AU728139B2 - Peptide fragment of the respiratory syncytial virus G protein - Google Patents

Description

0' P/00/0011I Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

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*555 *5 S S *5 Name of Applicant: Actual Inventors: PIERRE FABRE MEDICAMENT BINZ Hans, N'GUYEN Ngoc Thien, BAUSSANT Thierry, TRUIDEL Michel Address for service in Australia: Invention Title: CARTER SMITH BEADLE 2 Railway Parade Camberwell Victoria 3124 Australia PEPTIDE FRAGMENT OF THE RESPIRATORY SYNCYTIAL VIRUS G PROTEIN -la- PEPTIDE FRAGMENT OF THE RESPIRATORY SYNCTIAL VIRUS G PROTEIN The present invention relates to polypeptides which can be used especially in the preparation of immunogens and the obtainment of vaccine against respiratory syncytial virus (RSV) and to nucleotide sequences enabling them to be obtained. The invention likewise relates to an immune adjuvant protein extracted from Klebsiella pneumoniae, to compositions comprising the immunogenic polypeptides, possibly associated with such an adjuvant protein, and to their preparation process.

Respiratory syncytial virus (RSV) is the most S. 15 frequent cause of respiratory illnesses in the newborn: bronchopneumopathies (bronchiolites). The WHO estimates each year 50 million cases of RSV attacks, from which 160,000 die in the entire world. There are two subgroups of the virus (subgroups A and B).

20 RSV is classified in the Paramyxoviridae family, a type of pneumovirus comprising a nonsegmented RNA genome, of negative polarity, coding for 10 specific proteins.

There is at present no vaccine available against RSV. Inactivated virus vaccines have been shown to be inefficaceous and have sometimes even aggravated the infections of nursing infants. In the 60's, vaccination attempts with formalin-inactivated RSV resulted in failure: instead of conferring protection at the time of reinfection due to RSV, the vaccine had the effect of aggravating the illness in the child.

The Application WO 87/04185 proposed to use structural proteins of RSV with a view to a vaccine, such as the envelope proteins called protein F (fusion protein) or protein G, a 22 Kd glycoprotein, a 9.5 Kd protein, or the major capsid protein (protein N).

The Application WO 89/02935 describes the protective properties of the entire protein F of RSV, possibly modified in monomeric or deacetylated form.

2 A series of fragments of protein F have been cloned with a view to investigating their neutralizing properties.

However, the immune vaccines tested to date have been shown to be inefficaceous or have induced a pulmonary pathology (bronchiolitis or peribronchitis).

At the present time, there is no in-depth treatment of infections due to RSV.

Infections [lacuna] to RSV of the upper airways: treatment relies essentially on symptomatic medications identical to those for other viral infections.

Infections [lacuna] to RSV of the lower airways: treatment in nursing infants relies on the maintenance of correct hydration, the aspiration of the secretions and the administration of oxygen if necessary. A positive effect has been observed with ribavirin, a nucleotide which is active in vitro against RSV.

It is for these reasons that it is desirable to provide a polypeptide which is useful especially in immunogen production, characterized in that it is carried by the peptide sequence between the amino acid residues 130 and 230 of the sequence of respiratory syncytial virus protein G, or by a sequence having at least 80% homology with said peptide sequence. This sequence differs slightly for the subgroups A and B of human RSV, or for bovine RSV. The invention comprises the sequences originating from human RSV subgroup A and B, or bovine RSV.

Protein G is an RSV envelope glycoprotein, of 30 molecular weight of between 84 and 90 Kd, which is low in methionine.

The Applicant has demonstrated that the sequence between amino acids 130 and 230 of natural protein G is particularly appropriate for inducing an efficaceous protection against infection by RSV. The invention comprises the sequences originating from human RSV subgroup A or B, or bovine RSV.

SMore particularly, the present invention is relevant S to polypeptides, which are useful especially as 3 immunogenic element included in the above and which comprise the peptide sequence between the amino acid residues numbered 174 and 187 of RSV protein G (human, subgroups A and B, or bovine) or a sequence having at least 80% homology with the corresponding sequence.

Other peptide sequences adapted to the preparation of an immunogen included in said sequence of RSV protein G are formed by the sequence between the amino acid residues numbered 171 and 187 of human or bovine RSV protein G, or a sequence having at least homology with the corresponding sequence. Other peptides of interest according to the present invention are carried by the sequence between the nucleotides numbered 158 and 190 of RSV protein G or a sequence having at least 80% homology with the corresponding sequence.

According to another method of carrying it out, the invention is relevantto peptides useful for the preparation of an immunogen and which have a sequence corresponding to the sequence between the amino acid residues numbered 140 and 200 of human or bovine RSV protein G, or a sequence having at least 80% homology with the corresponding sequence. Sequences starting with amino acid 140 of said RSV protein G and whose C-terminal end corresponds respectively to the amino acid 198, 196, 25 194, 192 or 190, as well as sequences having at least homology with the sequence carried by these fragments, are particularly advantageous.

Among the' variants of the above sequences, 0oo polypeptides may be mentioned which comprise a sequence 30 in which: a) the Cys amino acid in positions 173 and/or 186 has been replaced by an amino acid not forming a disulfide bridge, in particular serine, and/or b) the amino acids in positions 176 and 182 are capable of forming a covalent bridge other than a disulfide bridge, especially aspartic acid and ornithine.

Thus the polypeptide sequence 130-230 of RSV RA1 subgroup A can be used complete, in its native form. This 7 sequence corresponds to the written sequence Seq id No.

4 1 (or G2A) In the same way, it is possible to use the complete polypeptide sequence 130-230 of RSV subgroup B in its native form. This sequence corresponds to the written sequence Seq id No. 2 (G2B).

The sequence id No. 1 will be written G2A in the remainder of the application.

The sequence id No. 2 will be written G2B in the remainder of the application.

Sequences having at- least 80% homology with G2A or G2B are also appropriate.

The sequence between the amino acids 130 and 230 can be modified by the replacement of the cysteine residue in positions 173 and 186 by serine residues to obtain a peptide retaining good immunogenic properties, owing to maintenance of the loop formed by the Cys residues in positions 176 and 182. The amino acid and nucleotide sequences of this polypeptide for subgroup A are represented in seq id No. 3 For subgroup B, the amino acid and nucleotide sequences are represented in seq id No. 4 (G2B6Cys).

The peptide sequences will be written G2A6Cys and G2B6Cys.

According to another aspect,also usefuiruwith'the 0 25 invention is a polypeptide which is useful for the preparation of immunogen, characterized in that it consists in the peptide sequence between the amino acid 0*e residues numbered 174 and 187 of RSV protein G or a 0eo sequence having at least 80% homology with said peptide 30 sequence.

In this last sequence the peptide 174-187 subgroup A can have the sequence: Seq id No. Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys.

The peptide 174-187 subgroup B can have the sequence: Seq id No. 6: Ser-Ile-Cys-Gly-Asn-Asn-Gln-Leu-Cys-Lys-Ser-Ile-Cys-Lys.

SThe Cys residue in position 186 can also be 5 replaced by a serine residue, so as to obtain the following sequence: Seq id No. 7 for subgroup A: Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys.

Seq id No. 8 for subgroup B: Ser-Ile-Cys-Gly-Asn-Asn-Gln-Leu-Cys-Lys-Ser-Ile-Ser-Lys.

In the sequence between residues 174 and 187 of the immunogenic peptide, according to one of the variants of the invention, the amino acid residues in positions 176 and 182 are respectively replaced by an aspartic acid and an ornithine, so as to obtain one of the following sequences: Seq id No. 9 for subgroup A: Ser Ile Asp Ser Asn Asn Pro Thr Orn Trp Ala Ile Cys Lys Seq id No. 10 for subgroup B Ser-Ile-Asp-Gly-Asn-AsAsGln-Le-Orn-Lys-Ser-Ile-Cys-Lys.

Seq id No. 11 for subgroup A: Ser Ile Asp Ser Asn Asn Pro Thr Orn Trp Ala Ile Ser Lys.

Seq id No. 12 for subgroup B: Ser-Ile-Asp-Gly-Asn-Asn-Gln-Leu-Orn-Lys-Ser-Ile-Ser-Lys.

The maintenance of the immunogenic properties is obtained owing to the replacement of the disulfide bridge (between the natural Cys residues) by an amide bridge between the positions 176 and 182.

25 Other sequences useful with the invention such as defined above appear in the annex of the present application under the names SEQ ID No. 14 to SEQ ID No.

73.

0 Desirably also, is likewise a o. 30 polypeptide which can be used as an immunogenic agent o having one of the preceding sequences and which additionally comprises at least one cysteine residue in the N-terminal or C-terminal position.

The invention likewise may use a polypeptide which consists of the peptide sequence between the amino acid residues numbered 130 and 230 of the RSV protein G sequence subgroup A and subgroup B, or of a sequence having 88% homology with said peptide sequence and which is in the form of a fusion protein with the receptor of 6 human serum albumin, called BBG2A6C or BBG2B6C, or another linking protein. The sequence of the complete BB protein appears in the annex (Seq ID No. 74).

The invention likewise may use the variants, for example glycosylated or sulfated, of the different peptides, whether these functions are natural or not.

The polypeptides can be prepared by peptide synthesis or by recombinant DNA techniques, which are known to the person skilled in the art.

In particular, the gene sequences coding for the epitope of approximately 100 amino acids can be prepared by solid-phase assembly of genes, and the corresponding protein- expressed, for example, in E. coli by the intracellular route.

The nucleotide sequences (RNA or DNA) coding for the proteins or the polypeptides defined above are useful for the invention.

According to one aspect, this invention p8F vi immunogenic agent which comprises a polypeptide such as defined above coupled to a carrier protein, in particular to an immune adjuvant protein.

Preferably, the polypeptide according to the invention is coupled to a carrier protein of the type OmpA of the external membrane of a bacterium of the genus 25 Klebsiella, preferably in the form of a soluble :conjugate.

The Applicant has been able to show that although the variants of the sequence 174-187 of the RSV protein G are weakly immunogenic, their coupling with such a 30 protein induces a specific immune response.

The intensity of the immune response has been compared to that obtained with conventional adjuvants, such as coupling to the carrier KLH (keyhole limpet hemocyanin) coadministered with Freund's adjuvant, or coupling to the carrier protein TT (tetanus toxoid).

Particularly advantageous results are obtained for compositions comprising an immunogenic polypeptide according to the invention coupled to protein p40 of 2!Da Klebsiella pneumoniae or a protein having 80% homology 7 with protein More particularly, said polypeptide is coupled to a protein comprising the peptide sequence written Seq id No. 13.

The nucleotide sequence (DNA or RNA) coding for the protein comprising the sequence id No. 13 is comprised in the invention.

The immunogenic polypeptide can be coupled to the immune adjuvant protein by methods known to the person skilled in the art, such as: Glutaraldehyde Carbodiimide EDC: 1-(3dimethylaminopropyl)-3ethylcarbodiimide[sic]).

Bis imido esters dimethyl adipimidate).

N-hydroxysuccinimidyl esters disuccinimidyl suberate).

For peptides comprising a supplementary cysteine in the N terminal or C terminal position: SMaleimido-N-hydroxysuccinimide esters MBS: maleimido benzoyl-N-hydroxysuccinimide ester).

N-succinimidyl bromoacetate.

The polypeptide can be conjugated to the carrier I protein by a linking protein, for example the human serum albumin receptor (BB).

25 According to another aspect, there is S. provided likewise a process for the preparation of a conjugated peptide inserted in a composition useful for prevention or treatment of infections with RSV, characterized in that: 30 a) the membrane lipopolysaccharides of bacteria of the genus Klebsiella are precipitated in the presence of a salt of a divalent cation and of detergents to recover the total membrane proteins in the supernatant, b) the proteins are submitted to anion-exchange chromatography to separate the fraction containing the immune adjuvant protein, RA c) the fraction containing the immune adjuvant protein is concentrated, 8 d) the immune adjuvant protein is conjugated with an immunogenic polypeptide such as defined above to form a soluble conjugate.

The divalent cation salt used in step a) is preferably a salt of calcium or of magnesium. After centrifugation, the proteins of the supernatant can be recovered in good yield by two precipitations with ethanol.

The membrane proteins, after resuspension, are separated on an anion-exchange column which can be used under industrial conditions. This chromatographic support is very stable and compatible with drastic pyrogen removal treatments, which was not the case with the chromatographic supports already described. On the other hand, elution of the protein can be carried out under isocratic conditions and not by application of an NaC1 gradient (as described previously), which is particularly advantageous under industrial conditions.

