Abstract:
The invention refers to a molecular presentation system in which viral proteins are foreseen as carriers for heterologous amino acid sequences. Hereby, the viral protein is derived from small insect viruses, primarily from Flock House Virus (FHV), with a known 3-dimensional structure and amino acid sequence, whereby heterologous amino acid sequences, for example epitopes, are inserted in the outwardly directed loops of the viral capsid protein. Moreover, the expression of the FHV capsid protein in insect cells can produce mature virus like particles (VLP) through a recombinant baculovirus.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims benefit to PCT/EP95/03114 filed on Aug. 4, 1995, which claims priority to Austrian Application No. A 1545/94 filed on Aug. 8, 1994, both of which are incorporated in their entirety. 
    
    
     TECHNICAL FIELD 
     The invention concerns a molecular presentation system in which viral proteins are being used as carriers for heterologous amino acid sequences. 
     BACKGROUND OF THE INVENTION 
     The possibility to identify and synthesize amino acid sequences from viral proteins, which are able to generate a protective immune response in animals, has stimulated the development of synthetic vaccines. Although it has already been shown that synthetic peptides in some cases can induce a good immune response, it has turned out that in general they were weak immunogens unless coupled to strongly immunogenic carrier molecules. They were frequently unable to induce protective immunity in vaccinated animals. Attempts to increase the immunogenicity of these antigens for use as vaccine have lead to the development of a series of antigen presentation systems. Many of these are designed to present the antigen as a polyvalent, particulate structure. The development of particulate vector systems for immunogenic epitopes provides a powerful approach for the presentation of antigens. Various systems were used to present foreign epitopes: the core antigen of Hepatitis B virus (HBV) (HBcAg)  1! and the surface antigen of Hepatitis B virus (HBsAg)  2!, the capside protein from Polio virus  3!, the yeast Ty protein  4!, the particles obtained after insertion of HIV 1-gag in Baculovirus  5!, rotavirus VP-6 protein  6!, core particles of the Bluetongue virus (BTV)  7!, and filamentous as well as icosahedral bacteriophages  8,9!. 
     It has been demonstrated that the immunogenicity of a peptide depends on its sequence as well as on the way it is presented to the immune system. By using a human rhino virus capsid sequence as a heterologous peptide and the particles of HBcAg as a carrier, it was shown that the internal location of the foreign sequence increases the immunogenicity of the epitope by 10 to 50 fold when compared to the amino terminus location  10!. Also the antigenicity (measured as reactivity to a monoclonal antibody (mAb)) was greatly enhanced by placing the foreign peptide in that position in the carrier. Furthermore, both constructs presented the epitopes considerably more efficiently to the mAbs than the free peptides. This was also the case when specific HIV-1 epitopes (the V3 loop) were introduced into different domains of the HBcAg  11!. Since the properties of a given epitope can be influenced by its conformation it was of great interest to have a carrier system with multiple entry sites conferring many possible conformations. This would increase the possibility of finding a conformation closer to the native one for a given sequence. In spite of the fact that, as mentioned above, various particulate systems have been developed for the presentation of epitopes, they were all based on the foreign epitope being inserted mainly in one position. This was partly due to lack of knowledge about the 3-D structure of the carrier particle. 
     SUMMARY OF THE INVENTION 
     With reference to the above, a new presentation system has been developed, characterized by the fact that the carrier protein is derived from small insect viruses, Flock House virus (FHV), with a known 3-D structure and amino acid sequence. Heterologous amino acid sequences, for example epitopes, are inserted into the outwards directed loops of the viral capsid protein. This carrier presents multiple possibilities for a conformationally suitable location of epitopes. Above all, the carrier system is characterized by the fact that the recombinant protein, or the virus like particles, are obtained from procaryotic or eucaryotic cells through the expression of the protein encoded by the appropriately modified RNA-2 gene of the FHV capsid protein. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 illustrates a crystallographic representation of the outward directed loops of the FHV capsid protein precursor with 3 Angstrom resolution showing the positions of insertions of the foreign genes (L1, L2, L3, I1, I2 and I3). 
     FIGS. 2A-2C depict the DNA and amino acid sequences of FHV RNA-2 with the individual loops (L1, L2, L3, I1, I2 and I3) shown in bold type. 
     FIGS. 3A-3F depict the full restriction map of the DNA sequence of FHV RNA-2. 
     FIGS. 4A-4B depict the number of restriction endonuclease usage sites in the FHV RNA-2. 
     FIGS. 5A-5D depict all of the restriction endonuclease sites in the FHV RNA-2. 
     FIG. 6 is a graphic representation of the unique endonuclease sites in the FHV RNA-2. 
     FIG. 7 depicts a stereo diagram of the FHV RNA-2 protein showing the positions in which HIV-1 specific sequences are inserted. 
     FIG. 8 depicts a representation of FHV capsomer virus-like particles produced in Baculovirus. 
     FIGS. 9A and 9B depict expression of the wild-type and the hybrid FHV capsomer. FIG. 9A shows SF-21 infected cell lysates analyzed in 10% SDS-PAGE and stained with Coomassie blue. FIG. 9B shows SF-21 infected cell lysates analyzed by Western blot with (1) an antiserum against FHV; (2) a human HIV-1 monoclonal antibody; (3 and 4) a HIV-1 positive sera and (5) a HIV-1 negative serum. 
     FIGS. 10A-10C depict the sedimentation profile and antigen composition of VLPs produced by three different baculovirus infected SF-21 cells. FIG. 10A depicts AcNPV-FHV which expresses the unmodified FHV capsid protein. FIG. 10B depicts AcNPV-FHV-V3/L1 expressing the same protein carrying the HIV-1 epitope in position L1. FIG. 10C depicts AcNPV-FHV-V3/L2 expressing the same protein carrying the HIV-1 epitope in position L2. 
     FIG. 11 depicts serum anti-V3 antibody titers measured against recombinant gp120. 
     FIG. 12 depicts the expression of hybrid FHV-HCVc proteins through recombinant Baculovirus AcNPV-HCVc. 
     FIG. 13 depicts the profile and FHV reactivity (ELISA test) after a sucrose gradient of VLPs formed by SF-21 infected cells with the recombinant Baculovirus AcNPV-HCV. 
     FIG. 14 depicts the distribution of the ELISA titer obtained by VLP based assay among 100 selected HCV core positive sera. 
     FIGS. 15A-15D depict the detection of HCV specific antibodies in a VLP based ELISA test and its correlation with alanine aminotransferase (ALT) levels and previous currently available test kits (RIBA II) values. FIGS. 15A-15D show the results of 4 out of 50 patients which were analyzed. The RIB II values are shown in the upper panel of each of the figures. The VLP-based ELISA test values are represented in the diagrams as circles. The ALT values are represented in the diagrams as a vertical line. 
