosis, enhanced cytokine and oxygen radical production, and antibody-dependent ceil cytotoxicity. ClqR also interacts with the collectins SP-A, MBL, CL43and conglutinin (Maihotra eta/(lQQO) J. Exp. Med. 172,955), resulting in enhanced phagocytosis of coiiectin-bearing particles via ClqR-bearing ceils (Gee&ma eta/(1994) Am. J. Physioi. 267, L578). The interaction between ClqR and Clq or the collectins involves a cluster of charged residues on the collagen tails of the iigands and is ionic-strength dependent. The region within ClqR that binds to these iigands has yet to be elucidated. ClqR has an almost complete amino acid sequence identity with calreticulin (CaR) (Malhotra et a/ (1993) immunology 78, 301). an abundant multifunctional protein with potential roles as an autoantigen and chaperone. Materials & Methods: in order to iocalise functional activity within Cl qR/CaR, we produced four recombinant CaR domains [N (residues 16-l 96), P (197-306), C (309-417), and S (160-283), which spans the intersection between the N and Pdomains] using the pTrxfus expression system. Purified domains were used to study the interaction with Clq and the collectins, using standard micmtitre plate binding assays and haemolytic assays. Reeults: Both the N and P-domains were implicated in Clq binding. 125i-Sdomain demonstrated concentration-dependent binding to immobiiised Clq. This interaction is ionic strength and pH-dependent. consistent with previous observations with native ClqR. Competitive inhibition studies of the S-domain-Clq interaction revealed that the Sdomain binds to Ciq tails and to the coiiectin proteins, SP-A, MBL, CL43 and conglutinin. Conclusion: We have demonstrated that the Clq and collectin binding site on ClqR is localised within the S-domain, a region spanning the N and P-domains of ClqWCaR. This region is being further chatacterfsed.

0.3.03.8

Characterixation of the C5a anphylatoxin effector domain and selection of a C5a-R antaaonist udns C5a phage libraries

M. Hennecke. A. Kda, M. Baensch, A. Klos, W. Bautsch, J. Kbhi. lnstitote of Medical Microbiolog)! Medical School. Hannovec Germany Introduction: C5a is a potent pminflammatory mediator generated upon activation of the complement system. The functional activities am mediated through specific interaction with the C5a receptor (C5aR). Peptide studies demonstrated that the last eight C-terminal amino acids H K D M Q L G R of hC5a represent the effector domain of the molecule. Site directed mutagenesis studies indicated C-terminal positions Lys68, Leu72,Arg74 and possibly Hiss7 as receptor binding residues. Recently we described a novel phage display based selection system to study CSa-CSa-receptorinteraction (Hennecke et al., Gene, 1996,164: 263). We have used this system to investigate simultaneous substitutions within the C5a effector domain. Material and Methods: Two different libraries were constructed by PCR on pJuFoC5aA’a27vector. in iibraty I positions 67, 68, 72, and 74 were randomly mutated using NNK codons (IUB nomenclature). in library II we performed random mutagenesis of positions 69-73. Selection of C5a receptor binding clones was performed on Bt2cAMP differentiated UQ37 ceils. Three (iibraly II) or four (library I) cycles of affinity enrichment were performed. After the last panning 55 (library I) or 36 (library II) colonies were randomly picked and the phagemid DNAs were sequenced. C5a mutants were purified by affinity chromatography using C5a specific mAb 561. Binding of C5a mutants was determined in competitive binding studies with differentiated UQ37 cells. Functional activity was assessed by determination of N-acetyi-fi-D-glucosaminidase release from the differentiated UQ37 cell line. Results: In more than 80% (position 72) or 90% of ail cases (positions 68 and 72) the original residues of hC5a were selected from library I. At position 67 in more than 90% of all clones a Trp residue was selected, a residue formerly shown in peptide studies to enhance biologic potency. None of the clones displayed the original His residue at position 67. In library II leucine was preferentially selected at positions 70-72 while at position 89 thirteen of the twenty possible amino acids were found. This result indicates that not only position 72 but also positions 70 and 71 interact with the C5a receptor, whereas position 69 does not. The most frequent amino acid which we found at position 73 was arginine followed by tyrosine. Those mutants with (i) Leu in positions 70 and 71 (ii) Ser or Leu in position 70 and (Iii) Arg or Tyr in position 73 showed a much higher binding affinity as compared to hC5a-(1-73) (about 10-20 fold). in fact the binding affinity was only two to threefold lower as for hC5a. One mutant was selected C5aA’827-(1a6)-FKRSLLR. with only a fourfold reduced binding affinity as compared to hC5a. Binding of this mutant to the C5a-R did not

20

Vaccine strategies

result in receptor mediated hydrolysis of guanosine 5’-triphopshate in membrane preparations of differentiated U937 cells and, in consequence, did not induce any measurable enzyme release up to a concentration of 1 PM. On the other hand incubation of U937 cells with this mutant resulted in desensitization of the cells for a subsequent C5a stimulus. Conclusion: The selection of a C5a phage libraries on cells confirmed postulated and revealed new binding residues of C5a demonstrating the power of the method. In addiion a receptor antagonist was found on the basis of the natural ligand. This molecule might prove useful to (i) investigate the mechanism of desensitization and (ii) to inhibit functional activities in vitro and in vivo.