Preferably, step c) is followed by a second chromatography step, on a cation exchanger, and the fractions containing the adjuvant protein are recovered and concentrated. This supplementary step allows a better elimination of the lipopolysaccharides. The adjuvant protein is then conjugated to an immunogenic polypeptide according to the invention.

According to another aspect, the invention relates to a composition useful for the prevention and/or treatment of infections provoked by RSV, characterized in 30 that it contains a polypeptide characterized above.

More particularly, the compositions additionally contain pharmaceutically acceptable excipients adapted •for administration by the injectable route.

In fact, the Applicant has demonstrated that the injection of such compositions affords protection, not by a neutralizing effect, but by a systemic immune response of-the body.

SThe humoral and cellular responses (IgM, IgG, IgA S and T cells) are provoked by the product which likewise 9 induces a long-term protection and an immunological memory against the RSV subgroups a and b.

With a view to the administration of the vaccine compositions by the subcutaneous route, it is desirable to have available soluble conjugate, which is difficult by the conventional methods.

It is for this reason that the invention likewise relates to a process for the preparation of a conjugate between an immunogenic peptide and a membrane protein of Klebsiella, in particular the protein p40 of K.

pneumoniae, in which the coupling is carried out in the presence of glutaraldehyde at concentrations lower than or equal to 0.05%.

This coupling process considerably reduces the 15 concentrations of glutaraldehyde in comparison with those usually used (2 times 0.01% instead of 1% approximately); the glutaraldehyde is added in 2 portions over a period of five days although the protocols described mention times of 24 hours.

20 These modifications have allowed the obtainment of a soluble conjugate, in a form adapted for subcutaneous administration.

The usual protocols (higher concentrations of Sglutaraldehyde and short times) are manifested by the S* 25 formation of a thick gel (due to P40-P40 conjugation reactions, very probably), a form unfit for administration and manipulation in general.

The conjugated peptide can be frozen and used as such or lyophilized.

The examples which follow are intended to illustrate the invention without in any way limiting the range thereof.

In these examples, reference will be made to the following figures: Figure 1: intensity of the immune response induced against G1A in different forms, Figure 2: kinetics of the immune response induced against GIA presented in different forms, Figure 3: kinetics of the immune response induced 10 against the carrier alone, Figure 4: cloning strategy by genetic amplification of Example 1: Synthesis and purification of G 1

A

The polypeptide of sequence Ser-Ile-Cys-Ser-Asn-Asn-Pro-Thr-Cys-Trp-Ala-Ile-Ser-Lys I s-s I written G 1 A is prepared by -solid-phase synthesis using Boc chemistry.

Assembly The assembly of the peptide is carried out by 10 solid-phase peptide synthesis on polystyrene (divinylbenzene starting with a Boc-Lys(2-cl-Z)phenylacetamidophenyl linking agent.

The Boc-benzyl chemical strategy was used with the following deprotection-coupling procedure: 1. 55% TFA in DCM (1 x 5 min) 2. 55% TFA in DCM (1 x 25 min) 3. DCM (2 x 1 min) 4. Isopropyl alcohol (1 x 1 min) DMF (2 x 1 min) 20 6. 10% DIEA in DMF (2 x 2 min) 7. Coupling 8. DMF (2 x 1 min) 9 DCM (2 x 1 min) In each step, 20 ml of solvent are used per gram of peptide resin.

The coupling is carried out in DMF with a preformed hydroxybenzotriazole ester for 30 min. It is verified in each step of the coupling if residual free amine functions are present by the ninhydrin test. If necessary, a double coupling is carried out.

For the synthesis of the G 1 A peptide, the following side-chain protection groups were used: 2-chlorobenzyloxycarbonyl for lysine, benzyl for serine and threonine, 11 4-methylbenzyl for cysteine, formyl for tryptophan.

Before the final deprotection/cleavage step, the formyl group is eliminated by treatment for 30 min with a 25% solution of piperidine in DMF. The peptide resin is washed with DCM and ether, and dried under reduced pressure.

Cleavage The peptide is cleaved from the resin and completely deprotected by treatment with liquid hydrogen fluoride. 10 ml of hydrogen fluoride per gram of peptide resin are conventionally used at 0 C for 45 min in the presence of p-cresol and ethanedithiol as a trap. After evaporation of the hydrogen fluoride, the crude reaction 15 mixture is washed with ether, dissolved in TFA, precipitated with ether and dried.

Cyclization and purification General conditions of purification by HPLC: Stationary phase: C 18 silica, 15-25 jim, 100 A Mobile phase: solvent A: water 0.1% TFA solvent B: acetonitrile/A, 60/40% (v/v) Linear gradient: 20 to 50% B in 30 min (first purification step) 15 to 40% B in 30 min (second purification step) Flow rate: 40 ml/min Detection: UV (210 nm) The crude peptide obtained after cleavage is purified under the conditions described above (gradient of 20 to 50% Fractions having a purity of greater than 70-80% (HPLC) are combined and lyophilized. The peptide is then purified in a mixture of acetonitrile water and DMSO (1 mg/ml) and stirred until the cyclization is complete (4 to 6 days). The progress of the reaction is checked by HPLC. The reaction mixture is finally concentrated on a preparative HPLC column and a 12 gradient of 15 to 40% of B is applied in 30 min so as to purify the peptide.

Generally, after lyophilization, a second purification under the same condition is carried out to attain the degree of purity required.

The purity and the identity of the final product are checked by analytical HPLC, amino-acid analyzis and FAB mass spectrometric analyzis.

In the peptide thus obtained, the serine residue in position thirteen replaces the Cys residue of the natural peptide, thus avoiding heterogeneity in the formation of disulfide bridges, which can be harmful to the immunogenicity.

Example 2: Preparation of the epitope G 2 ABCys Gene construction: materials and methods In an Eppendorf microtube, 300 gg of beads are washed with washing/binding buffer (1M NaC1, 10mM Tris- HC1 pH7.5, 1 mM EDTA) before adding 0.2 pmol of biotinylated oligonucleotide; 15 minutes' incubation at ambient temperature for binding. The beads with the immobilized oligonucleotide are rinsed and sedimented.

0.2 pmol of the following oligonucleotide is added in 60 P1 of hybridization/ligation buffer (50mM Tris-HCl pH7.6, 10 mM MgCl 2 ImM ATP, 1 mM 1,4-dithiothreitol [DTT], polyethylene glycol [PEG] 8000]. The hybridization mixture is incubated at 70 0 C for 5 min and allowed to come to 37 0 C before adding 3 units of T4 DNA ligase (BRL) followed by 15 min incubation at 37 0 C. The reaction mixture is rinsed before adding 0.2 pmol of the following oligonucleotide. The hybridization/ligation procedure is repeated as many times as a new complementary oligonucleotide is added. At the end, the DNA duplex immobilized on magnetic beads can be separated from the support by cutting with the appropriate restriction enzymes.

The DNA corresponding to the sequence G2A6Cys and 13 to the sequence G2A6Cys attached to the linking protein to human serum albumin (BB) written BB-G2A6Cys is prepared.

The nucleotide sequence is expressed in E. coli to recover the corresponding proteins.

Expression vector: pVABBG2A6C is an expression vector of the intracellular type, it contains a promoter of E. coli origin, the tryptophan (Trp) operation [sic], followed by the gene coding for the receptor of human serum albumin BB (P-A Nygren et al., J. Mol. Recognit., 1988, 1, and finally the gene coding for G2A6C of RSV. The expression of the heterologous gene can be induced in the presence of IAA (3-6-indoleacrylic acid) The fusion 15 product BBG2A6C can be purified by affinity on an HSAsepharose column, after having liberated the cytoplasmic proteins of E. coli.

Examples of purification of proteins starting from 500 ml of culture: 20 The strain E. coli RV 308 (Maurer et al., J. Mol.

Biol., 1980, 139, 147) transfected by the plasmid pVABBG2A6C was selected on agar containing ampicillin i (100 jg/ml) and tetracycline (8 ig/ml). The strain was 0* inoculated into an Erlenmeyer flask containing 100 ml of TSB culture medium (Tryptic Soy broth, Difco) (30 g/1), supplemented with yeast (Yeast Extract, Difco) (5 g/1), ampicillin (100 gg/ml), tetracycline (8 yg/ml) and tryptophan (100 pg/ml). Incubate at 32 0 C for 12 hours with stirring (190 rpm). Transfer the culture into another erlenmeyer flask (5 liters) containing four times the initial volume (400 ml of TSB yeast the same antibiotics at the same concentration). When the optical density of the medium (at 550 nm) has reached an O.D. of approximately 1.5, the production of the proteins is induced by adding IAA to the medium to a final concentration of 25 ;g/ml. Culturing is stopped after incubation for 5 hours, with stirring (190 rpm) at 32 0

C.

After centrifugation, the bacterial plug is resuspended 14 in a vessel comprising approximately 60 ml of cold TST solution (50 mM TrisHCl, pH 8.0, 200mM NaC1, 0.05% Tween 0.5 mM EDTA).

A standard sonicator probe (VIBRA-CELL, Somics Mat, USA) is introduced into the vessel. Sonication is carried out at a power of 5 for approximately two minutes. The supernatant of the solution after centrifugation is filtered at 0.45 ym, and passed into a column containing approximately 3 ml of HSA-sepharose gel (STAHL et al., J. Immunol. Meth., 1989, 124, 43).

The purified proteins are analyzed by SDS-PAGE on a Phast System apparatus (PHARMACIA) or on Mini Protean BIORAD. The gels are visualized by Coomassie Blue. The protein BBG2A6C, representing more than 90% purity, 15 corresponds well to the expected size (39.3 Kda) with respect to known molecular weight standards.

The immunotransfer of this protein to a Problott membrane (ABI) allows anti-BB and/or antiprotein G of RSV (ss-group A) to be identified with specific antibodies.

The yield of purified soluble proteins starting from the cytoplasm of E. coli is approximately 50 mg/liter of culture.

In a 2-liter fermenter, it is possible to obtain 500 to 800 mg of BBG2A6C proteins per liter of culture S" 25 under optimum culture conditions.

Example 3: Isolation and purification of the natural protein The process of purification of the P40 protein starting from the biomass of Klebsiella pneumoniae, strain 1-145, was developed with one main objective: to develop a process allowing transposition to a large scale and industrial extrapolation. This process successively brings into play the preparation of a fraction enriched in membrane proteins and the purification of the protein by chromatography.

MATERIALS AND METHODS The biomass of Klebsiella pneumoniae (strain 15 1-145, 40 g of dry cells) is adjusted to pH 2.5 with the aid of pure acetic acid.

After addition of volume of a solution comprising 6% cetrimide, 60% ethanol, 1.5 M CaC12 [sic] whose pH is adjusted to 2.5 with acetic acid, the mixture is stirred for 16 hours at ambient temperature.

After centrifugation for 20 min at 15,000 g at 4°C, the proteins of the supernatant are precipitated with ethanol. Two successive precipitations with intermediate centrifugation (10 min, 10,000 g, 4°C) are carried out: from 20 to 50% then from 50 to The plugs obtained after the second precipitation are resuspended in a solution of zwittergent 3-14, 1%.

After stirring for 4 hours at ambient 15 temperature, the pH is adjusted to 6.5 with the aid of 1 N NaOH.

Centrifugation of the mixture for 20 min at 10,000 g at 4 0 C allows a fraction enriched in membrane proteins (MP fraction) to be obtained.

The proteins of the MP fraction are dialyzed against a 20 mM Tris/HCl buffer pH 8.0; zwittergent 3-14, The dialyzate is applied to a column containing a support of the strong anion exchanger type (column of S*diameter 50 mm x H 250 mm, Biorad Macroprep High Q 25 gel) equilibrated in the buffer described above. The protein is eluted by an NaCl concentration of 50 mM in the equilibration buffer.

The fractions containing the P40 are collected and dialyzed against a 20 mM citrate buffer pH zwittergent 3-14, The dialyzate is applied to a column containing a support of the strong cation exchanger type (dimensions of the column diameter mm x H 160 mm, Biorad Macroprep High S gel) equilibrated in the 20 mM citrate buffer pH zwittergent 3-14, The P40 protein is eluted by an NaC1 concentration of 0.7 M. The fractions containing the are collected and concentrated by ultrafiltration with the aid of a Minitan Millipore tangential-flow filtration system used with membrane sheets having a 16 kDa cutoff threshold.