     FIG. 16 depicts the detection of HCV core-antibodies in human sera by dot-blots using recombinant (VLP-HCV) antigen or free peptides. Lanes 1-6 show patients&#39; sera and lane NC shows the negative control serum. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Characteristics of the Carrier Particle 
     Flock House Virus (FHV) 
     FHV is a non-enveloped icosahedrical insect virus with a bipartite RNA genome and belonging to the Nodaviridae family. These viruses are among the smallest and simplest known. The FHV genome consists of two single stranded mRNA molecules (RNA-1 with 3.1 kb and RNA-2 with 1.4 kb), both encapsidated in the same particle. RNA-1 carries the information for the viral RNA-polymerase and RNA-2 codes for the coat precursor, alpha protein. Upon synthesis the coat precursor alpha is rapidly assembled with both RNAs, whereby immature, virion-like particles (provirions) are formed. These are slowly processed to mature particles by autocatalytic cleavages  12!. 
     X-ray diffraction studies have shown the structure of the viral particles at 3.0 Angstrom resolution  13! (see FIG. 1). The virion has 60 icosahedrical, asymmetric units each consisting of three quasiequivalent protomers forming a protein shell around the inner RNA genome  14!. The protomers consist of 1) a basic, crystallographically disordered aminoterminus, 2) a Beta-barrel structure, 3) an outer protrusion composed predominantly of Beta sheets and formed by three large insertions between the strands of the Beta-barrel, and 4) a carboxyterminal domain composed of two distorted helices lying inside the shell. The external zone of the virion, which is the least conserved, has many sequence differences which essentially contain all the deletions and insertions of the different strains  12!. The variations in the loops, directed outwards from the segments of the Beta-barrel structure, define serologically distinct viral strains. 
     These loops were selected as the regions to be manipulated for the insertion of the foreign epitopes. The positions for these insertions (L1, L2, L3, I1, I2, I3,) are given by the following amino acid regions of the RNA 2 gene: 
     Loop L1 amino acids 195-219 
     Loop L2 amino acids 263-277 
     Loop L3 amino acids 129-138 
     Loop I1 amino acids 107-110 
     Loop I2 amino acids 152-165 
     Loop I3 amino acids 304-310 
     FIGS. 2A-2C show the DNA sequence of FHV RNA-2 and the corresponding amino acid sequence (SEQ ID NOS:2 and 3), the individual loop regions are bolded. 
     FIGS. 3A-3E show the full restriction map of the DNA sequence of FHV RNA-2 (SEQ ID NO:1). 
     FIGS. 4A-4B represent the number of cutting sites of the endonucleases. 
     FIGS. 5A-5D show all sites in which the endonucleases cut FHV RNA-2. 
     FIG. 6 is a graphic representation of the unique cutting sites of the endonucleases. 
     FHV grows vigorously in cultured cells and produces yields of 20% of the total cell protein  14!. In addition, FHV grows well in several Lepitopteran larvae. The viruses of this family show a considerable resistance to inactivation by heat, detergents and other denaturants  14!. It was shown recently  15! that the expression of the capside protein FHV RNA-2 in insect cells via a recombinant baculovirus produces virion like particles (VLP) similar to authentic virions. It was shown by the present inventors that this can also be achieved by the expression of a modified gene carrying insertions for the expression of foreign amino acids within the capsomer structure. The VLPs generated by this procedure in insect cells are mature particles since the precursor protein, which is present in provirions, is cleaved. This system allows the production of 1-2 mg of purified synthetic virions (VLPs) in 50 ml of cultured cells  16!. 
     Another method for the production of particles carrying foreign epitopes is by recovery of infectious virions after cotransfection of the genomic RNA-2 (obtained by in vitro transcription of modified cDNAs) with purified RNA-1  17!. This is only valid for genomes which carry alterations that do not change the replicative cycle or the assembly of the virus. The RNA-1 can be purified by several cycles of autonomous replication in DM-1 cells (Drosophila Melanogaster) taking advantage of the fact that RNA-1 behaves as an autonomous replicon in transfected cells  18!. 
     Detailed Description of the Construction of Recombinant Baculovirus Carrying the Wild Type or Modified Capsomer Gene 
     FHV was grown in DM-1 cells and purified in sucrose and CsCl gradients as described in  19!. The genomic RNA was extracted from the purified virions by treatment with proteinase K and by phenol-chloroform extraction. A single stranded cDNA was made with reverse transcriptase using 20 bases long oligonucleotides complementary to the 3&#39; end sequence  12! (see FIG. 2). A double stranded cDNA was made with standard PCR techniques  20! amplifying the single stranded cDNA using a 20 bases long oligonucleotide, complementary to the 5&#39;-end of the RNA-2, together with the first primer. Both primers carried extra bases coding for selected restriction enzyme sites (Bam-HI site for the 5&#39;-end and Xba-I site for the 3&#39;-end). After the PCR amplification, the double stranded DNA was gel-purified and ligated to pUC18 (Sma I site). For the in vitro transcription of RNA-2 the corresponding cDNA was inserted into the plasmid pBluescript SKII (Stratagene) under the control of phage T7 polymerase. Examples of the above mentioned modifications of FHV RNA-2 are shown in Table 1. 
     
                       TABLE 1______________________________________Posi-Restrictiontion Sites     Comments______________________________________L1   Kpn I     a) Aminoacids 205 to 209 (ATDPA) deleted from(after    the original sequence.mutagenesis)          b) Val 204 mutagenized to Gly (GTT to GGT) to          create kpn 1 site:    G.sub.204         T.sub.205    GGT  ACC    CCA  TGG          c) After oligo insertion, GT duplicatesL2   Pst I     a) Aminoacids 270 to 273 (GSTG) deleted from(after    the original sequence.mutagenesis)          b) Mutagenesis of the codon usage in L.sub.269 (CAG to          CTG) and Q.sub.274 (CAA to CAG) to generate Pst I site:    L.sub.169         Q.sub.274    CTG  CAG    GAC  GTC          c) After oligo insertion, LQ duplicates.L3   Nhe I     a) Aminoacids 128 to 134 (VPAGTFP) deletedSpe I     after doble digestion with Nhe I-Spe I:(original)       A.sub.126                      S.sub.127 T.sub.135                                     S 136          NheI   GCT  AGC  Spe I                                ACT  AGT                 CGA  TCG       TGA  TCA          b) S.sub.127 and T.sub.135 are regenerated after oligo          insertion. There are no duplications of aa.I3   Bsu36 I   a) There is no loss of aa in the original sequence.(original)          b) Oligo insertion duplicates aa P.sub.304 and E.sub.305    P.sub.304         E.sub.305                G    CCT  GAG    G    GGA  CTC    CI2   BamH I    a) Aminoacids 154 and 155 (TT) are deleted(after    after mutagenesis.mutagenesis)          b) Change of codon usage in S.sub.156 (TCA to TCC)          to generate BamH I site:    G.sub.153         S.sub.156    GGA  TCC    CCT  AGG          c) GS duplicates after oligo insertion.I1   Bsu36 I   a) Mutagenesis of G.sub.108 and Q.sub.109 to generate(after    Bsu36 I site:mutagenesis)    G.sub.108                     Q.sub.109                GGA  CAG          to:   P.sub.108                     E.sub.109                CCT  GAG  G (of D.sub.110)______________________________________ 
    
     The cDNA of RNA-2 was also inserted into the vector pVL-1393 (Invitrogene) (Bam-HI/Xba-I sites). In this vector the gene is placed under the control of the polyhedrin promotor and flanked by sequences of the Autographa californica Nuclear Polyhedrosis Virus (AcNPV) which allow in vivo production of recombinant virus after cotransfection with AcNPV genomic DNA. 
