Complexes of immunogens derived from RSV surface glycoprotein G covalently coupled to a support molecule

FIELD OF THE INVENTION

RSV is the most frequent cause of the hospitalization of unweaned infants under one year old for acute respiratory infections. Infants suffering from laryngotracheobronchites, bronchiolites and pneumonias require hospital care, and the incidence of mortality in unweaned infants exhibiting congenital cardiac diseases is greater than 37%. Other disorders such as bronchopulmonary dysplasias, renal diseases and immunodeficiency are equally much factors which are responsible for increased mortality. Infections with RSV can also be a cause of death in old people.

BACKGROUND OF THE INVENTION

In temperate countries, the RSV epidemic occurs during the winter period from November to April, and the highest incidence of serious diseases is found in the unweaned infant of 2 to 6 months. A distinction is made between two types of RSV, RSV-A and RSV-B, on the basis of the antigenic variation of the G glycoprotein of RSV: subgroup A and subgroup B, which circulate concurrently. A recent study which was carried out in France from 1982 to 1990 demonstrated the alternation of one subgroup with the other over a period of 5 years. Strain A is often the cause of infections which are more serious than those caused by strain B.

In the 1960's, an unsuccessful attempt was made to develop conventional vaccines, that is using formolinactivated RSV, in analogy with anti-measles vaccines. Instead of conferring protection on the vaccinated infant, this type of vaccine had the effect of potentiating the natural viral disease.

Human RSV belongs to the genus pneumovirus, which is a member of the Paramyxoviridae family. The genome of the virus consists of an RNA strand which is of negative polarity, is nonsegmented and encodes 10 distinct proteins: NS1, NS2, N, P, M, SH (or 1A), G, F, M2 (or 22K) and L.

Numerous published experiments have demonstrated that the main proteins involved in protection are: F, G and N. The fusion glycoprotein F, which is synthesized as precursor F

0

, is cleaved into two subunits F1 (48 kDa) and F2 (20 kDa) which are bound together by disulphide bridges. The F protein is conserved between RSV-A and RSV-B (91% homology). Conversely, the attachment glycoprotein G varies greatly from one subgroup to the other. Only one region of 13 amino acids (aa 164 to aa 176) is highly conserved and four cysteine residues (173, 176, 182 and 186) are preserved in each subgroup. It has been shown in animal models that the two glycoproteins F and G play a major role in the immunology of RSV. Monoclonal antibodies directed against G and F are able to neutralize the virus in vitro and, when administered passively, they protect the cotton rat from RSV infection.

Current treatments for aggravation of the disease caused by RSV in unweaned infants are clearing the respiratory tract of congestion by aspirating mucus and respiratory assistance provided by ventilation. An antiviral agent, ribavirin, appears to be effective in seriously affected cases. However, its use in paediatric therapy is still poorly defined. Passive immunization with anti-RSV immunoglobulins represents an alternative route in the treatment of serious RSV infections: no undesirable side-effect has been observed. Nevertheless, this type of treatment is very costly and difficult to extrapolate to a large scale.

Different approaches have been taken to vaccinating against human RSV: either the vaccine protects against RSV infection in animals (rodents and primates) but induces pulmonary pathology or else the vaccine is not sufficiently immunogenic and does not provide protection (Connors et al., Vaccine 1992; 10: 475-484).

BRIEF SUMMARY OF THE INVENTION

For this reason, the present invention relates to a process for improving the immunogenicity of an immunogen, in particular an antigen, or a hapten, when it is administered to a host, independently of the mode of administration, characterized in that the said immunogen or hapten is coupled covalently to a support molecule in order to form a complex, and in that this support molecule is a polypeptide fragment which is able to bind specifically to Tmaimalian serum albumin.

Administration can, in particular, take place enterally, parenterally or orally.

The immunogenicity of the complex between the immunogen and the support molecule is found to be improved as compared with that of the immunogen alone, in the absence of any other immunostimulant.

A complex which is particularly suitable for implementing the present invention is obtained by using a conjugate with a polypeptide which is derived from the G protein of Streptococcus; this protein has been characterized by Nygren et al. (J. Mol. Recognit. 1988; 1:69-74).

The invention relates to a process in which the support molecule exhibits the amino acid sequence denoted sequence ID No: 74 or a sequence which exhibits at least 80%, and preferably at least 90%, homology with the said sequence ID No: 74.