RESULTS

The fractions obtained after each chromatographic step are analyzed by SDS-PAGE so as to collect those containing the P40 protein.

The quantities of proteins are measured by the method of Lowry (Table The purity and homogeneity of the P40 protein are estimated by SDS-PAGE, in the presence of molecular weight standards.

After the cation exchange chromatography step, -the P40 protein is devoid of the major contaminant present in the MP fraction (the protein having an apparent molecular weight of 18 kDa) and has a degree of purity of greater than 15 The electrophoretic profile of the P40 reveals several bands. These bands are identified after immunoblot with P40 monoclonal antibodies obtained in mice. The upper major band corresponds to the denatured protein (by treatment at 100 0 C, 15 min in the presence of SDS), and the lower minor band to the protein in its native form.

P40 is in fact a "heat-modifiable" protein, and we have been able to verify this property with the aid of heating kinetics at 100 0 C in the presence of SDS. Without heating, the protein in native form has an a-helix structure which fixes more SDS and thus migrates further toward the anode than the denatured form (denaturation complete after 5 min at 100 0 C) which has a #-pleated sheet structure KELLER (1978) J. Bacteriol. 134, 1181-1183).

The contamination with lipopolysaccharides (LPS) is estimated by determination by gas-phase chromatography of P-hydroxymyristic acid, the fatty acid marker of LPS of Klebsiella pneumoniae (Table I).

ft*S ft ft ft ft C. ft ft ft ft ft ft ft ft ft ft ft ft ft. ft ft.

Cpft ft ft.. ft ft ft ft ft ft.. ft ft ft ft ft S ft ft ft ft ft ft ft ft ft ft ft ft ft. ft. ft.

ft. ft ft. ft. ft ft ft..

C ftC ft ft ft ft ft ft ft. ft ft ft. ft. ft. S Table 1: Table of the quantities of protein and LPS of the fractions obtained for the different steps in the process for the purification of the p40 protein not determined).

f r PROTEINS YIELD

LPS

BIOMASS 40 g d.

MP FRACTION 900 mg 2.25% n.d.

FRACTION ENRICHED IN P40 400 mg 1% PROTEIN 130 mg 0.3% 1% i 18 This method is used to approximate the content of LPS in the samples from the different purification steps.

The quantity of 9-hydroxymyristic acid present in the P40 fraction after cation-exchange chromatography being lower than the quantification threshold of the determination, it is possible to estimate that the quantity of residual LPS is lower than 1%.

Example 4: Cloning of the p40 protein and expression of 10 BACTERIAL STRAINS e coli: RV 308: ATCC 31608 strain (MAURER R.,

*I

MEYER PTASCHNE J. MOL. BIOL, 1980, 139, 147- *e 161).

K. pneumoniae: IP 145: C.I.B.P.F-strain *o

VECTORS

pRIT 28 (Hultman et al., 1988,7: 629-638): cloning and sequencing vector containing the ampicillin resistance gene, the replication origins of E. coli and of the phage F1 as well as a portion of the lac-z gene of E. coli (g-galactosidose [sic]).

SpVABB: gene fusion expression vector.

SOLUTIONS

Genetic amplification: Lysis buffer: 25 mM Taps pH 9.3 2 mM MgC12 [sic] Amplification buffer: 25 mM Taps pH 9.3 2 mM MgC12 [sic] tween 20 0.1% 200 mM dNTP.

Purification of proteins: TST (20X): Tris base 0.5 M HC1 0.3 M 19 NaCI 4 M Tween 20 1% EDTA 20 mM Washing buffer: Tris HC1 50 mMpH MgC12 [sic] 5 mM Denaturation solution: Gua-HC1 7.8 M Tris-HC1 28 mM pH Renaturation solution: Gua-HC1 0.5 M Tris-HCl 25 mM pH 10 NaCI 150 mM *I Tween 20 0.05%.

*MATERIAL AND METHOD Synthesis of oligonucleotides The nucleotide primers were determined starting 15 from the published part of the sequence of the OMPA of Klebsiella pneumoniae (LAWRENCE, et al., Journal of general microbiology, 1991, 137, 1911-1921) of the consensus sequence from the alignment of the sequences of OMPA of enterobacteria coli, S. tryphimurium [sic], S. marcescens, S. dysenteriae, E. aeroginosae as well as sequences of peptides obtained by manual sequencing.

The oligonucleotides were synthesized according to the phosphoramidite chemical method on the "Gene Assembler Plus" apparatus from Pharmacia.

Genetic amplification by PCR of the P40 gene The DNA of OMPA of Klebsiella pneumoniae was amplified in the following manner.

A colony of Klebsiella pneumoniae is lysed in 10 p1 of lysis buffer by heating to 95°C for 5 minutes.

1 1l of this solution serves as a source of DNA k 20 for the amplification reactions.

These are carried out in 100 zl of amplification buffer (cf. annex), with 5 pmol of each primer and one unit of Taq polymerase enzyme (Perkin Elmer Cetus). Each cycle comprises one denaturation step of 30 seconds at 0 C followed by a hybridization of the primer to the DNA and an extension of one minute at 72 0 C. 30 cycles are thus carried out with the aid of a Perkin Elmer Cetus 9000 "Gen Amp PCR" thermocyclizer.

The following PCR are prepared starting from the DNA fragments amplified above.

The amplified DNA fragments are then digested, purified and ligated to the vector pRIT 28.

SEQUENCING

15 The fragments cloned in this way are sequenced on an Applied Biosystem 373 DNA Sequencer automatic sequencer. The sequencing reactions are carried out with the aid of the "dye Terminator" kit according to the recommendations of the supplier (Applied Biosystem) either on double-stranded DNA obtained after genetic amplification or from maxiprep or on single-stranded DNA from denatured PCR fragments (Hultman et al., Nucleid [sic] acids res.; 1989, 17:4937-4946).

EXPRESSION OF THE PROTEIN The entire P40 gene is cloned in the expression vector pVABB. This vector allows an affinity tail "BB" to be attached to P40; B being the part of the streptococal G protein which ligates serum albumin (Nygren P.A. et al.; Journal mol. Recognit. 1988; 1, 69-74).

The strains of E. coli RV308 transformed by the vector pVABBP40 are cultured for one night at 37 0 C with stirring, in 100 ml of TSB supplemented with yeast extract, ampicillin (200 pg/ml) tetracycline (8 pg/ml) and tryptophan (100 pg/ml). The next day, a culture of OD 1 for a wavelength of 580 nm is prepared in TSB 21 yeast extracts ampi tetra.

After culturing for 10 minutes, expression of the protein is induced by addition of IAA at (25 pg/ml) to the medium. The culture is centrifuged at 4°C at 2460 g for 10 minutes.

The plug is taken up with 20 ml of TST 1 x pH 7.4, and the solution is then centrifuged at 4 0 C at 23,000 g for 30 minutes.

The supernatant is filtered through Sepharose which allows proteins termed soluble to be isolated. The plug is washed with washing buffer and then centrifuged at 23,000 g at 4°C for 30 minutes. The plug containing the inclusion body is then taken up with 900 il of a denaturing solution 100 pl of 10mM Diothiothreitol 15 [sic] and incubated at 37 0 C for 2 hours.

The solution is then incubated at ambient temperature for 1 night, with stirring, [lacuna] in 100 ml of renaturation buffer at 2300 g for 1 hour.

The supernatant is filtered through HSA 20 Sepharose.

In the two cases, the immobilized proteins are eluted with 0.5 M acetic acid pH 2.8 and collected in 1 ml fractions.

The fractions collected are then analyzed on SDS- PAGE electrophoresis gel and by Immuno blot.

RESULTS

The cloning of the gene was carried out in three stages according to the strategy presented in Figure 4.

In a first stage, we confirmed the published part of the sequence with the exception of a T in the place of an A in position 103.

Then we determined the 3'-sequence of the gene and finally the The entire gene was obtained by fusion of the two parts 8/4 and 3/14 and then cloned in the vector pRIT 28.

The sequence corresponds to SEQ ID No. 13.

The protein is expressed in the form 22 It is essentially obtained starting from inclusion bodies. For a 200 ml culture, fifteen milligrams of protein are purified.

The electrophoretic profile shows that obtained after denaturation, is of high purity. The apparent molecular weight corresponds to the calculated theoretical weight which is 63 kDa.

The Immuno blot characterization shows that the purified protein is indeed recognized by a rabbit anti- P40 serum.

Example 5: Coupling of the p40 protein to the G 1 A peptide p40 (5 mg/ml, 40 mg) is dialyzed against 300 volumes of 0.1 M sodium phosphate buffer pH 7, zwittergent 3-14, 0.1%.

15 The dialyzate is adjusted to a concentration of 2 mg/ml with the aid of a 0.1 M carbonate buffer pH 9; zwittergent 3-14, Sodium dodecyl sulfate (SDS) is added to obtain a final concentration of 4%.

The G 1 peptide (10 mg/10 ml of 0.1 M carbonate 20 buffer pH 9; 0.1% zwittergent 3-14) is added to the solution. The pH is checked (between pH 9 and pH Add 220 pl of glutaraldehyde in water) and stir for 24 hours at 4*C.

Add 5 ml of 0.1 M carbonate buffer pH 9; 0.1% zwittergent 3-14; check the pH (between pH 9 and pH stir for 72 hours at 4°C.

Add 220 il of glutaraldehyde in water), check the pH, stir for 24 hours at +4 0

C.

The reaction is stopped by addition of 100 yl of 1 M lysine. The solution is dialyzed for 24 hours at 4°C.

The SDS is eliminated by double KCl precipitation.

The solution containing the p40 conjugate is frozen and used as such or lyophylized.

Sii 23 Example 6: Activity Material and methods C57BL/6 mice are immunized on day 0, day and day 20 by the subcutaneous route with 10 yg of Gl, optionally coupled to a carrier, in the presence or absence of an adjuvant. The serum is collected and tested by ELISA. The anti-Gl or anti-carrier Igs are isolated on a BSA-G1 support and on a "carrier" support (KLH or TT or The Igs are visualized with the aid of an anti-Ig 10 rabbit peroxidase conjugate. The optical density is read at 450 nm and the anti-Gl antibody titer is given by the reciprocal of the last dilution giving twice the background noise. The results represent the mean standard deviation of the titers of 5 mice.

15 RESULTS Induction of an immune response against G1A The mice are immunized with G1A in different forms according to an identical immunization scheme. The antibody responses induced by the different forms of G1A are compared 28 days after the start of the experiment.

The synthetic G1A peptide administered pure does not induce any immune response even if it is coadministered with Freund's adjuvant. Presented with the carrier KLH, G1A induces a weak response which is significantly increased by the coadministration of Freund's adjuvant Presented with p40, G1A induces a greater response than that obtained in the conventional KLH/G1+AF, p40 immunization scheme to "self-adjuvant carrier" properties.

The results are presented in Figure 1.

Kinetics of the immune response to G1A The mice are immunized with G1A in different 24 forms according to an identical immunization scheme. The antibody responses induced by the different forms of G1A are compared at the times: 7, 17, 28, 35, 42 days after the start of the experiment.

The anti-G1A response is significantly higher and more rapid when the mice are immunized with p40/G1A than the more conventional TT/G1A and KLH/G1A+AF immunizations. A single injection of p40/G1A allows an anti-G1A antibody titer of 1000 to be obtained in 7 days. This titer is obtained with TT/G1A or KLH/G1A+AF in 28 days.

The maximum response (titer 1/380 000), obtained after three injections, in 28 days is approximately 30 times greater than that obtained with KLH/G1A+AF and 70 times greater than that obtained with TT/G1A. The anti-G1A 15 antibody titer holds steady without weakening until day 42.

The results are presented in Figure 2.

0* Kinetics of the immune response to the carrier The mice are immunized with G1A coupled to a 2 carrier according to an identical immunization scheme.

The antibody responses induced by the different carriers are compared at the times 7, 17, 28, 35 and 42 days after the start of the experiment.

The anti-p40 response (titer close to 10,000) is higher than the anti-KLH response but not significantly different to the anti-TT response.

The results are presented in Figure 3.

CONCLUSION

The chemical coupling of the G1A peptide to the p 4 0 protein allowed a significantly more important and more rapid anti-G1A response to be induced than that provoked by the KLH/G1A+AF or TT/G1A reference models.

Coupling of the GIB peptide ought to induce similar responses.