     Introduction of Foreign Sequences in the cDNA of RNA-2 
     Small insertions/deletions in the sequences of RNA-2 were carried out by the PCR technique  20!. The epitope specific sequences were inserted into one or more of the selected sites either using restriction enzyme sites (when available) or by the PCR technique. The stereo diagram of the FHV capsid protein precursor in FIG. 7 shows the sites where the specific HIV-1 sequences &#34;IGPGRAF&#34; (SEQ ID NO:10) were inserted. Those amino acids were inserted into the positions L1, L2, L3, and I2, whereas the aminoacids &#34;IGPGRAFE&#34; (SEQ ID NO:19) were inserted into position I3. In all positions, except in position I3, certain amino acids were deleted: In position L1 amino acids 205-209 were deleted and aa 204 was mutated to create Kpn I site; in position L2 amino acids 270-273 were deleted and aa 269 and 274 were mutated to create Pst I site; in position L3 amino acids 128-134 were deleted after digestion with Nhe I-Spe I. In position I2 amino acids 154-155 were deleted. See Table 1. 
     Examples of the insertion of foreign sequences into the recombinant FHV capsomer are listed in Table 2. 
     
                                           TABLE 2__________________________________________________________________________Examples of foreign sequences included in FHV recombinant capsomer      Amino acids sequenceSequences Sites      and their characteristics                              Expressed in__________________________________________________________________________HBV-PreS1 I3   (SEQ ID NO:4) MGTNLSVPNPPAFGANST-                              E. coliNPDWDFNPGGMQWNSTAL      Tcell epitope.      Receptor binding site.HBV-PreS2 I3   (SEQ ID NO:5) MQWNSTALDPRVRGL                              E. coli      B cell epitopeHBV-S L1,L2,L3      (SEQ ID NO:6) CTTPAQGNSMFPSCCCTKPTDGNC                              E. coli I2,I3      B cell epitope          BaculovirusHCV-core L1,L2,L3      1. (SEQ ID NO:7) TNPKPQRKTKRNTNRRPQD                              E. coli I2,I3      2. (SEQ ID NO:8) VKFPGGGQIVGGVYLLPRR                              Baculovirus      B cell epitopes.HIV-1 gp120 L1,L2,L3      (SEQ ID NO:9) IQRGPGRAF                   (IIIB) I2,I3      (SEQ ID NO:10) IGPGRAF                   (MN)       E. coli      (SEQ ID NO:11) FGPGQAL                   (Mal)      Baculovirus      (SEQ ID NO:12) IGPGRTL                   (NY5)      (SEQ ID NO:13) KGPGRVI                   (RF)      (SEQ ID NO:14) IGLGQAL                   (Z2)      V3 loop.B cell epitope.      Neutralyzing epitopeHIV-1 gp120 L1,L2,L3      1. (SEQ ID NO:15) GKAMYAPPI I2,I3      2. (SEQ ID NO:16) NMWQE(K)VGKA                              E. coli      (C4).B cell epitope.    Bacufovirus      Neutralyzing epitopeHIV-1 gp41 L1,L2,L3      (SEQ ID NO:17) ELDKWAS  E. coli I2,I3      B cell epitope          Baculovirus      Neutralyzing epitopeHIV-1 gp41 L1,L2 L3      (SEQ ID NO:18) IEEEGGERDRDR                              E. coli I2,I3      B cell epitope          Baculovirus      Neutralyzing epitope__________________________________________________________________________ 
    
     Production of Recombinant Baculovirus Carrying the RNA-2 Gene 
     The cDNA of RNA-2 (wild-type or after genetic manipulation) was inserted into the transfer vector pVL-1393 under a polyhedrin promotor (sites Bam HI and XbaI of the polylinker). This pUC9 based vector carries a segment of AcNPV in the sequence flanking its polylinker and allows the transfer of the foreign gene to a baculovirus genome after in vivo recombination (see FIG. 8). Insect cells (Spodoptera Frugiperda SF-21 cells) were co-transfected (LIPOFECTIN-cationic liposomes) with linear genomic DNA (non-viable) of AcNPV (BACULOGOLD--Autographa Californica Nuclear Polyhedrosis Virus from PharMingen) and with the transfer vector carrying the FHV gene. After 4 days the virus progeny was harvested and titered. Thereafter, several recombinant viruses were plaque purified (3 to 4 times from well isolated plaques). These recombinants were denominated AcNPV-FHV. In some cases the VLPs can tolerate the insertion of up to 20 amino acids without alteration of the assembly process. In other cases, where the insertions prevented the formation of VLPs, this could be circumvented by coinfection with both the wild-type and the modified baculovirus. Thereby, mosaic VLPs were generated carrying both types of capsomer structures. 
     Production and Purification of VLPs From Insect Cells Infected With AcNPV-FHV 
     In order to obtain purified antigens for immunological antigen studies, Sf-21 cells (in suspension or as monolayer) were infected with recombinant baculoviruses at a multiplicity of infection of 10. Two to three days after the infection 0.5% nonidet P-40 and 0.1% Beta Mercaptoethanol (2-ME) were added to the medium. After 15 minutes on ice, the cell debris were removed by centrifugation for 10 minutes at 12000 g. The VLPs in the supernatant were pelleted through a 30 wt/wt % sucrose cushion (50 mM HEPES, 0.1% 2-ME) at 40.000 rpm in an SW41 rotor for 3 hours at 4 C. The pellet was resuspended in 50 mM HEPES, 0.1% 2-ME and laid on a 10 ml 5-20 wt/wt % continuous sucrose gradient in the same buffer. The particles were sedimented in an SW41 rotor at 40.000 rpm for 1 hour at 11 C. The fractions of the gradient were collected from the bottom and aliquots of each fraction were run on a 10% SDS poyacrylamid gel in order to localize the particle peak. The fractions containing the VLPs were pooled, pelleted by centrifugation and resuspended in the same buffer. The protein content of these preparations was determined by Micro BCA Protein Assay Reagent (Pierce). 