10:00-l

2:oo

0.4.01 0.4.01 .l

Vaccine strategies lnductlon of FlV-speciflc CTL responses and protective Immunity by Immunlsation with repllcatlon defective FIV provlral DNA

J.N. Flynn, M.J. Hosie, C.A. Cannon, MA. Rigby, A.H. Mackay, D.A. Argyle, T. Miyazawa, 0. Jarrett, D.E. Onions, J.C. Neil. MRC Retrovirus Laboratory, Department of Veterinary Pathology; llnksity of Glasgow Bearsden, Glasgow, UK The potential of DNA immunisation to induce virus-specific humoral and cellmediated immune responses, and the effect of co-immunisation with feline interferon (IFN) y to act as an adjuvant was evaluated. Cats were immunised intramuscularly at weeks 0, IO and 23 with defective provtral DNAfrom the F14 clone of FIV containing a 33 codon in-frame deletion centered on the unique Pat 1 restriction site in the RT (FIVART). FIV-specific CTL responses were detected using autologous or allogeneic skin fibroblast target cells labelled with 51Cr and infected with recombinant vaccinia viruses expressing either FIV Gag or Env. Following vaccination, both FIV Gag- and Env-specific effector CTL were elicited in the peripheral blood of all vaccinated cats, and could be detected without any requirement for prior in vitro restimulation. This response peaked at 3 weeks post vaccination, declined by 6 weeks post vaccination, and was undetectable by 9 weeks. Boosting at week IO had negligible effect on the levels of detectable CTL in the peripheral Mood. Co-immunisation with another vector expressing feline IFN y resulted in higher levels of virus-specific lymphocytototicity. However, co-administration of IFN y also induced high levels of non-specific cytotoxidty and non-MHC-restricted CTL activity. No FIV-specific humoral immune responses were detected in the vaccinated cats at any timepoint examined. The cats were challenged intraperitoneally with 25 CID% of the homologous F14 clone of FIVPFT -. at week 26 and the oertpheral blood examined at 3 week intetvals for the presence of virus. One oit of&e cats immunised with FIVART, and three out five cats immunised with a combination of FIVART and IFN y were protected from challenge. FIV was detected in the peripheral blood of all control cats following challenge. Thus. immunisation with FIVART DNA induces FIV-soecific cell-mediated. but not humoral, immune responses in viw and confers pr&ctive immunity to a proportion of vaccinated cats. Co-administration of feline IFN y DNA enhances this immune response and the levels of protection observed, indicating the potential of this cytokine to act as an adjuvant.

Monday, 23 June 1997 - Oral presentations

tokines. Serum antibodies were assayed by haemagglutination-inhibition (HI), neuraminidase neutralization and ELlSA; DTH was determined by footpad thickness; and cytotoxidty was measured by a 51Cr release assay. Resuftr~ (a) The efficiency of encapsulation was 95%, QO%and 50% for the antigen, IL-2 and GM-CSF respectively. (b) Upon storage at 4”c, the liposome canter was fully stable at 1 year, while the entrapped agents retained 75-95% of their initial activity at 3-6 months. (c)The antibody response of mice immunized with Lip-As was up to IO times stronger and its duration was 3-5 times longer than that of mice immunized with AI-AS/F-Ag. (d) The antibody level in mice injected with any of the 3 antigen preparations combined with free cytokine was 2-20 times greater than that in mice vaccinated with antigen atone, and the two cytokines had an additive effect. (e) Liposomal cytokines were 2-20 times more effective than soluble cytokines and their stimulatory effect was more durable. (f) Co-injection of Lip-Ag and liposomal cytokines, but not the free antigen, elicited a high titer of IgGl, lgG2a, lgG3 and IgM antibodies, as well as strong DTH response and CTL activity, indicating Thl and Th2 responses. (g) In mice infected intranasallv with the virus Q-14 months post immunization, the level of protection following vaccination with Lip-Ag + r$tokines was 7O-100% versus O-25% in mice immunized with AI-As alone. Conclusion: Cur novel influenzavaccine is cleatly superior to the currently used influenza vaccines. The data suggest that liposomal entrapment of antigen and either GM-CSF or IL-2 appreciably improves the efficiency of subunit vaccines.