25 Example 7: Evaluation of the protective potential of peptides and of recombinant proteins of glycoprotein G of respiratory syncytial virus (RSV) subgroup A coupled to carrier protein BALB/c mice were immunized with the following different preparations: 1) G1A synthetic peptide coupled to KLH (keyhole limpet hemocyanin) KLH.G1A.

2) G1A synthetic peptide coupled to p40 carrier protein p40.G1A.

3) p40 control alone.

4) Recombinant protein produced in E. coli: BBG2A6C S coupled to p40 carrier protein p40.BBG2A6C.

5) G1A synthetic peptide coupled to tetanus toxin (TT) carrier protein TT.G1A.

6) TT control alone.

7) BB control alone.

8) Long RSV control (subgroup A).

20 The mice received 3 intramuscular doses (200 Ag/mouse) with aluminum hydroxide as adjuvant (used currently in man) The results of the protection tests as well as the immunological profile of the sera are found in Table 2.

The following preparations confer complete protection following challenge with long RSV (strain A): p40.G1A, p40.BBG2A6C, with respect to TT.G1A which also confers very good protection comparable to the peptide KLH.G1A. In the ELISA test, they all recognize RSV antigen with the highest titer for p40.G1A 1/12800.

As for the neutralization test, none of the preparations possess any neutralizing activity in vitro.

2* Protection Recombinant prtisad DICT 5 0 loglO/g lungs Elisa titer Neutralizprtischallenge with long RSV (1.5 X 10 5 /mouse) versus long ation log (subgroup A) RSV 2/25 ys1 56 days 7-8Bdays 2.45 2.45 KLH.G1A 2.45 s2.0±0.4 2.15 s2.0+0.4 (100 to 157 tig) <1.7 P<0.001 <1.7 P<0.001 4000 <1.7 <1.7 <1.7 <1.7 <1.7 <1.7 .G1A <1.7 <1.7±0 <1.7 <1.7±0 12800 (200 p~g) <1.7 P<0.00l <1.7 p<0.O0l <1.7 <1.7 4.7 4.7 controls 4.45 4.5±0.1 4.45 4.5±0.1 300 (200 jsg) 4.45 P<O.O0l 4.45 P<O.O0l 4.45 4.45 <1.7 <1.7 P40.BBG2A6C <1.7 <1.7±0 <1.7 <1.7±0 1700 (200 jig) <1.7 P<O.O0l <1.7 P<O.00l <1.7 L <1.7 Table 2: Protection conferred and immunological profile of the sera after challenge with long RSV following immunization of BALE/c mice with different recombinant proteins. (3-4 weeks after 3 doses i.m. with Altuminumi hydroxide) Protection pecotienand

DICT

5 loglO/g lungs Elisa titer Neutralizproteins challenge with long RSV versus long ation log x 10 5 /mouse) RSV 2/25 ,pl (subgroup A) -6 days 7 8days <1.7 <1.7 TT.GlA <1.7 <1.9±0.3 <1.7 <1.9±0.3 (200 pg) <1.7 p<O.OO1 <1.7 p<O.00l 7200 <1.7 <1.7 2.45 2.45 4.45 4.7 TT controls 4.2 4.2±0.3 4.2 .4.2±0.4 (200 p~g) 4.2 p=0.0 22 4.2 p=0.05 3 250 4.45 4.45 3.7 .3.7 2.95 2.95 BE controls 4.2 3.7±0.5 4.2 3.8±0.5 (200 jig) 3.95 p=0.853 4.2 p=0.760 150 3.7 3.7 3.7 13.7 1 Table 2 (continued) Protection conferred and immunological profile of the sera after challenge with long RSV following immunization of BALB/c mice with different recombinant proteins. (3-4 weeks after 3 doses i.m. with Aluminum hydroxide) 0 Protection Recombinant IT lg/glnsEiattr Nuaiz peptides and sIT 5 ogOglnsEiater euriprtenschallenge with long RSV versus long ation log prtis(1.5 X 10 5 /mouse) RSV 2/25 Al~ (subgroup A) 6 days 7 8 days <1.7 <1.7 Long RSV <1.7 <1.7±0 <1.7 <1.7±0 controls <1.7 p=O.O0l <1.7 P=O.O0l 76800 6.6 <1.7 <1.7 <1.7 3.95 3.95 IControls, 3.95. 4.2 nonimmunized, 3.7 3.7±0.2 3.7 3.8±0.3 150 challenged 3.45 3.45- 3.95 3.95 3.45 Controls, nonimmunized, No virus No virus 150 unchallenged Table 2 (continued) :Protection conferred and immunological profile of the sera after challenge with long RSV following immunization of BALB/c mice with different recombinant proteins. (3-4 weeks after 3 doses i.m. with Aluminum hydroxide) 29 Example 8 Evaluation of the protective potential of peptides of glycoprotein G of respiratory syncytial virus (RSV) subgroup A and subgroup B coupled to KLH.

Protection against a challenge carried out with the two subgroups of RSV.

BALB/c mice were immunized with the following different preparations: 1. C1A synthetic peptide coupled to KLH (keyhole limpet hemocyanin) KLH-G1A 2. GIB synthetic peptide coupled to KLH (keyhole limpet hemocyanin) KLH-GlB. The GIB peptide corresponds to the sequence G (174-187)6Cys of the subgroup B whose sequence is: Ser-Ile-Cys-Gly-Asn-Asn-G In-Leu-Cys-Lys-Ser-Ile-Ser-Lys I s-s_ 15 3. KLH control 4. Long RSV control (subgroup A) 8/60 VRS control (subgroup B) The mice received 3 intramuscular doses (200 gg/mouse) with Freund's adjuvant. The results of the 20 protection tests as well as the immunological profile of the sera are found in Table 3.

The preparation KLH-G1A allows complete protection against RSV subgroup A but not against RSV subgroup B. On the contrary, the preparation KLH-GlB allows complete protection against RSV subgroup B but not against RSV subgroup A. The ELISA test reflects the same situation.

Peptides coupled PROTECTION ELISA titer to KTH DICT 1 0 log 10/g lungs Challenge Challenge Versus Versus 8/60 Long RSV 8/60 RSV long RSV RSV (B) (subgroup A) (subgroup B) X 105/8 (50/pl) 0.6 X 105/s (50/1 41) GlA sl1.8 ±0.3 3.3 n =11 p 0.001 n =10 p =0.237 29 866 266 GlE 3.8 ±0.8 s 2.1±0.5 n =7 P 0.517 n 8 p <0.001 S 100 7 200 KLH control 3.7 0.3 3.4 0.3 n =11 p =0.01 n 10 p 0.6 s 200 133 VRS control s 1.7 ±0 s 1.7 0 n =11 p 0.001 n 11 p 0.001 s 68 266 51 200 VRS control s 1.7 0 s 1.7 0 n =10 p 0.001 n 10 p 0.001 s 76 800 68 266 Table 3: Protection conferred and immunological profile of the sera after challenge with long RS [sic] (subgroup A) or with RS [sic] 8/60 (subgroup B) following immunization of BALE/c mice with the peptides GlA and.GiB.

31 Example 9: Veterinary application Evaluation of the protective potential of GIvAC peptide derived from protein G of the bovine strain of Respiratory Syncytial Virus (RSV) Lerch et al., 1990, J.

Virol. 64:5559 coupled to KLH carrier protein.

174 187 Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Ser His having a disulfide bridge in position 176-182.

The peptide prepared by solid-phase synthesis using Boc chemistry is coupled to KLH using glutaraldehyde (Schaaper et al., Mol. Immunol. (1989) 26:81-85).

2..6 Two calves were immunized by the intramuscular route with 500 Ag of G1vAC-KLH with incomplete Freund's adjuvant 3 times at intervals of 3 weeks. One calf was immunized with KLH without GIVAC peptide and with an incomplete Freund's adjuvant.

The animals are challenged with the Snook strain, 21 days after the last inoculation, by the intranasal and 20 intratracheal route each with lml of virus titrating at 2 x 105/ml.

The virus titrated on calf kidney cells according to the plaque method is determined in nasopharyngeal washings 3 and 2 days respectively after challenge and 7 days in the lungs of the sacrificed animals.

32

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C. CIRCULATING ANTIBODY RESPONSE: Calf 3432 (KLH FIA): loglO ELISA titer Date Treatment Peptide Peptide KLH BRSV KLH (Snook) 23/li Day 0 vaccination 1.0 1.0 1.0 14/12 Day 21 vaccination 1.0 1.0 3.0 04/01 Day 42 vaccination 1.0 1.0 4.7 01/02 Day 70 VRS IN/IT 1.0 1.0 5.7 08/02 Day 77 sacrifice 1.5 1.0 4.8 Calf 3440 (Peptide KLH FIA) loglO ELISA titer Date Treatment Peptide Peptide KLH BRSV KLH (Snook) 23/11 Day 0 vaccination 1.0 1.0 1.0 14/12 Day 21 vaccination 1.6 1.0 1.0 04/01 Day 42 vaccination 3.8 2.6 1.7 1.9 01/02. Day 70 VRS IN/IT 2.7 2.8 2.6 3.7 08/02 Day 77 sacrifice 4.1 2.6 1.7 3.1 Calves to which 500 yg of GivAC KLHen incomplete Freund's adjuvant was [sic] administered on three occasions at 3 week intervals.

RESPONSE TO THE VIRUS CHALLENGE Nasopharyngeal Day 7 widening pulmonary Calves Vaccina- No. of max. LBA Lung %pneumtion days titer titer hom- oniae (pfu/- 0g.

ml)_ 3432 KLH+qFIA 3 5 .13X10 3 1.4x,0 2 3/3 12 3440 peptide 2 5.5SX10 2 <0.7 0/3 <1 KLH+FIA III RKPLACMUMT SEET (RULE 26) 33 CIRCULATING ANTIBODY RESPONSE loglO ELISA titer (Snook BRSV) Calves vaccination Day 0 Day 24 Day 42 Day 68 Day 4138 KLH FIA 1.5 1.5 1.5 1.5 2.4 410 *Peptide KLH <1.5 <1.5 3.0 2.5 2.9 4140 FIA *Calf to which 500 jug of BP 4006 KLH in incomplete Freund's adjuvant was [sic] administered on three occasions at three week intervals.

RESPONSE TO THE VIRUS CHALLENGE Nasopharyngeal Day 7 widening pulmonary virus Calves Vaccina- No. of max. LEA Lung pneumtion days titer titer horn- oniae (pfu/- og.

ml)_ 0 4138 KLH+FIA 5 4x10 1 6.5x102 2/3 27 4140 peptide 4 2X10 3 7 .Ox10 1 3/3 2 KLH+FIA 04.@ 4* 9*

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SHEET (RULE 26) 34 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

(A)

(B)

(C)

(E)

(F)

NAME: PIERRE FABRE STREET: 17, AVENUE CITY: CASTRES COUNTRY: FRANCE POSTAL CODE: 81106

MEDICAMENT

JEAN MOULIN AGENT, PHARMACEUTICAL COMPOSITION AND PREPARATION PROCESS.

(iii) NUMBER OF SEQUENCES: (iv) COM4PUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS

C.

*7.

ft.

(vi) DATA

(A)

(B)

OF THE LAST APPLICATION: NUMBER OF THE APPLICATION: FR 94 04009 FILING DATE: APRIL 06 1994 Information for SEQ ID NO: 1 G2A 20 SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 101 amino acids, 303 nucleotides STRANDEDNESS: single TOPOLOGY: linear

I.