     Recovery of Modified FHV Containing Exogenous Sequences 
     Live, recombinant FHV viruses can be recovered when the heterologous amino acid sequences which are inserted into the capsomer do not alter the virion assembly process. The recovery was carried out by co-transfection of DM-1 cells with in vitro made transcripts of modified RNA-2 and authentic RNA-1 purified of RNA-2 by multiple transfection passages at the limiting dilution as described by Ball  18!. 
     EXAMPLE 1 
     Production of VLPs Carrying the HIV-1 (Human Immunodeficiency Virus, Type 1) Specific Sequence IGPGRAF (SEQ ID NO:10) 
     Several domains of the HIV-1 gp-120 can induce the production of neutralizing antibodies. One of them is the hypervariable region 3 (V-3 loop). This is a linear, immunodominant epitope known as the &#34;Principal Neutralizing Determinant&#34; (PND)  21, 22!. Although the entire domain varies greatly among different isolates, it was recently found that, may be due to conformation restraints, the amino acid sequence on the tip of the loop is well conserved. Sequence data from 245 isolates from the USA showed that the V3 loop sequence &#34;GPGRAF&#34; (SEQ ID NO:20) was present in more than 60% of the isolates  23!. In addition, it was found that animals immunized with peptides containing this sequence produced sera which could neutralize several diverging isolates, although with a low titer  24!. This sequence was inserted into five different positions on the surface of the FHV structure and in some cases in two sites of the same molecule. The positions selected were the outwards directed loops mentioned above (see FHV structure FIG. 1). In one case (position I3) the foreign sequence was introduced directly as an insert in the original sequence of RNA-2. In order to obtain this, the cDNA coding for the FHV capsid protein was digested with Bsu 36I (cuts the DNA at nucleotide 934), and a synthetic oligonucleotide was inserted coding for the HIV-1 specific sequence. As a consequence of this procedure an additional glutamic acid was inserted at the carboxy terminus of the HIV-1 sequence. The structures of all these recombinant proteins are shown in FIG. 7. 
     FIGS. 9A and 9B show the proteins induced in Sf-21 cells after infection with the recombinant baculovirus carrying the HIV-1 epitopes in the positions shown in FIG. 7. Cells infected with these recombinants, mock infected cells and cells infected with baculoviruses without inserts were lysed and analyzed in a 10% polyacrylamide gel. Coomassie staining of the gel showed, in the lysates from cells infected with recombinant viruses, the presence of bands with a molecular weight similar to the expected molecular weight for FHV capsomer precursor protein (alpha protein) or its cleavage product. These bands were not present in the case of lysates from cells infected with baculovirus without the insert (AcNPV-RP6). Western blots from similar gels, analyzed with rabbit hyperimmune anti-FHV serum, confirmed the identity of the chimeric proteins. In addition to the alpha precursor, its cleavage product (the mature beta protein) was seen in all cases. This probably indicates that the modified capsomers are still capable of assembling and autocleaving. However, in some cases the percentage of mature protein seemed to be low (e.g. L3; I3; I2), probably indicating that the presence of the insert affects the autocleavage process. When a similar blot was analyzed, either using sera from HIV-1 positive patients or HIV-1 specific human monoclonal antibodies, a quite different pattern of recognition developed. The patients&#39; sera mainly recognized the epitope in the L2 position or in those combinations where this position was used. On the contrary, the monoclonal antibodies strongly recognized the position L1 or combinations derived from that position. Position L3 was also extensively recognized by patients&#39; sera though consistently less than L2. The other positions were barely detectable by these sera. On the other hand, certain human sera detected preferentially proteins carrying the inserts in the positions L3 or I3. This suggests a difference in the specificity of the individual immune response to the same sequence. However, until now the strongest signals were always obtained when the proteins carried the inserts in the positions L1 or L2. Coomassie staining of the gels showed that the differences do not depend upon the amount of induced protein in the insect cells. This confirmed the hypothesis that the antigenicity of the epitope is influenced by its localization. Until now the reason for the differences in the patterns of recognition in different patients could not be explained. Further investigations are now to be carried out to explain these data. For example concerning the origin of the infecting strain, the neutralization titer of the sera, the differences in the idiotypic answers, the difference in the patients&#39; prognosis, etc. 
     Purification of VLP-V3 From Recombinant, Baculovirus Infected Sf-21 Cells 
     FIGS. 10A-10C show the sedimentation profile and the antigen composition of VLPs produced by three different baculovirus: AcNPV-FHV which expresses the unmodified FHV capsid protein; AcNPV-FHV-V3/L1 expressing the same protein yet carrying the HIV-1 epitope in position L1; and AcNPV-FHV-V3/L2 carrying the insert in position L2. See FIG. 7 for details on insert locations. In all cases it was found that the particulate material, obtained as described above, migrated to the same position in the gradient as the FHV particles. The particulate nature of these components was further confirmed by electron microscopy. Aliquots from each peak were run on a polyacrylamide gel and probed with HIV-1 positive serum after transfer to nitrocellulose paper. In all cases the detected proteins migrated to the same position as did the FHV capside protein or its precursor. In the case of wild-type or L1-derived particles the main band corresponded to the mature protein, whereas in L2-derived particles a large quantity of immature protein (alpha protein) was present in addition. However, there seemed to be an increase of mature protein in VLPs when these were compared with the input material prior to the purification. Similar results were also obtained after analysis of the products of those recombinant baculoviruses expressing the FHV capside proteins with the inserts in the other positions described above. The only differences found were the yield of particulate material and the percentage of VLPs carrying immature protein in that material. 
     The experiments were carried out as follows. Four days after the infection the cells and the medium were processed as previously described and the presence of the particles was analyzed by sucrose density gradient sedimentation (SW50.1 rotor at 45000 rpm for 30 minutes at 20 degrees C.). The fractions were collected from the bottom of the tubes. In order to detect the distribution of the FHV along the gradient, aliquots of each fraction were tested for FHV reactivity by means of an ELISA assay. The distribution of HIV-1 specific reactivity along the gradient was measured through western blotting of samples from the peak fractions. The western blots were probed with HIV-1 positive sera and the resulting bands are shown at the bottom of each graph. FIG. 10A shows the reactivity of AcNPV-FHV-derived particles, FIG. 10B shows particles derived from AcNPV-FHV-V3-L1 and FIG. 10C shows particles derived from AcNPV-FHV-V3-L2. The arrows indicate the migration of FHV run in a parallel gradient. 