10.4.01.3

[ Vaccine strategies for the delivery of multlple CD8+ cytotoxic T cells epltopes

A. Suhrbier, S. Thomson, S. Elliott, M. Shenitt, S. Pye. CooperaW Research Cenbe Ibr Vaccine Technology,Queensland Institute of Medical Research, PO Box RBH, Brisbane, Old, Australia Introduction: Development of an effective epitope-based CD8 @ cytotoxic T lymphocyte (CTL) vaccine requires a strategy capable of co-delivering large numbers of epitges. Recently we reported-ihat each epitope within an a& ficial polyepitope (poiytope) protein containing nine minimal CD8+ CTL epitopes in sequence, was processed and presented to appropriate CTL dories. Natural flanking sequences were thus not required to direct processing and flanking sequences comprising of other CTL epitopes did not interfere with processing’. Materlal8 and Methods: To illustrate that a polytope could also prime for multiple CTL responses, mice were immunised with a recombinant polytope vaccinia coding for ten contigous murtne CTL epitopes, which were restricted by five MHC alleles and derived from nine antigens. In a separate series of experiments mice were immunised with a DNA vaccination plasmid coding for the same polytope. To illustrate that the CTL raised by these polytope vaccines mice were functional in vivo, mice were challenged in virus and tumour challenge models. Reaufts: The polytope vaccinia and the polytope DNA plasmid were both capable of inducing primary CTL responses against each epitope, and of protecting against two viral and one tumour challenge systems2r3. Conclusions: The ability of every CTL epitope in a protein containing only CTL epitopes to be immunogenic is likely to find application in the construction of CTL based vaccines in several diseases, particularly melanoma, ESV and HIV where CTL epitopes are well charactertsed and CTL responses have been shown to be protective. [I] Thomson, et al., 1995. PNAS 92; 5845. [2] Thomson and Elliott et al., 1996. J. Immunol. 157: 822. [3] Thomson and Sherritl et al., 1996. (submitted)

( 0.4.01.2 ] A novel influenza vaccine composed of encapsulated H/N antigens and cytoklnes elicits strong humoral and cellular responses In mice

1O-4.01.4 ] In vivo englneerlng of a cellular immune response by co-admlnlstratlon of IL-12 expresslon vector wlth a DNA lmmunogen

I. Babai ‘, S. Samira I, Y. Barenholz *, 2. Zakay-Rones3, E. Kedar‘. ’ Dept.of lmmunolo~ Hebrew University-Hadassah Medical School, Jerusalem, Israel, *Dept. of Biochemistry. Hebrew University-Hadassah Medical School, Jerusalem, Isreel, 3Dept. of Vhvlog~ Hebrew UniversityHadassah Medical School, Jerusalem, Israel

Jong J. Kim 1,2,Velpandi Ayyavoo l, Mark L. Bagarazzi I, Kesan Dang I, Michael A. Chattergoon I, Bin Wang ‘, Jean D. Boyer l, David B. Weiner ‘, ’ Department of Pathology and Laboratory Medicine, University of Pennsyh9nia. Pennsyivania. USA, *Department of Chemical Engineering, Univetsity of Pennsylvania, ~nnsylvania, USA

Introduction: The aim of this study was to improve the potency of the currently used influenza vaccines. Materials and Methods: Influenza A virus haemagglutinitineuramlnidase (Shangdong, H3N2) and the cytokines rm-GM-CSF and m-IL-2 were encapsulated, each separately. in large (mean diameter, 1.5 pm) multilamellar vesicles composed of dimyristoylphosphatidylcholine (DMPC). BALB/c mice were immunized once, i.p. or s.c., with 0.5 pg H/N administered either as free antigen (F-Ag), adsorbed to alum (AI-AS), or encapsulated in liposomes (Lip-Ag), separately or together with 1 x 103-5 x 104 units of free or encapsulated cy-

Introduction: To engineer the enhancement of cellular immune response in viw and to direct antigen-dependent immune response, we investigated the role of co-delivery of genes for IL-12 and GM-CSF along with DNA vaccines for HIV-l antigen. Materialsand Methods: The genes for murtne IL-12 (bath ~35 and p40 chains) and GM-CSF were individually cloned into expression vectors under control of a CMV promoter. The gene plasmid expression cassettes were then injected into mice along with DNA vaccine cassettes for HIV-I. We then analyzed the immunological effects of the co-immunization with these genetic adjuvant