MOLECULE TYPE: protein 130 N 'D Val Lys 7h Lys Asn fw M~ flW M=r Cn M= l~ n Pro Ser 4's P=o 7r Mlr 4 A= GMG AA AM AJ'A APC AM A= AM G A= Ca XC AAA CM X= A= AP aln krg M Amo 1.)s Po Pzo Am~ 4's Pro An Am AV~ Pe His Rhe Glu Val. Rn Asn Pte G G AAC APA CM AAC MA CC A;C AAC GT TIC OT TIC GIA GMG TIC APC TIC 171 173 1"7613 6 %tl P=o Cys S= le Cys Ser Asx Asn Pro *flr Cys 'Zp Ala Il~e Cys LYS Akg le PMo Am GIm a a A= A XC A~ caG AC 7mC 7w 1G Alc mA anr Apc 192 4'.s Lys pro Caly 45, Ly's Jh. M- 'flr 4s Pro 2x 1)5 L's PrD Mx Re m' fL Mr Mw Ls ApA AMA aX arAA AM XC ).10 XC MA M AM AAA AM CM TIC A AC AX AA 213 230 Lys Asp His .,Is Pro aln flw flw 4's Pr 14' MuI Val Pio M- "flr 1s Pro C pA GC =T MA cm A= AMA CMA GA GIG CMA ACC AA M 31 35 a a. a. Information for SEQ ID NO: 2 G2B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 101 amino acids, 303 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 130 N M-1Ala Cnn MWr Ls Gy Arg le Mw M= Ser M= Gin T Mr AME Ls Pro Se Dr 'Z I S- AT m G ~cCu pi AA or AC .e .ur rr TG C APC A 7Gi YrATP AAA 150 Se Ag Se Ls AmPi Pro Pro Ls Ls Pro Lys Asp Asp Iyr is Fe au al Re Am Ph P PX AA APC APA APA CZ A14A GAT GC CC GIG TIC CPbC TIC 171 173 176 2M 156 %t1 Pro Cys Ser nle Cys aly Am As Gn Ll CS L5 Se le Cs 14s frw ne Pro Ser GI Xr AM ar- AAC APC C G IG 7W AtA A= 7M AAA AM CM ;GC 192 Am 14S Pro 14s 14s Ls Pr M lie Ls Pro w AmI LW Pro 11,x L s 11f lM* Am A;C A;A anA ApG AA CM p= ;CC APA CM; P= APC AAA w AA AMA A= ;C AbIC 230 21323 1's Ag AV Pro 14s M= Pro Ala Ls Mtt Pro 14's 4s a~u lie ne fr Asn C ;AA r GT X a O GM AbAk A C AAPG AG GCA Auc PX APC 3 Information for SEQ ID NO: 3 G2A6Cys SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 101 amino acids, 303 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 1.30 N V al 14s 1 An m= l 7hr M Gl n Sw Gin PRl Ser 1W Pro D-W 11W Ls GIG APA XC AAA AbC A PCC M A C* 7 A= m PA=AA 36 150 Mn -g Can Am ys Pro Pro Am Ls Pro Am Asm Awam Pais Re alu vt Re AM Re aPG ar CG AAC AAA an AC AAA CM APC AC TIC T TIC CA GG TIC AC TIC 171 173 176 1am 186 Val Pro Sr Sr ne Cis Sm Am Am Pro Ph Cys Tzp Ala lie Ser Lys Arg fe Pro As, ;CC A7 C 7W PXL APC AP^C M A O f C CC 7W GM A PX AAA a APC 132 14s I. Pro miy L?,s 16s M x M= 71 Ls Pro Mi Lys 14s Pro D R Lys T= 72 4S AA APA C AA AaA ACC AM AAA CM O AAA APA TIC AA PXa A= APA 213 230 I.s Asp His Lys Pro Cn Mr M- Lys Pro 14's alu Val Pro M= Mr Ls Pro C AA 7T AACC ACAAA= AAA @AA GIG r PC AA CM 31 Information for SEQ ID NO: 4 G2B6Cys SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 101 amino acids, 303 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 130 1N Ala Gin M= Lys My Arg ne M= M= Ser Gin Ma Am Ls Pro Se '2=ry x= a; G AM AAA G I A PCC PX A= C1 X= APC AAA CM XC P= APA ,*150 Ser krg Ser Ls Am PRo Pro 16s 14s P= s Asp Asp Tyr is P H Mu 1al Re Am~ Re Xr X C ADA A AA AAA aX; AAA TX MC CPC T @PA GMG TIC XCT 171 173 176 1B2 156 Val Pro Ser Ser lie Cys aly Asi Am Gin Leu Cs Lys Ser lie Ser 1s L l ne Pro Ser GIM Cm xr ArC A AC A UG CG 7W AP A;XC A= WAA= m aC Am 192 Am 14s Pro 1's 14s 14s Pro l lie Lys Pro 1w As Ls Pro It Mw4s M- x T=r AAC AAA ADA APG AAA G A A CM ;M APC AAA CI X A X= A PX APC 213 230 Lys krg AV Pro Ls M= Pro Ala s Vt1 Pro 14s Lys cu lie lie w Am C AAA GAT Ca AAA A= G AA APG AG GA A=C ICC X= APC 31 -37 Information for SEQ ID NO: 5 GlACys SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 14 amino acids, 42 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 176 182 186 187 N Ser le Cys Se- Am~ Asn Pro Mw Cys Tp Al' fle C~s Lys C A A7C hr APC APC ar AM 7W 7MG APA 3 Information for SEQ ID NO: 6 GlECys SEUEC TYE.*ioais n uloie SEQUENCE TYET: 1 amino acids, nd nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 1.76 im 196 187 N ser le Cys CZ~y As Asa Can leu C Lys Sr fle C~s L5 s C AX- 7W G=AAC (3G C7G APA XC XC= -3 Information for SEQ ID NO: 7 GIA SEQUENCE TYPE: amino acids and nucleotiLdes SEQUENCE LENGTH: 14 amino acids, 42 nucleotides STRANDEDNESS: single TOPOLOGY: linear 38 MOLECULE TYPE: peptide 174 176 182 186 187 N Ser le Cys Se Asi Asn Pro CyS T~p Ala le S= L~rs C 51 PXAM7WP APC AC GC A7C AMA 31 Information for SEQ ID NO: 8 GiB SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 14 amino acids, 42 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOECL TYE:pptd 99.SEMOECE TYPE: aemideacd 174 176 22 186 157 N Sr Ile Aso SIe An As n T Cms Lws Ala le Cs- lys C -39- Information for SEQ ID NO: 10 Gl'E SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 14 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 176 182 196 187 :N Ser e Asp My Am Am Gan LIB C' L~js Ser Ile Cys L C Information for SEQ ID NO: 11 Gl'A6C 0 edb *SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 14 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 176 18B2 196 187 N Sr 32e. Asp Ser As Asn Pxo M= O' -p Ala .le Sc- 1~s C Information for SEQ ID NO: 12 G1B6C! SEQUENCE TYPE: amino acids LENGTH: 14 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 176 132 18W 2B7 N Se lie Asp Cay Asn Asi Gan Leu (zi Lys Ser Ile Se I. C Information for SEQ ID NO: 13 SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 335 amino acids, 1005 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 0o N -Ala Pro Liys Asp Am M- L-Tp Tyr Ala Gly Gly Ls Leu aly Tp Se- 00 17 Co Can 7w His Asp m= My P Tyr Gy Am My Ft n As Am As My Pro C;G C;C GC A= TIC VC =rAPC fC ArC APC AAC= CG io. 7r- Arg Asm As o n Leu Gly Ala Cay Ala Re My ly Ty r n Val Asi Pro P= r APC GC UG r (r G= M r Gd VC C G Grr A C 53 7 Leu My Rm Glu m- Gly Tyr Asp f-p Leu Gay Arg Mt Ala Tyr Lb's aly :c cic TI c tA Au ar mc ?D =C cG G Am mkT A~AkOX 71 Val Asp Aw G y Ala Re L4 Ala Mn Cay Val Gln 1eai fl- Ala L,'s LeI ;X Grr 7C AAC GGr G= TIC AAA CPG cGrr CU; G PC e r APA CU Cay Tyr Pro Ile Aso Asp tu Asp 31e 7w f2r Arg Lei MIy y t Val Ar 7c AC GC GT CU GC C VC ;a M MC CU PG Grr ****107 Arg Ala Asp Se ys Mly Am Tyr Ala Ser -11 .xly Val Ser Axg Sr Gi 7M GT G;C AAA GGC APC MC r XC G or MT =I C T 125 His Asp mi ly vAl Ser Pro Val RE Ala Cy My Val Mu Trp Ala Val Ma CP C o Ar rr Ga P= MCrr Car GM A G =a Wr GT P= .14.3 kig A Ile Ala 7r: kg Lex iu 7r Mn Mrp Vhl Asn As le Gly Asp Ala CGT G A7C GT AM C r CM G A WC C;G 7 =r APC APC CC GC 161 My Va -l My M- Arg Pr Asp Asn MLy fft Le Ser lax My Val Ser Ty- Ca: A= GIG Gr X r Mr G' APC CE PG C GC aG CIG0T =TM 179 Arg Re My Gan Gu Asp Ala Ala Pro Val Val Ala Pro Ala Pro Ala Pro Ala DG TIC GA OK T GC CIT Grr cr- r G= ar CEG cm a' 41 197 Pro Mu Val Ala M= Ls His P-e Mr Laf LS SFer -A Val Leu Re Am Fte cmGA GmG GIA CC TIC AT IG NC aTC @C AIT CI TIC C TIC 215 AMn Ls Ala 7 Lea Ls Pro aLu Cay Mn Cn Ala Lai Am Mn La I rr C NA G' A= CM AA CPA aIT CCG CCG Gc GG aG GTC G C MC 233 Mn aIx Ser Asi a't Asp Pro bi. As ply Se Ala Val Val Leu Cay Tyr -L CC C1G AX NC =T CC an A CC 7M rr CmT Cu G C AT 251 Asp Arg le CGy S= Gu Ala Tr Am Gn Cn Lax Ser Gau L js Arg Ala Cmn CC A T GA M C APC CtC; CPcr CC NPA ar cr CC 269 Ser tVl Val Asp r Lax Val Ala Lxs Cy ne Pro Ala Cay Ls Ile Ser Ala iw CiT rrC TC aCI G=r AAA =AC CA CA GA C 287 Akg Cy t Cy cu Sew Am Pro Val M= Cy Am T= Cys Asp As Val Ls A c=i= CGAA 7=FC C CT x= a: XC AC= rG CAC APC GIG NA 305 Ala Axg Ala Ala Lwn fle Asp Cs Lax Ala Pro ASI Arg Arg Val Mu fle alu CIC G G= C aC AT C CG =U CU GkT Cr AC CA 323 335 Val ,ys Cay Tyr Ls Gu Vl Val G Cmn Pro Ala cy 7M C CT NAP C MC AAA CA GiT G A CC C C T MA 3' Information for SEQ ID NO: 14 G2A6CF SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 101 amino acids, 303 nucleotides 0 .0 0 STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 130 N M= Val Lys 7r lys An PZ Mw Mi Ga n M- Can Pro Ser Ls Pro Mx frD Ls AT GIG AA AT AAA C C= A T A A=C XT CG CX AT A A= ;C CAA 150 10 165 168 170 Gan kgan Am Lys Pro Pro Asn LW Pro Asn An Asp Ser Ms Ser Cau Val Ser Asn Ser CG CG NAC AAA cc anU AC AA CU AAC C GX 1 C= 01= A GIG n NC TIC 171 173 176 1Z 16 val PRoSer Ser Ile Cys Ser Asn Am Pro M=r Cys rp Ala ne Se kg Ile Pro Ami GI C ;M T AT 'C 7WAC AC CM AC GM 'I CU AT AAA CTC Ca; APC 192 I6s 16s Pro My L6s Lay5s M= fl= L Is Pro Ly Ds Lys Pro 'Dr Fe Lbs fr= flw Lys APA AA al C AP AA AT AM AT NPA CG ;a AAA AAA CM ATC TC AAA A=T AT A 42 22230 I. 's His L~s P=o Mn M= M= Ij;'s Pmo Lys Mu Pr-o U= Ls P=o C AAAT CAT APA PGA AM AX; CAA GIG CMAMP AA -3 Information for SEQ ID NO: 15 G4A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 17 amino acids, 42 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 1'76 121661B7 N %tl Pro Cys Ser le Cys Ser Asn Asn Pro M= qys TTp Ala le Cys5 Lys C Information for SEQ ID NO: 16 G4A6C SEQUENCE TYPE: amino acids and nucleotides 0 SEQUENCE LENGTH: 17 amino acids, 42 nucleotides *.0 STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 186 187 N Val Pro Ser Sr le Cys SL- Am Am Pzo Mlr Cys -frp Aia lie Ser D's C MCG; G WA P AC=A MG =X P 31 -43- Informiation for SEQ ID NO: 17 G4B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 17 amino acids, 42 nucleotides STRA2NDEDNESS: single linear MOLECULE TYPE: peptide 171 173 176 182 1S6 187 N Val P=o Cys Sex fle C:ys aly Asn Asn an 12u~ Cys Lys Se-nIe Cys D~s C a C7 P CM AAA C A A- 31 Information for SEQ ID NO: 18 G4B6C SEQUENCE TYPE: amino acids and nucleotides *SEQUENCE LENGTH: 17 amino acids, 42 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide :17. 173 176 182 166 187 NJ~ Pro SL-- Ser Ile Cys aLy Am~ Am an Lai Cys Ly's Ser lie Ser Ls C Information for SEQ ID NO: 19 G4'A SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 17 amino acids STRANqDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 186 197 N Val Pro Asp Ser fle As se Asa Am Pro flr Cmi 'fp Ala flae an Lys -c -44- Information for SEQ ID NO: 20 SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 17 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide- 171 173 176 182 186 187 N Val Pzo Ser ser lie As e Am Asn Po Cm 71p Ala lie s= Lys -c Information for SEQ ID NO: 21 G4'B SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 17 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide *171 173 176 182 186 187 Val Pro As Ser lie Ap Cly Ami Asn (nn Leu QM Ly's Ser lie QM Ly's -C Information for SEQ ID NO: 22 G4'B6C SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 17 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 1S2 186 187 N Val Pro Ser SL---fle Asp Gly Asn Asn Can Lai QM Lys Ser le Ser 14s C Information for SEQ ID NO: 23 G200A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 61 amino acids, 183 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N a.n Mn Pro Sr I3s Pro M M I4s Gan Az gn Asn L5 Po Pro Aa -P G AM CG AZA CC Xr PA CP G D A-C A C a Ac 158 173 176 Lys Pro Asn Asa Aso Re His Pe Glu Val Pie Asn Pte Val Pzo Cys S= ne C~s APA CG APC C T TIGC =AT TIC GPA TC CNC 7W AM AIC 7 177 lB 186 S= Am An P= Mw CTp Ala Cy Lys Axg Ile Pro Asn s L s Pro Cy XZ AC cUai; A= 7m a AC U AAA cmCA== APC AA A C;G 196 200 Lys 1 s MW M= 7rx C AAA ADA AMXI; A= 3' Information for SEQ ID NO: 24 G198A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 59 amino acids, 177 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Gn M= C~n Pro S= Ls P=o M= 7r Lys aln Arg cn As Ls Pro pro Asi G~ A= C; C MA =U AM ACC AAA CWr OG AC AAA C APC 158 173 176 1ys Pro As Am Asp Re His Pe Gn Val fl- Asn Re Val Pro Cys S= nle Cys APA C AC NC =CA~ TI= (A GIG T1C NPC TIC GIG =C W ;WAC A7 177 22 186 Se Asn Am Pro flr Cys fp Ala Ile Cys Ly kg Ile Pro Am 14s Lys Pro Mly An CU A= aC C nA =U Ai CU N NA Ap A C G 196 196 1s I,'s flr C AAPA AfY 3' -46 Information for SEQ ID NO: 25 G196A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 57 amino acids, 171 nucleotides STRANDEDNESS: single linear MOLECULE TYPE: protein 140 N n 'DL Gan Pro Ser ljs Pmo M= f= Js Gan Arg Mn Am~ L~s pro Pro As 158 173 176 .Is Pro Am~ Ami Am Pip- His Fjj au Nt1 pi As 1Ro Cys Ser Ie Cys AAA Ac A~c Gi 0;1 =T TICA GMU =I Atc TIc =I ax 7w; A=C ~177 l 8 Sa .w sn Pro Mi oys .rp Ala fle Cys 4s Akg fle Pro Ami 4s L~s Pro Gly 196 *Lys C Information for SEQ ID NO: 26 G19.4A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 55 amino acids, 165 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Gan Mwu Can Pro Ser Lbjs Pro M= 2r Lys can Arg Gin Asni L~s Pro Pro ps- =X AAA AM AMA Ak CG Cr -P AAA C_ AAC 15 1773 176 Lys Pro As' As Asp Re Is Pe Cu %IReAs Phe %l Pro Cys Ser 3ae Cys AAACM AC AC T C TC 4A GIG =I APCC GIG C± WXCX G 177 2B2 3B6 194 SF-.s Am Pro Tw Cys Mfrp Ala fle cys 1bs kg fle Pin Asn Lys 15s pro C ;X AP =A =7MG 77X ArAC AIC CM PC AAA APA CG- 3' 47 Information for SEQ ID NO: 27 G192A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 52 amino acids, 156 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N G~n M= Gan P= Ser Ls Pro 7 k=T Lys Gn Arg Gin Am Lys Pro Pro A DG AM CPG =C APA A= PCC APA a a APC AAA Ax 158 173 176 Ly Pro Asn AsAs P e His FReaiuu l Re As Rn V1 Pro Cs se fle Cqs S177 322 186 132 Sr As-I Am- Pro 7r Cys frp Ala fle Cys Ls Axg fe Pro Am Lys C AM qZ AZ C 70 =GM A7C 7W APA aT F AAC AA 3 Information for SEQ ID NO: 28 G6A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 51 amino acids, 153 nucleotides 0 0010 STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Can h Gin Pro S ys Pro M= T= Lys Gin Azg can As- Lys Pro Pro Asn C43 AC C;G ;C AAAA =ACr AMCAG AC AAA MZ; AAC 158 173 176 Lys Pro Asm Asm Asp R His Phe Gau val nx Am- Re %l Pro Cs Se fle Cys AM AAC AC TIC TC GIG TIC AC TIC GIG CM =X mC I= 177 1m 186 190 Ser Am Asm Pro ft Cys frp Ala fle Cys I, sg fle Pro C WC AJC CM AM A2O C GMA= AP AT C 3' 48 Information for SEQ ID NO: 29 G7A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 33 amino acids, 99 nucleotides STRANDfEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein lw 173 N 14,s Pro Asi AmAsp R-e His Phe Mun %I fl AMi Re Val Pro Cys Se lie APA CM AAC AAC CA TIC T TIC @PA G-IG TIC APC =I GIG CM PG C S1*76 182 186 190 :::Cys Se s s iPro Mwys cp Alanle Cys D,s Arg fle Pro C 7G A A~c A~C CUr x==7MG A7C =I A aCU AC 3 Information for SEQ ID NO: 30 G200AbC *~.SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 61 amino acids, 183 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Can 7h aln Pro Sez- Lys Pro Mir *flr Lys Can Axg Gn A~x LYS Pro Pro Asn 7 CM ;X AA ax; AmAC AAA C;G CGT C; AAC AA =U CI;AP 158 173 176 L ,s Pro Ami Am~ A Re His Re Mun Val Phe Asi Pte Val P=o Sr Ser lie Cys AA ax; Apc Apc G.TTI Tic G;A GiG T= APC =I GIm ax; ;m ArC 7W 1712 2.86 Ser Asx Asi Pro Cys .fp Ala le Ser Ly's Azg Ile Pro Asn P=o My ;XAC APC CaUAC w7 ax; ;GC AAA aU ACU C AA AA =G 196 200 Ily~s flr MW~ w C -49 Information for SEQ ID NO: 31 Gl98A6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 59 amino acids, 177 nucleotides STRANDEDNESS: single linear MOLECULE TYPE: protein 140 4.N N- Mn Mwn Gan P=o Ser Lys P=o M= MI L~.s Can rg Can Ami bys pmo p= Am C;G A= (7G an .G APA A= A APA c car cG Ap AAA an= ***158 173 176