     Immunogenicity of Chimeric VLP-V3 Particles 
     In order to determine whether the seven HIV-amino acids inserted into the VLP structure were capable of inducing an immune response, three groups of guinea pigs, each consisting of three animals, were immunized with purified VLPs carrying the HIV-1 insert as described in the following. The first group of animals was inoculated with the insert in position L3, the second group with the insert in position I3 and the third group was inoculated with the insert in both positions L3 and I3. All three groups were immunized subcutaneously with 500 microliters PBS containing 50 microgrammes of the respective VLP preparations. For the first immunizations on day 0 the antigens were formulated in complate Freund&#39;s adjuvant (CPA). For the boosters on days 14 and 28 the same amount of antigen was formulated in Freund&#39;s incomplete adjuvant (IFA). Blood was taken from each animal 35 days after the first inoculation by cardiac puncture. Sera from the immunized animals were tested for specific anti-V3 and anti-FHV antibodies in an ELISA test. The data represent reciprocal dilutions at OD 492. For the anti-FHV titer the ELISA plates were coated with CsCl-purified viruses (200 nanogrammes per well). The titers against the HIV-1 inserts were analyzed on plates coated with recombinant gp120 (ABT-Baculovirus produced, 100 ng per well) as a capture antigen. The data in FIG. 11 show that these preparations had elicited a good antibody response specific for the V3 sequence. As shown by this test, no major differences existed among the various constructs. However, the rest of the positions are yet to be analyzed and an evaluation is to be made of the differences in the affinity shown to the native gp-120 by the immune sera, a parameter known to be associated with their neutralizing capacity. 
     EXAMPLE 2 
     Hepatitis C Virus (HCV) 
     The transfusion induced Hepatitis, which can neither be attributed to Hepatitis A virus nor to Hepatitis B virus (NANBH), belongs to the main group of transfusion transmitted diseases  25!. The cloning and expression of HCV has allowed the development of antibody screening immunoassays for the detection of HCV infections, using as solid phase antigen a fusion polypeptide expressed through recombinant yeast. Initial studies using this protein confirmed that HCV was the predominant agent of NANBH. However, these and subsequent studies demonstrated a series of shortcomings with this serological test due to low sensitivity and specificity. The tests currently used are mostly based on the detection of antibodies against the non-structural proteins NS3-NS4 which, however, do not appear in infected patients until the disease is in an advanced state (4-6 months after the onset of the Hepatitis). Later, it was demonstrated that most immunodominant epitopes are located within the aminoterminal parts of the core protein  25, 26, 27! and that antibodies against these epitopes appear early after the infection. This was shown either by using recombinant HCV-core protein produced in bacteria or baculovirus, or by using synthetic peptides corresponding to these sequences. Moreover, the HCV-core protein is considered as the most significant single antigen for the detection of antibodies in infected patients. Among all positive samples 80-85% are found to be core positive and in most of them this was the only antigen recognized. 
     Production of FHV-VLPs Carrying an HCV-core Epitope 
     With reference to these considerations, the epitopes of the HCV core protein were tested in the molecular presentation system of the invention here reported. A 20 amino acids long sequence was selected corresponding to the amino acids 20-40 in the original sequence about which it had already been shown that they were very effective for diagnostic purposes  26!. The 20 amino acids long epitope was inserted in the I3 position on the Bsu-36I site of the RNA-2 gene contained in pVL-1393. The recombinant Baculovirus was produced and purified as described. The resulting recombinant was denominated AcNPV-HCVc. FIG. 12 shows the expression of hybrid FHV-HCV proteins through the recombinant baculovirus AcNPV-HCVc. The chimeric capsomer was produced through the recombinant baculovirus as follows. Sf-21 cells were infected with recombinant baculovirus AcNPC-HCVc (lane 3, 6, and 7), with a baculovirus carrying an unmodified FHV capside protein AcNPV-FHV (lane 2 and 4) and with a polyhedrin-minus baculovirus carrying no insert AcNPV-RP6 (lane 1), respectively. The whole cell extracts were run in a 10% SDS-Page gel. Purified FHV was included as a marker (lane 5). Lanes 1-3 were stained with Coomassie blue. After the running, the proteins in lanes 4-7 were blotted on Nitrocellulose paper. After staining with Poinceau red, paper strips corresponding to each well were cut out and probed with a specific serum. Lanes 4 and 6 were probed with serum from a patient who was core positive in a RIBA-II test. Lanes 5 and 7 were probed with rabbit-anti-FHV serum. 
     The insect cells were infected with the recombinant virus and four days after the infection the cells were lysed and analyzed on a 10% SDS-PAGE gel. After the running, the gel was stained with Coomassie brilliant blue. The introduction of HCV-sequences apparently had no influence on the protein production. However, all the detected protein migrated with the molecular weight of the precursor (alpha protein). This indicates that the maturation process is somewhat impaired by the sequence alteration. In order to confirm the identity of this protein, the lysates of the infected cells were run on a similar gel, transferred to Nitrocellulose paper and probed with specific antisera. As expected, the protein reacted strongly with the specific rabbit-anti-FHV antiserum (dilution 1:2000) as well as with HCV positive human serum (dilution 1:200). 
     FIG. 13 shows the FHV reactivity (measured in an ELISA test) after sucrose sedimentation of VLPs produced by infection of Sf-21 cells with the recombinant baculovirus AcNPV-HCVc. The running conditions were identical to those described in connection with FIG. 10. Aliquots from each peak fraction were western blotted and probed with HCV positive human sera. A photo of the developed Western bands is inserted at the bottom of the graph. The arrow indicates the position of FHV run in a parallel gradient. 
     As in the case of the particles carrying HIV-1 specific sequences, the particles migrated somewhat slower than the wild-type FHV particles. Western blots of aliquots from the peak reacted with HCV-positive sera. This indicates that the unprocessed protein is not impaired in its ability to autoassemble into a particulate structure. 
     To asses the capability of the antigen to detect specific antibodies, purified VLPs were used for the ELISA test. Wells of ELISA plates (Nunc) were coated with 100 microliters of purified VLPs diluted in PBS buffer (100 nanogrammes per well). After blocking with PBS containing 5% BSA, 100 microliters of serum dilution were added to each well and the plate was incubated for two hours at room temperature. The bound antibodies were detected by a second incubation with a horse radish peroxidase conjugate of the IgG fraction of goat anti-human immunoglobulin for one hour at room temperature. The enzyme activity was measured using o-phenylendiamine as a substrate. The absorbance of each well was measured at A=490 nanometer. To test the sensitivity of this antigen, 100 sera, known to be core positive in a commercial test (RIBA II-Chiron Corp.), were analyzed. Almost 85% of the samples gave titres higher as 1:1000 which indicates a very good sensitivity when detecting anti-core antibodies. These results demonstrated that these 20 amino acids from the HCV core sequence represent a very reliable antigen for the detection of HCV infections, when introduced in the carrier system of the present invention. 
     The results are represented in the block diagram in FIG. 14. 
     Comparison of a VLP-based ELISA Test With Current, Commercially Available Tests 
     In order to test the sensitivity of this ELISA assay, a collection of serially drawn blood samples from infected patients, encompassing the period of seroconversion, were analyzed for specific HCV core antibodies. At that stage, all patients already have a high level of the specific liver enzyme Alanine aminotransferase (ALT). In four out of 50 patients which were analyzed (see FIGS. 15A-15D) the test subject of this invention showed seroconversion earlier than in the currently available testkits (RIBA II). These results show that the antigen is extremely suitable for detection of contaminated samples in blood banks. The serially drawn blood samples from selected patients, taken for the RIBA-II test before the seroconversion, were analyzed by using plates coated with VLPs carrying an HCV core-specific epitope. The serum dilution was 1:100 for RIBA-II as well as for the ELISA test of the invention here reported. The RIBA-II values are shown in the upper panel. The VLP-based ELISA test values are represented in the diagram as circles, the ALT values as a vertical line. 
     A Comparison Between Antibody Detection by VLPs, Carrying HCV Core Sequences, and Antibody Detection by the Free HCV Peptide 
     It has been shown that short peptides are very efficient when used as capture antigens for detection of specific antibodies in human as well as animal sera especially in the form of branched peptides  29!. It has also been reported that they react better than the corresponding recombinant antigens  30!. In transfusion induced Hepatitis-C cases it was established that by using peptides as capture antigens, positive sera could be detected as early as one month after the first transfusion. This coincides with the first increase in the specific liver enzymes and would make short amino acid sequences a useful marker for detecting acute specific HCV infections  27!. For this reason it was decided to compare in a dot-blot assay the HCV specific antigens described in the invention at issue with the corresponding free peptides (HCc-2p), with a peptide encompassing the first 20 amino acids of the core (HCc:1p), and with peptides corresponding to other HCV proteins (NS peptides), respectively. 
     FIG. 16 shows a photograph of these dot-blots which were carried out as follows. Aliquots of 10 microliters of purified VLPs (5 microgrammes per ml), carrying an insert of 20 amino acids from the core of HCV, and solutions containing peptides representing different areas of the HCV genome (100 microgrammes per ml) were blotted on nitrocellulose paper. After blocking with 5% fat free and dry milk, each strip was incubated with a 1:100 dilution of human sera. After washing, each filter was incubated with anti-human antibodies conjugated to horse radish peroxidase (Dako, dilution 1:5000), and finally incubated with diamino benzidine (DAB). Lanes 1-6 show patients&#39; sera. Lane NC shows the negative control serum. 
     As can be seen, already very low levels of antigen (50 nanogrammes corresponding to 2.5 nanogrammes of the specific HCV peptide) are strong enough, in the form of VLPs, to elicit a good signal with a positive sample. The corresponding free peptide (HCc-2p) gave only a very weak signal although it recognized the same number of positive samples. In this case, the amount of antigen loaded onto the nitrocellulose paper was 400 times higher as in the case of the VLPs, based on a molar ratio. The peptide corresponding to the first 20 amino acids (HCc-1p) gave stronger signals, but failed to detect one positive sample and gave an indeterminate result with another positive sample. Peptides which corresponded to other HCV proteins and which were designed on the basis of published results  31! are far less efficient for detection of HCV positive sera. 
     
         __________________________________________________________________________#             SEQUENCE LISTING- (1) GENERAL INFORMATION:-    (iii) NUMBER OF SEQUENCES: 20- (2) INFORMATION FOR SEQ ID NO:1:-      (i) SEQUENCE CHARACTERISTICS:#pairs    (A) LENGTH: 1400 base     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- GTAAACAATT CCAAGTTCCA AAATGGTTAA TAACAACAGA CCAAGACGTC AA - #CGAGCTCA  60- ACGCGTTGTC GTCACAACAA CCCAAACAGC GCCTGTTCCA CAGCAAAACG TG - #CCACGTAA 120- TGGTAGACGC CGACGTAATC GCACGAGGCG TAATCGCCGA CGTGTGCGCG GA - #ATGAACAT 180- GGCGGCGCTA ACCAGATTAA GTCAACCTGG TTTGGCGTTT CTCAAATGTG CA - #TTTGCACC 240- ACCTGACTTC AACACCGACC CCGGTAAGGG AATACCTGAT AGATTTGAAG GC - #AAAGTGGT 300- CAGCCGAAAG GATGTCCTCA ATCAATCTAT CAGCTTTACT GCCGGACAGG AC - #ACTTTTAT 360- ACTCATCGCA CCTACCCCCG GAGTCGCCTA CTGGAGTGCT AGCGTTCCTG CT - #GGTACTTT 420- TCCTACTAGT GCGACTACGT TTAACCCCGT TAATTATCCG GGTTTTACAT CG - #ATGTTCGG 480- AACAACTTCA ACATCTAGGT CCGATCAGGT GTCCTCATTC AGGTACGCTT CC - #ATGAACGT 540- GGGTATTTAC CCAACGTCGA ACTTGATGCA GTTTGCCGGA AGCATAACTG TT - #TGGAAATG 600- CCCTGTAAAG CTGAGTACTG TGCAATTCCC GGTTGCAACA GATCCAGCCA CC - #AGTTCGCT 660- AGTTCATACT CTTGTTGGTT TAGATGGTGT TCTAGCGGTG GGGCCTGACA AC - #TTCTCTGA 720- GTCATTCATC AAAGGAGTGT TTTCACAGTC GGCTTGTAAC GAGCCTGACT TT - #GAATTCAA 780- TGACATATTG GAGGGTATCC AGACATTGCC ACCTGCTAAT GTGTCCCTTG GT - #TCTACGGG 840- TCAACCTTTT ACCATGGACT CAGGAGCAGA AGCCACCAGT GGAGTAGTCG GA - #TGGGGCAA 900- TATGGACACG ATTGTCATCC GTGTCTCGGC CCCTGAGGGC GCAGTTAACT CT - #GCCATACT 960- CAAGGCATGG TCCTGCATTG AGTATCGACC AAATCCAAAC GCCATGTTAT AC - #CAATTCGG1020- CCATGATTCG CCTCCTCTCG ATGAGGTCGC GCTTCAGGAA TACCGTACGG TT - #GCCAGATC1080- TTTGCCGGTT GCAGTGATAG CGGCCCAAAA TGCATCAATG TGGGAGAGAG TG - #AAATCCAT1140- CATTAAATCC TCCCTGGCTG CTGCAAGCAA CATTCCCGGC CCGATCGGTG TC - #GCCGCAAG1200- TGGTATTAGT GGACTGTCAG CCCTTTTTGA AGGATTTGGC TTTTAGAAGC AT - #CCGGACGC1260- CAACCTAACC GGGCAAGTAT CCGAACAATC GGACATTTGG CCACAATAAG CC - #CAATTTGG1320- TTGAAGATTA AAGTAGTGAG CCCCCTTAGC GCGAAACCGG AATTTATATT CC - #AAACCAGT1380#                 140 - #0- (2) INFORMATION FOR SEQ ID NO:2:-      (i) SEQUENCE CHARACTERISTICS:#pairs    (A) LENGTH: 1378 base     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..1221-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- ATG GTT AAT AAC AAC AGA CCA AGA CGT CAA CG - #A GCT CAA CGC GTT GTC  48Met Val Asn Asn Asn Arg Pro Arg Arg Gln Ar - #g Ala Gln Arg Val Val#                 15- GTC ACA ACA ACC CAA ACA GCG CCT GTT CCA CA - #G CAA