L?

7 s Pro Asi As Ap P His El Gu Val Fir- Asi pR J val pro Sce Ser le Cys .9..CC A TC AA X=O TIC G A GM~ TIC I GM~ PXA AMCI 06*177 3M 1B6 0~A Am S~sAmPro *Z Cys frp Ala lie SL- L ,s Axg fle p=o xc~i I.s L 's p=o a~y xr AA= APC aZ Am Ma mX AAA cr ~r Apc Am m Information for SEQ ID NO: 32 G196A6C SEQUENCE TYPE: amino acids and nucleotides .**SEQUENCE LENGTH: 57 amino acids, 171 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 14D N Mn M=Cn Pro Ser Lys Pzo M= 7hr Lys Mn Arg Mn Asn Ls Po Pro Am z'cr PX AA CC;A MAA rCGAp p 50 9 I.

e* *9 158 173 176 bys Pro Am )s Am FIP His R e.u Val RP Am RE l Pm SeL Ser Lie Cys A C AAC AAC GT t 3 T TIC GA GMT T1C AP TC GMG -P A;CC C iXA 177 1m 186 Ser As AX Pro M-w Cys 7p Aa Lie Se rixs -g nle Pro Am L s PRo aLy AMC AC _PC ACE p Z C AAA CM AP C A k A C 196 ls- C AA -3 Information for SEQ ID NO: 33 Gl94A6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 55 amino acids, 165 nucleotides STRANDEDNESS: single 5 TOPOLOGY: linear MOLECULE TYPE: protein 140 N Gn Mx Gin Pro SL- Lys Pro M M= Ls Gin Ag cn Asn Ls Pro Pro Asi DG-A= OG =C PXAA X= =A CG =r CG APC A4A CM APX 158 173 176 19 Pro Ami Am Am- ER His Bk niu Val Phe Re Val Pro Ser S le Cs AAA APC APC GC TIC 1 TIC GDA GIG TC AC TIC GIG A AMC 177 1S2 1B6 1% Ser Am Am Pro w Cs Mrp Ala nle L.s Axg Ile Pro An 14s Lys Pro C AM APIC A P= 7M GM A C Xr APA Cr A7C (11 APC AWh APA CM 31 Information for SEQ ID NO: 34 G192AbC SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 52 amino acids, 156 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Can 7r .Gn Pro Se l. Pro -2 Mw Lys Wn kxg aln Am 1&s Pro Pro Asi CAG A= C .AG AAA X= A AFk G DGAP AA r; AAC 51

IS

C

158 173 176 iys Pro Asm Am Av a eis Pi -p 1 a P Ami 2a Pro S= S rle C~s AAA CM APX A7C GA TI C CPA GIG T C TC GIG AC CM ;X PC 177 1S 156 152 Ser Am As Pro flw Cs Mfp Ala ne S Lys kg ne Pro Asm 4ys C AC APC CM A= 7 GI =Pa AAA C;r A= CM APC AA 3' Information for SEQ ID NO: 35 G6A6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 50 amino acids, 150 nucleotides STRANDEDNESS: single 5 TOPOLOGY: linear MOLECULE TYPE: protein 140 N Qn M= Qn Pro SIys Pro T= Lys GIn Azg GIn Asn 1s Pro Pro Asn CPG AM:_CC CAM AN CG A= PC AAA GG Cr.T GC APC AAA 0Z APC 158 173 176 Lys Pro Asi Asn Asp R His me G\u v1 Phe A9. ne val Pro S S lie C~s AM A APC T TC G= TIC @GA GIM TIC AC TIC G= CG a AX p 17 182 156 190 sce Am Am Pro w Cys Mcp Ala lie Ser Lys Arg Le Pro C Information for SEQ ID NO: 36 G7A6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 33 amino acids, 99 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 158 173 N Lys Pro Asn Asn Asp Phe His R eu Vl PT' Asn Pe Vl Pro s= S= le AAA APC AAC GAT TIC C7X TIC GA4 GIG TIC AAC TXC GIG CM 3C C C. C.

C.