AAC GTG CCA CGT  96Val Thr Thr Thr Gln Thr Ala Pro Val Pro Gl - #n Gln Asn Val Pro Arg#             30- AAT GGT AGA CGC CGA CGT AAT CGC ACG AGG CG - #T AAT CGC CGA CGT GTG 144Asn Gly Arg Arg Arg Arg Asn Arg Thr Arg Ar - #g Asn Arg Arg Arg Val#         45- CGC GGA ATG AAC ATG GCG GCG CTA ACC AGA TT - #A AGT CAA CCT GGT TTG 192Arg Gly Met Asn Met Ala Ala Leu Thr Arg Le - #u Ser Gln Pro Gly Leu#     60- GCG TTT CTC AAA TGT GCA TTT GCA CCA CCT GA - #C TTC AAC ACC GAC CCC 240Ala Phe Leu Lys Cys Ala Phe Ala Pro Pro As - #p Phe Asn Thr Asp Pro# 80- GGT AAG GGA ATA CCT GAT AGA TTT GAA GGC AA - #A GTG GTC AGC CGA AAG 288Gly Lys Gly Ile Pro Asp Arg Phe Glu Gly Ly - #s Val Val Ser Arg Lys#                 95- GAT GTC CTC AAT CAA TCT ATC AGC TTT ACT GC - #C GGA CAG GAC ACT TTT 336Asp Val Leu Asn Gln Ser Ile Ser Phe Thr Al - #a Gly Gln Asp Thr Phe#           110- ATA CTC ATC GCA CCT ACC CCC GGA GTC GCC TA - #C TGG AGT GCT AGC GTT 384Ile Leu Ile Ala Pro Thr Pro Gly Val Ala Ty - #r Trp Ser Ala Ser Val#       125- CCT GCT GGT ACT TTT CCT ACT AGT GCG ACT AC - #G TTT AAC CCC GTT AAT 432Pro Ala Gly Thr Phe Pro Thr Ser Ala Thr Th - #r Phe Asn Pro Val Asn#   140- TAT CCG GGT TTT ACA TCG ATG TTC GGA ACA AC - #T TCA ACA TCT AGG TCC 480Tyr Pro Gly Phe Thr Ser Met Phe Gly Thr Th - #r Ser Thr Ser Arg Ser145                 1 - #50                 1 - #55                 1 -#60- GAT CAG GTG TCC TCA TTC AGG TAC GCT TCC AT - #G AAC GTG GGT ATT TAC 528Asp Gln Val Ser Ser Phe Arg Tyr Ala Ser Me - #t Asn Val Gly Ile Tyr#               175- CCA ACG TCG AAC TTG ATG CAG TTT GCC GGA AG - #C ATA ACT GTT TGG AAA 576Pro Thr Ser Asn Leu Met Gln Phe Ala Gly Se - #r Ile Thr Val Trp Lys#           190- TGC CCT GTA AAG CTG AGT ACT GTG CAA TTC CC - #G GTT GCA ACA GAT CCA 624Cys Pro Val Lys Leu Ser Thr Val Gln Phe Pr - #o Val Ala Thr Asp Pro#       205- GCC ACC AGT TCG CTA GTT CAT ACT CTT GTT GG - #T TTA GAT GGT GTT CTA 672Ala Thr Ser Ser Leu Val His Thr Leu Val Gl - #y Leu Asp Gly Val Leu#   220- GCG GTG GGG CCT GAC AAC TTC TCT GAG TCA TT - #C ATC AAA GGA GTG TTT 720Ala Val Gly Pro Asp Asn Phe Ser Glu Ser Ph - #e Ile Lys Gly Val Phe225                 2 - #30                 2 - #35                 2 -#40- TCA CAG TCG GCT TGT AAC GAG CCT GAC TTT GA - #A TTC AAT GAC ATA TTG 768Ser Gln Ser Ala Cys Asn Glu Pro Asp Phe Gl - #u Phe Asn Asp Ile Leu#               255- GAG GGT ATC CAG ACA TTG CCA CCT GCT AAT GT - #G TCC CTT GGT TCT ACG 816Glu Gly Ile Gln Thr Leu Pro Pro Ala Asn Va - #l Ser Leu Gly Ser Thr#           270- GGT CAA CCT TTT ACC ATG GAC TCA GGA GCA GA - #A GCC ACC AGT GGA GTA 864Gly Gln Pro Phe Thr Met Asp Ser Gly Ala Gl - #u Ala Thr Ser Gly Val#       285- GTC GGA TGG GGC AAT ATG GAC ACG ATT GTC AT - #C CGT GTC TCG GCC CCT 912Val Gly Trp Gly Asn Met Asp Thr Ile Val Il - #e Arg Val Ser Ala Pro#   300- GAG GGC GCA GTT AAC TCT GCC ATA CTC AAG GC - #A TGG TCC TGC ATT GAG 960Glu Gly Ala Val Asn Ser Ala Ile Leu Lys Al - #a Trp Ser Cys Ile Glu305                 3 - #10                 3 - #15                 3 -#20- TAT CGA CCA AAT CCA AAC GCC ATG TTA TAC CA - #A TTC GGC CAT GAT TCG1008Tyr Arg Pro Asn Pro Asn Ala Met Leu Tyr Gl - #n Phe Gly His Asp Ser#               335- CCT CCT CTC GAT GAG GTC GCG CTT CAG GAA TA - #C CGT ACG GTT GCC AGA1056Pro Pro Leu Asp Glu Val Ala Leu Gln Glu Ty - #r Arg Thr Val Ala Arg#           350- TCT TTG CCG GTT GCA GTG ATA GCG GCC CAA AA - #T GCA TCA ATG TGG GAG1104Ser Leu Pro Val Ala Val Ile Ala Ala Gln As - #n Ala Ser Met Trp Glu#       365- AGA GTG AAA TCC ATC ATT AAA TCC TCC CTG GC - #T GCT GCA AGC AAC ATT1152Arg Val Lys Ser Ile Ile Lys Ser Ser Leu Al - #a Ala Ala Ser Asn Ile#   380- CCC GGC CCG ATC GGT GTC GCC GCA AGT GGT AT - #T AGT GGA CTG TCA GCC1200Pro Gly Pro Ile Gly Val Ala Ala Ser Gly Il - #e Ser Gly Leu Ser Ala385                 3 - #90                 3 - #95                 4 -#00- CTT TTT GAA GGA TTT GGC TTT TAGAAGCATC CGGACGCCA - #A CCTAACCGGG1251Leu Phe Glu Gly Phe Gly Phe           405- CAAGTATCCG AACAATCGGA CATTTGGCCA CAATAAGCCC AATTTGGTTG AA - #GATTAAAG1311- TAGTGAGCCC CCTTAGCGCG AAACCGGAAT TTATATTCCA AACCAGTTTA AG - #TCAACAGA1371#        1378- (2) INFORMATION FOR SEQ ID NO:3:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 407 amino     (B) TYPE: amino acid     (D) TOPOLOGY: linear-     (ii) MOLECULE TYPE: protein-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:- Met Val Asn Asn Asn Arg Pro Arg Arg Gln Ar - #g Ala Gln Arg Val Val#                 15- Val Thr Thr Thr Gln Thr Ala Pro Val Pro Gl - #n Gln Asn Val Pro Arg#             30- Asn Gly Arg Arg Arg Arg Asn Arg Thr Arg Ar - #g Asn Arg Arg Arg Val#         45- Arg Gly Met Asn Met Ala Ala Leu Thr Arg Le - #u Ser Gln Pro Gly Leu#     60- Ala Phe Leu Lys Cys Ala Phe Ala Pro Pro As - #p Phe Asn Thr Asp Pro# 80- Gly Lys Gly Ile Pro Asp Arg Phe Glu Gly Ly - #s Val Val Ser Arg Lys#                 95- Asp Val Leu Asn Gln Ser Ile Ser Phe Thr Al - #a Gly Gln Asp Thr Phe#           110- Ile Leu Ile Ala Pro Thr Pro Gly Val Ala Ty - #r Trp Ser Ala Ser Val#       125- Pro Ala Gly Thr Phe Pro Thr Ser Ala Thr Th - #r Phe Asn Pro Val Asn#   140- Tyr Pro Gly Phe Thr Ser Met Phe Gly Thr Th - #r Ser Thr Ser Arg Ser145                 1 - #50                 1 - #55                 1 -#60- Asp Gln Val Ser Ser Phe Arg Tyr Ala Ser Me - #t Asn Val Gly Ile Tyr#               175- Pro Thr Ser Asn Leu Met Gln Phe Ala Gly Se - #r Ile Thr Val Trp Lys#           190- Cys Pro Val Lys Leu Ser Thr Val Gln Phe Pr - #o Val Ala Thr Asp Pro#       205- Ala Thr Ser Ser Leu Val His Thr Leu Val Gl - #y Leu Asp Gly Val Leu#   220- Ala Val Gly Pro Asp Asn Phe Ser Glu Ser Ph - #e Ile Lys Gly Val Phe225                 2 - #30                 2 - #35                 2 -#40- Ser Gln Ser Ala Cys Asn Glu Pro Asp Phe Gl - #u Phe Asn Asp Ile Leu#               255- Glu Gly Ile Gln Thr Leu Pro Pro Ala Asn Va - #l Ser Leu Gly Ser Thr#           270- Gly Gln Pro Phe Thr Met Asp Ser Gly Ala Gl - #u Ala Thr Ser Gly Val#       285- Val Gly Trp Gly Asn Met Asp Thr Ile Val Il - #e Arg Val Ser Ala Pro#   300- Glu Gly Ala Val Asn Ser Ala Ile Leu Lys Al - #a Trp Ser Cys Ile Glu305                 3 - #10                 3 - #15                 3 -#20- Tyr Arg Pro Asn Pro Asn Ala Met Leu Tyr Gl - #n Phe Gly His Asp Ser#               335- Pro Pro Leu Asp Glu Val Ala Leu Gln Glu Ty - #r Arg Thr Val Ala Arg#           350- Ser Leu Pro Val Ala Val Ile Ala Ala Gln As - #n Ala Ser Met Trp Glu#       365- Arg Val Lys Ser Ile Ile Lys Ser Ser Leu Al - #a Ala Ala Ser Asn Ile#   380- Pro Gly Pro Ile Gly Val Ala Ala Ser Gly Il - #e Ser Gly Leu Ser Ala385                 3 - #90                 3 - #95                 4 -#00- Leu Phe Glu Gly Phe Gly Phe           405- (2) INFORMATION FOR SEQ ID NO:4:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 36 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:- Met Gly Thr Asn Leu Ser Val Pro Asn Pro Pr - #o Ala Phe Gly Ala Asn#                15- Ser Thr Asn Pro Asp Trp Asp Phe Asn Pro Gl - #y Gly Met Gln Trp Asn#            30- Ser Thr Ala Leu   35- (2) INFORMATION FOR SEQ ID NO:5:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 15 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:- Met Gln Trp Asn Ser Thr Ala Leu Asp Pro Ar - #g Val Arg Gly Leu#                15- (2) INFORMATION FOR SEQ ID NO:6:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 24 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:- Cys Thr Thr Pro Ala Gln Gly Asn Ser Met Ph - #e Pro Ser Cys Cys Cys#                15- Thr Lys Pro Thr Asp Gly Asn Cys       20- (2) INFORMATION FOR SEQ ID NO:7:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 22 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:- Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Ar - #g Asn Thr Asn Arg Arg#                15- Pro Gln Asp Pro Arg Arg       20- (2) INFORMATION FOR SEQ ID NO:8:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 19 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:- Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gl - #y Gly Val Tyr Leu Leu#                15- Pro Arg Arg- (2) INFORMATION FOR SEQ ID NO:9:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 9 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:- Ile Gln Arg Gly Pro Gly Arg Ala Phe1               5- (2) INFORMATION FOR SEQ ID NO:10:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 7 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:- Ile Gly Pro Gly Arg Ala Phe1               5- (2) INFORMATION FOR SEQ ID NO:11:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 7 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:- Phe Gly Pro Gly Gln Ala Leu1               5- (2) INFORMATION FOR SEQ ID NO:12:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 7 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:- Ile Gly Pro Gly Arg Thr Leu1               5- (2) INFORMATION FOR SEQ ID NO:13:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 7 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:- Lys Gly Pro Gly Arg Val Ile1               5- (2) INFORMATION FOR SEQ ID NO:14:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 7 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:- Ile Gly Leu Gly Gln Ala Leu1               5- (2) INFORMATION FOR SEQ ID NO:15:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 9 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:- Gly Lys Ala Met Tyr Ala Pro Pro Ile1               5- (2) INFORMATION FOR SEQ ID NO:16:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 10 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:- Asn Met Trp Gln Glu Lys Val Gly Lys Ala#                10- (2) INFORMATION FOR SEQ ID NO:17:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 7 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:- Glu Leu Asp Lys Trp Ala Ser1               5- (2) INFORMATION FOR SEQ ID NO:18:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 12 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:- Ile Glu Glu Glu Gly Gly Glu Arg Asp Arg As - #p Arg#                10- (2) INFORMATION FOR SEQ ID NO:19:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 8 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:- Ile Gly Pro Gly Arg Ala Phe Glu1               5- (2) INFORMATION FOR SEQ ID NO:20:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 6 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:- Gly Pro Gly Arg Ala Phe1               5__________________________________________________________________________