52 176 1i 186 q~s Ser Am Asn Pzo hr Cys rp Ala Ile Se- 4s Arg le Po C Am AC A C AM 7W 7Wr A 7 AA t Information for SEQ ID NO: 37 G200B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 61 amino acids, 183 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N e rr in Mw Asn Ls Pro M Lys S Ag Ser Ls Asn Pro Pro 1ys Ls Pro NE A= CG A= ApC AAA CC 3 .X AC AA .k AMr M AAA C C AAA A C 160 173 176 s sp Asp Tyr His RrE Mu Val R-e sn Pe Vl Pro Cys Ser fe Cys My Asn Asn Gin PA G CGCT 7C C TC GkGM=I TIC C TI 7 WCC A7C N AC P CPG 186 200 Lau Cys Ls Se lie Cs Lys M= lie Pro Ser Am Lys Pro Lys L~s Ls Pro M= Ile- C CIG APA GC AAA P= AC APC AAk CM APA AG AA AM X C- 3' Information for SEQ ID NO: 38 G198B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 59 amino acids, 177 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Ser M= Gin Mlw Asn Lys Pmo Se r= Lys Ser Azg S= Dys Asn Pro Pro 1.o L Ys Pro C P G C AC APA AAA C AEX= AAA r AAA APC CM A APA C 160 173 176 Lys Asp Asp yr His Pe Glu Vl A Asn Fl tVa Pro Cys Ser Ile Cs My. -n Ai Gin AAA G;C GAT 7;C C;C I GPA TIX NC =I eG NC NCC 7W OA A im 186 198 iaw Cs Lys se lie Cys Lys M Ile Pro Sr Am~ L s Pzo I ,s Ls Lys Pro C CM 7X AAA ;M AA A CMC ;GC AC APA C APAA. APA C 31 53 Information for SEQ ID NO: 39 G196B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 57 amino acids, 171 nucleotides STRA±NDEDNESS: single S TOPOLOGY: linear MOLECULE TYPE: protein 140 N Ser n M= Am Lys Pro Sr Mw L~s A Axg Ser- L3,s Am, PMo P=o L)JS Ds PMo 60173 176 1 s sp sp yT is tv- MuV~i Ph AmPhela PmCys Se= fle ys aly Asn m a ~~AAA GC~ GAZ JJL. CPL. =LL b4A LiLG =LL iAL. 7M.. AM~ A7C G APC AAC CC Im 2M LB 96 **lau Cys 1~s Ser fle Cys Lys mi fle Pro Se Asn Ls Pro Lys 1 s C 7C C AAA Xr A7C 71 APA XC A= ax J~ APC AN A1A P 3 Information for SEQ ID NO: 40 G194B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 55 amnino acids, 165 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Ser 7lr Can Asn bs Pro Ser fl= Lys L~s Asn Pro Pro 4ys 4s Pro :Ar O AM AP AAA CM PG PX AAA r =z A~ Pc; A 160 173 176 1LW Asp Asp Iyr His Am Mu Val Phe As Pe a Pro Cys S= Ile Cys Mfly An Asn Can APA GA T C7 =i GA GM T= APC =TG CC 7W PM GOC AC APC OG Im186 194 Leu Cys Lys ser fle Cys I ,s fw Ile Pro Ser Asn L.ys Pro C CM AAA XC A7C APA ACE A7 CM AX AX AAA~ C( 3' 54- Information for SEQ ID NO: 41 G192B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 53 amino acids, 159 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Ser 7h Can -n Ami I.s P=o Ser M= Lys Ser Azg SLer L~s Ami Pro Pro Lys L~s Pro F- XC ;XCGA cAAC r A= A.h PM=r AA AA __PAA a 160 1773 176 Lys Asp Asp Tyr His Re MVa AaRzalPC~s se-- le Cys Miy As snM A* G% GA P CC=GA M=AC TIC GMC Ar A7C aX AP 18 26 192 *Leu cys L~s ser lie C)ys I. s 7h Il~e Pro Ser Am C Information for SEQ ID NO: 42 G6B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 51 amino acids, 153 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Ser 7h Gan 7hr Asn t Pro Ser 7h Lys Ser Arg Ser Lys Asx Pro Pro Lyqs 1lbs Pro IO173 176 Lys As Asp 7y His he Mun Val Re Am Phe 'Al P=o C~s Ser le Cys Gly Asn An Gin AA GC G C C TIC G A GIG T= APC TIC GMt CC 7= ATC GM APC APC CPG 32186 190 Le Cys I Se 33.e C~s Lys m= fe Pro C 55 Information for SEQ ID NO: 43 G7B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 33 amino acids, 99 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 158 173 176 N 14's Pro LVs A Asp 'lyr His Re Gu Wl RE Am Re Val Pro cys Ser Ile Cys Gly AMA ca; MA GAT W VC DC TIC Gkk GIG T=C APC TC GIG AC *I 182 186 l9O Awx Am Cn Lai Cys Lbs Ser le C)'s 1's m=r lie Pro C APC APC CPG AA?=r u IA AM A7# 7W AG 31 Information for SEQ ID NO: 44 G200B6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 61 amino acids, 183 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Se flM Gn 7r Asi LW Pro S flh Lys Ser Arg Ser 14s A Pro Pro 14's D's Pro p0 Xr CG X= APC APA =G ;X AWI C A APA APC CM CM A AM 160 173 176 1w Asp Asp Tyr His e au vil Pe Am n val Pro Ser Ser ne Cys Gly Am Am Gin MA GC GaT C TC A IG TIC PC TI GI 7 IP C APC APC OG 182 16 200 Ieu Cs 14s Ser ne Ser 14s s wle Pro Ser Am Lys Pro Lys Ls Ls Pro 2 wl e- C CIG A Mr ACC AM AA XC Ma CCG KC AAC AAA AAA APG MDA anG A 3' -56- Information for SEQ ID NO: 45 Gl98B6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 59 amino acids, 177 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 *160 173 176 Ds Asp Asp Tyr His Ri Miu Val e Am~ Phe~1 P=o ser sr fe Cyjs GLy Am Asi Gin AAA GT GAT VC CAC TI @A GIM =I NC CM =i XE PM=7 N-C OC C;G 18 1B6 196 Lai cys Lys ser fe Ser 1 s -2w nle Pro Ser Asn LWs Pxo L~s Lys L6,s Pro C CIG =I AZA ;?CrC APA A= A= C X3 AC AAA aZ APAAP AA A CM -3 Information for SEQ ID NO: 46 G198B6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 57 amino acids, 171 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Ser 'D Gn 7h Asn Lys Pro Sefr MWLy Ser Azg Ser Lyjs Asn Pro Pro 1 ,s 1. Pro A= A=D N=AC AP CM ;X AMA r D P C X A P 160 173 176 LsAspAs Tyr HiS Rv- Gu Wl Rr Am Ph Val P=o Ser S=r Ile Cys GIly Asn Asn Gin AAA GC GCM C GA GIG T= AC TIC G7G=; Am mc 7w aZ AC APC CPG lm. l86 196 Lau )s lys Ser fle Ser 1,,s Tw fle Pro Ser Am DIs Pro I s L4s C Cm AAA ;C C MAA ACE Xt Ca; AMC AAM NAA AP -3 57 Information for SEQ ID NO: 47 G194B6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 55 amino acids, 165 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Se 'Dx Gin Mw Am L Pro S M= Lys Ser Arg Ser bjs Am Pro Pro Ls L6s Pro AM ACXCF AM APC AAN =3G IC P AM AA k ;M ;m ApA Ap Ca al AAA AA M 160173 176 L z Psp T1 His Phe Gau Vl he As Re il Pro Ser Se- le Cs Cly Asn As Gn AA T GATGC c TIC ;A GMG T= AAC TIC GIG =PX A A7C aC 3 APC APC CG 216 _1 lea Cys Lys er le SerLs M wle Pro Ser Am Lys Pro C CM1= AA. ;IC PaA Am C G p'C AAA cco 3 Information for SEQ ID NO: 48 G192B6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 53 amino acids, 159 nucleotides 10 STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Ser hr Mn M As1 Lys Pxo Ser M =Ls Ser A19 Ser Lbs AI Po Pro Ls Lys Pro ;GC A= AG AAC AAk CM ;X A Ar~k P= C d Mr ApA ApC C ApPA ApA03 160 173 176 Ls Asp Asp 7r His heCu %I Re Asn RPe Val Pro Ser Se -le Cys Cy Asa sn MGn AAA GC GC MC CC =I GA GG TIC MA TIC GIG cc ;X ;M =C ~AApC 010 186 132I ei Czs 1ys Ser 3le e 1s fr lie Pro Ser As C CM AM XC AM AAA AC XC AA 3' 58 S.

S.

S. S

S..

4' S.

C.

S

S

S Information for SEQ ID NO: 49 G6B6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 51 amino acids, 153 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Ser M= Gan MWr Ami Lys PRo Set rh Lys S Axg S= Lys Am Pro Pro Ls Ls Pro A= C;G A= AAC AA JA CG ;CC X AA PX CGr aM AAA AA CM A A C; 160 173 176 Ivs Psp Ap Ty His PR GiCu Vl Phe An Pte Val Pro Ser SL Lie Cys Gly Asn Asn (in APA GAT WL CXC 4A G TIC AAC T=C GI G C M; Q APC AC ?G Im 186 190 Lai Cys Lys Ser Lie s. iL-s m fl e Ro C A=IG 1A PA XC A PG A C CCZ 3' Information for SEQ ID NO: 50 G7B6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 33 amino acids, 99 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 158 173 176 N Lys Pro ys Asp Asp 7yr His RE Gu Val Re Asn RE Val Pzo Ser Ser le Cys Miy A~k CCC AAA GAT G W CC TIC GA GIG TIC A TIC IU GM AC= =0cZC m 16 190 Ami Asn Gn Lu Cys L 's Ser 32e Ser 1As m wle Pro C AAC APAC GG CIG 7M A~h ;M 7C JAC APA AM XC =3 3'

S

-59- Information for SEQ ID NO: 51 G2V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 101 amino acids, 303 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 1.30 N Cnn A&si Ag L, fle LYS aY Cn Ser M- Le. Pr-o Ala Mir kg Lys Pzo P=o le An M Aa ;G AAA A7C Ah Wr MA 7M AM Crk CM G AA% CM~ AT AAT 150 Pzo Se Gly SL- Ile Pro P=o aLu Asn His Gin Asp His Asn Am pre Can M= Li pro T *172. 173 176 122 186 a' l Pro ser m= cy cay Asni Isi Ala Cys Lei ser Leu Cys xis lie cfn fw M~u a' g Ala P=o SL- Ag Ala Pro Mw Ile Mw Lim 14s Lys fnr- pro 14s pro Ly's jlw mm LY'S *a ma;L x;ka a_ PC .ATC C AA AP Ama AAACAA Xr XA _A 213 230 Lys Pro frhr 14's 7h M- le His His krg M= Se Pro Cau M- 14's La.i aln C Information for SEQ ID NO: 52 G2V6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 101 amino acids, 303 nucleotides STRANqDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 130 N an Asn kg 14s lie 1's Gly Can SerM 1E~ ax o Ala M~ Ak~ LW' pzo Pxo lie Asn CA AA AN AA AC AAA Mr CP 70k A~k C Co N M;kAA Mk MkI- MT

I

150 Pro SL- my S= nle PMo Pro Gu As His Gin Asp His Asi An hp Gin M= Lax Pro 7 ZA 7M AC M A GAA T D (9 GC CPC AC APC TIC CA PCA W CC 171 173 176 182 186 Val Pro S S= 7hr C~s Gn Cy Am Leu Ala C~s Lai Se- L Se His lie aLu Mr Mun X- Gr AM 7Gr GkA ar AAT C GC7 70k 0C Xr C: r G C A MCA 192 Arg Ala Pro Se Arg Ala Pro 7h- ne 7r Lau Lbs Lys law Pro Ls Pro Dys f= Lys ;Gk G: CA Ok 0::k AM A= CrC AAA APG Pm aA AAA M% AAA A3k AM 213 230 L Pro 7lr Ls n= l e His His Arg M= S& Pro Mu 7= Ls Lai Gn C APG C3k A= APP ACA A C CAT C ;Gk Xr P. X G ;X AAA CG 0 31 Information for SEQ ID NO: 53 G200V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 61 amino acids, 183 nucleotides V* STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Lse Pro Ala mw Ag 1vs Pro Pro nle As Pro Ser my Ser -le P= Pro GCu A His 51 CIA C(k GCC P~kXA AP A k aFTIX AAr Xk =A G. CA CCA CA AP Ia 160 173 176 Gn Asp His Asi As R Gin 'flw Le Pro Tr V1 Pro COs Ser M Cs Gn M~y Asa leu @0A @PC APC APC TIC OA PM CC CIC T Grr GCC r XA =r @A Wr AAT Cr 182 186 200 Ala Cys Lax Ser Lai Cys His Ile Gau n2 Glu Arg Ala Pro Ser Arg Ala Pro fl- lie C GM T7M 7I =D M C T GG A A XA Ga (A XE PA GA CCA PMAC 31 61 Information for SEQ'ID NO: 54 G198V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 59 amino acids, 177 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Leiu Pro Ala w krg 1.5. P=o Pro ie Am ser my sL- lie Pro P=o Cu X~ His C~ a:LG AM AAAn P 03T pX ACC GA ApC CAT 160 173 176 Mn Am His Am~ Am- R~e Mn M= La Pro Tlyr Val p=o Cys Se Mwl Cys Glu Gly Am 101, ;C ~C AAC APC TX 0A A CC GIT CE'lGr X:LC GA a~ MT irr 182 156 198 :Ala Oys Leu Ser 101 Cys His lie Gau 7h GMu g Ala pro ser Ag Ala pro c GC l T7A 70kCI~~ ATT G G G A AM G: :Ljj 7 3' Information for SEQ ID NO: 55 G196V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 57 amino acids, 171 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Lzea Pro Ala M=l Axg L~s Pro Pro Ile Asn Pxo Ser Cay se lie Pro PMo Glu As His -3 C E G= AM MkAAA CM =T A~r CI 7M G3 XCACC% APC T 150 173 176 Gan Asp H=s Ami Ami Re~ Mn Le Pro Tyr Val Pro Cys Ser 'D Cys Mu May As-i to- GAGCCCAP PC C A AM CC =E 7P G7T C= =l 'lC1 X A h r AMT C2T 2M15 196 Ala Cy Leu sL- Lei C~s His lie Cau 7h au Pzq Ala Pro sF-- Ag c GM TM 7M CC 7W CA An' GP AM GA ;.AGkO XX 3' -62- Information for SEQ ID NO: 56 G194V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 55 amino acids, 165 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Liu PR Ala MW Ag L~s Pro PX0 le A~n P=o Ser (y SF- lie Pro Pro Glu Asmi kLis G=A-G h C C AAT =a 7c a~ ;x=CAC~kGA APC C,- 160 173 176 alnAspHisAs As ;h G~ 7x Li Po yr 141 Pzo C: s Ser 2 Cs Mu M Asni 1M826 194 Ala C~ 1 s Lai Ser Laei Cy.s His fle Mu 7rx a Aig Ala Pro c Inorato for SEQ ID NO: 57 G192V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 52 amino acids, 156 nucleotides :00%10 STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N I P= Ala MW Ag 1 s x' Il s =SrCyS-IeP 1 .AR.

10173 1.76 Cnn As Is Asn An Fn (M 7hre Pro Tyr %I Pro Cs Sr 2w C Glu M~y Asn Leu M GIC AAC AAC TIC CPA AC CIC i Ta 'IW AM AW Ala Cys Lar sLz- br Cys His fle mui mw mu~ kg c -63- Information for SEQ ID NO: 58 G6V SEQUENCE LENGTH: 51 amino acids, 153 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Lau Pro Ala zw Axg b.)s P=o Pro fle Ami Pmo Ser Cay Se le P=o Pro alu Am~ is 1 MG MX AAA C: CM AAT C~ 10 A CM CA k APC OT 160 .173 1,76 Gln Am Ris Ami Am R~e Gn Mwl Leni Pro 7y %tl Pro q5s S= M= Cys nu aly Am1 Lei CA GC CC AAC APC (;IC XX C7C CiT ;M G~ G= AA ~~CI 156 190 Ala Cys La.i Se Leu Cys His le M.u fl C GM 7,3 M~ 7M CIC Ck 3- Information for SEQ ID NO: 59 G7V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 33 amino acids, 99 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 158 173 176 N Asn His Can Aso His Asn Asn R- GOn M= J.c PrO 'lYr Val. Pro CyS Se 7 Y APC CC CA rC ac A~c TIC m Am c Th. ;r.T Gr 2M 26 190 Miu MIy Asn Leu Ala Cys La Ser Iae Cys His le Glu fr~ C (;AW A I GM 'TP. 7M C= =I O.T XT C-G; 3' -64- Information for SEQ ID NO: 60 G200VbC SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 61 amino acids, 183 nucleotides STRANDEDNESS: single linear MOLECULE TYPE: protein 140 N Li~ Pro Ala n= Ag Lys Pro Pro lie Am~ P=o Sr my se* lie P=o Pro a.u Am His G= AM- AAATT AAT CIA G~k Ar=aL A AAC CAT 160 173 176 la )sLuS- 186 Se HI n u MWr a.U Akg Ala Pto Ser Arg Ala Pro m=ne- TM7 S;GP HPA li ck;;GM~k~A -C3 Information for SEQ ID NO: 61 G19BV5C SEQUENCE TYPE: amino acids and nucleotides 0o SEQUENCE LENGTH: 59 amino acids, 177 nucleotides o 10 STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Leix Pro Ala M= Ag Z~s Pmo Pro le An Pro Ser aly Ser 11e PMo Pro Clii Asn His' 160 173 176 Can Am, His Asn Asn Phe Cn 'Z leu Pro Tyr Itl Pxo Ser Ser Sw C~s Mui Mfy Asn Leu CA C aC; AAC AAC T= A A~ C GIT =C A Gr AM cWr A~rC1 1IV 186 I Ala Cys leu Ser I~e. Ser His le Glii 7h Clii kg Ala Pro S= Mg Ala Prm C L TI 7 C ;GC CAT AT GG Aa GA ;G G Mk ;X ;GA:Z Mk -3 Information for SEQ ID NO: 62 G196V5C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 57 amino acids, 171 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N La~i P=o Ala T=a Axg Lys Pro Pro le Ami Pmro a ly sr tie Pro p=o aLu Asn His CA aA GC:A AAA MkCC ATr AAT a~ 7M=P Al CA MkCA p 160 173 1'76 t CAA GPC CC APC APC TI 0AA CIC G7T Ca Xr p TGT AM =uMAr MT C 2M 3B6 196 Al Cys Lgw S~ lau Sw His fle Glu 7hr Gl Ag Ala pro Ser kg C G~ 7M =m P= CAT GGA A AG' X 3 information for SEQ ID NO: 63 G198V6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 55 amino acids, 165 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N tI.i Pxo Ala 7h Arg Lys PMo Pro Il~e Ami Pro Ser My Se- Il~e Pro Pro Glu. Asn His CI CCA GC M G P CACA;TT Co m-n Mkp kM P AAC CPX 160 173 1'76 Mn As His As-k Am i e Gin 7h Lei Pro 'lyr %I1-Mr SW Se M- Cys Mlu Gly Asn Lai M GP CC AAC AAC =ICPA AM MC AM ;Gr ~7r AM GA GGr AAT CI 1B 26 194 Ala Cys Leu Ser Lea S=r His Ile Glu 7 Giu Azg Ala Pro C GM 77h 7M C1C r CU aT GG AMGkA kM 3 66 information for SEQ ID NO: 64 Gl92V6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 52 amino acids, 156 nucleotides STRANDEDNESS: single- TOPOLOGY: linear MOLECULE TYPE: protein 140 N L=s Pro Ala M= Arg Lys P=o Pro Ie Ami Pro Ser M~y Ser 3l1e pro pro Mu Asn His CI 0: G=A AAa C A7T AAT a; a 2C co Mk GA A' C T 160 173 176 CanAsoHisAwiAmPheCanM= euProTyrJ roSer S= M= C~s Mu Mly sLo 12186 :Ala Cys I= SeriaL S=r His fle alu 7 Mun kg C information for SEQ ID NO: 65 G6V6C SEQUENCE LENGTH: 51 amino acids, 153 nucleotides STRANDEDNESS: single :10 TOPOLOGY: linear MOLECULE TYPE: protein 140 N Iai Pro Ala M= Axq ly Pro Pro fl* Asn Pro Ser Gly SE-- fle Pro Pro Mun Asi Ihis 173 176 Gix. As Hs Am Asn Ph- Gin Tw Lu Pro Tyr %I1 Pxo Se= S=r Mw Gin Mu y Asni Leu CA GC MC APC AAC T= Mh A CI MDX G1T .C AM X7 AM 7=GAr AAT CI 282 18690 Ala C~s Lai S= Lou Ser His 32~e Gn C Go T7m 7M C AM CC ;aT G 3' -67-.

information for SEQ ID NO: 66 G7V6C! SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 33 amino acids, 99 nucleotides STRA24DEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 128 173 176 N Ami His Gn As His Asn Asn R-e Mn 12lw Lei P=o lyr Val Pzo Ser Ser M Cys AAC CA DA GC CAC APC A'C =I CA AC% Cr IT XC P0 7= 122 186 190 MyA*liX a e e e i l D o Information for SEQ ID NO: 67 G4V *SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 17 amino acids, 51 nucleotides STRANDEDNESS: single TOPOLOGY: linear.

00 0060 MOLECULE TYPE: peptide 171 173 176 122 166187 N Val Pzo Clys SL- '2 Cys Cau M~y Asi ILeu Ala Cys Leai SF- Lai Cys His C -68- Information for SEQ ID NO: 68 G4V6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 17 amino acids, 51 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 166 187 N Vl Pro Se Se M= Cs Mu ay As Lua Ala Cys La Se Laz Se- His C GIT C AE r AG A rCAGT A aG AAT CIT TIA Ta CIc xe CT 31 Information for SEQ ID NO: 69 G4'V

T

SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 17 amino acids :10 STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 186 187 N 1l Pro Asp Se M= Asp Glu My Asn Las Ala Cm La Ser leu On His C Information for SEQ ID NO: 70 G4'V6C SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 17 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 186 187 N Val Pro Se Se Mu- Asp Qu Gly AsI Lau Ala Qm Lan Se Lae Ser His C 69 Information for SEQ ID NO: 71 G1V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 14 amino acids, 42 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 176 22 186 187 N Se r Cys Gl Gly Asm Le Ala Cys Li Ser Leu Cys His C ;Gr AA Gr GA GaT AAT CIT G Xk= Tm S CiC I7'T 3' 0* Information for SEQ ID NO: 72 G1V6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 14 amino acids, STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide *174 176 182 186 187 N Se hr Cys Gu Gly As lz Ala Cys Lal Se Le.1 Sex His C PGr AM 7= CA Wr AATT CIA 7W TIA CIC A CAT 3' Information for SEQ ID NO: 73 Gl'V6C SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 14 amino acids STRANqDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 176 182 186 157 N Ser 7hr Asp Mu Miy Am Lai Ala Om Lau Ser Le. Ser Mis -C *~.Information for SEQ ID NO: 74 BE SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 219 amino acids, 657 nucleotides STRANDEDNESS: single ****TOPOLOGY: linear *So.

MOLECULE TYPE: protein 17 M= Val Glu Gly Val 1ijs Asp Lei Can Ala Gin Val 'Jai aiu Ser Ala 1&s Iys Ala Arg fle Ser alu Ala Mw Asp Giy Ia3 SL- Asp Ri Leu by Ser ain 'D S3 P o Ala Gilu Asp M Val Lys Ser fle Giu Leu Ala Mun ;aa Lys Val LIe Ala G= GA~ GU A=GTAA7 TGATAa~ A A I I 71 71 Am Arg Mu Lea Asp 1s Tr My Wd1 Ser A 7Lr His Lys Am La Lie Am APC AA G A =r GAC AAA G~ G33k PT G T GC APC C=%A A 89 An Ala Lys Mw Val Mu Gly '1 Lys Asp Lea Gn Ala Cn Vl Vl Clu Se AAT AAA X7 Gr CPA ar G3A AAA G; CT CA Dk GC T CIT @A 7M 107 Ala Ls 1S Ala Arg lie Ser al Ala 7hr Asp My ai.I .Se As Pte Lu Ls a AA AAAlGm ad T C G GXk Am GP c a r Tn. T TM AAA 125 Ser Mn Mr Pro Ala Glu Asp 'fr Val Lys Ser nle Glu Leu Ala Cau Ala Ls 7% A 0A 7T= Gr GA G ACr GIT APA 7%.A XT G;A TIA GA CET AAA 143 Ntl Lax Ala Asn Ag Glu Lax Asp Lys 7 T Gy i Ser Asp Tyr 7 L Asn G1 TM AC XP GDA Crr TGC AAA= GA GM ;ACr GC =T VC cG APIC 161 Iwax Ile Asx As Ala Ls M= V1 GLu Cy Val Lys Ala La ile Asp Giu lie CPJC V~C AkT AAA CPIT A Wr GM AM G~ CIG A AT 179 Lai Ala Ala Lax Pro Lys DY Aso -Dr Iw Tyrs Lei lie La Ami Gly LW Mr 7'm a~r Gok T MT =X A=Y C AC TC AA TM AM CIT AAl CT AM ACk 197 Leu Lrs y Cu M= Drx Mr w u Ala Wl Asp Ala Ala Tw Ala Arg Ser I-e TM AA GOC GDA A P CIT ;CT C.r G4% AGT GC TIC 215 219 Asn 2E Pro ne Leu C TIC CT AlC d 3' Information for SEQ ID NO: 75 6BB fragment of BE SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 108 amino acids, 324 nucleotides STRANDEDNESS: single TOPOLOGY: linear 09 004 a* FP 0 tJ 84 q,