This sequence can be attached to linking sequences which promote its expression in a host.

According to the invention, use can also be made of a support molecule which exhibits one of the sequences ID No: 75 or No: 78, as well as of molecules which exhibit at least 80%, and preferably at least 90%, homology with the said sequences.

The peptide sequence ID No: 78 exhibits the following characteristics:

Sequence ID No: 78

Molecular weight: 26529

Gly: 10 (4.08%);

Ala: 30 (12.24%);

Ser: 14 (6.12%);

Thr: 16 (6.53%);

Val: 20 (8.16%);

Leu: 23 (9.39%);

Ile: 12 (4.90%);

Pro: 4 (1.63%);

Cys: 0 (0.00%);

Met: 1 (0.41%);

His: 2 (0.82%);

Tyr: 9 (3.67%);

Asp: 19 (7.76%);

Glu: 19 (8.16%);

Lys: 27 (11.02%);

Arg: 5 (2.04%);

Asn: 16 (6.94%);

Gln: 8 (3.27%);

Phe: 7 (2.86%);

The complex between the support molecule and the compound whose immunogenicity it is desired to improve can be produced by recombinant DNA techniques, in particular by inserting or fusing the DNA encoding the immunogen or hapten into the DNA molecule encoding the support.

According to another embodiment, the covalent coupling between the support molecule and the immunogen is effected chemically using techniques known to the person skilled in the art.

The invention also relates to a gene fusion which renders it possible to implement the process for improving the immunogenicity, characterized in that it comprises a hybrid DNA molecule which is produced by inserting or fusing the DNA encoding the immunogen or hapten into the DNA molecule encoding the support molecule and which is fused with a promoter; the invention also comprises a vector which contains such a gene, it being possible for the said vector to have, in particular, as its origin a DNA vector which derives from a plasmid, a bacterio-phage, a virus and/or a cosmid.

A vector which exhibits the sequence ID No: 76 or 77 belongs to the invention, as does the corresponding polypeptide. These polypeptides exhibit the following characteristics:

Sequence ID No: 76

Molecular weight: 38681

Gly: 11 (3.15%);

Ala: 31 (8.88%);

Ser: 18 (5.16%);

Thr: 37 (10.60%);

Val: 25 (7.16%);

Leu: 23 (6.59%);

Ile: 15 (4.30%);

Pro: 19 (5.44%);

Cys: 4 (1.15%);

Met: 2 (0.57%);

His: 4 (1.15%);

Tyr: 9 (2.58%);

Asp: 22 (6.30%);

Glu: 22 (6.30%);

Lys: 48 (13.75%);

Arg: 7 (2.01%);

Asn: 26 (7.45%);

Gln: 13 (3.72%);

Phe: 12 (3.44%);

Trp: 1 (0.29%);

Sequence ID No: 77

Molecular weight: 39288

Gly: 12 (3.37%);

Ala: 31 (8.71%);

Ser: 22 (6.18%);

Thr: 37 (10.39%);

Val: 26 (7.30%);

Leu: 23 (6.46%);

Ile: 15 (4.21%);

Pro: 21 (5.90%);

Cys: 2 (0.56%);

Met: 2 (0.56%);

His: 4 (1.12%);

Tyr: 9 (2.53%);

Asp: 23 (6.46%);

Glu: 22 (6.18%);

Lys: 48 (13.48%);

Arg: 7 (1.97%);

Asn: 26 (7.30%);

Gln: 13 (3.65%);

Phe: 12 (3.37%);

Trp: 1 (0.28%);

The DNA molecule which encodes the complex between the immunogen and the support molecule can be integrated into the genome of the host cell.

In one of its embodiments, the novel process includes a step for producing the complex, by genetic manipulation, in a host cell.

The host cell can be of the prokaryote type and be selected, in particular, from the group comprising:

E. coli

, Bacillus, Lactobacillus, Staphylococcus and Streptococcus; it can also be a yeast.

According to another aspect, the host cell is derived from a mammal.

The gene fusion which encodes the complex having an improved izmunogenicity can, in particular, be introduced into the host cell by the mediation of a viral vector.

The immunogen which is used preferably derives from bacteria, parasites and viruses.

This immunogen can be a hapten: peptide or polysaccharide.

The novel process is particularly suitable for a surface polypeptide from a pathogen. When this polypeptide is expressed in the form of a fusion protein, using recombinant DNA techniques, the fusion protein is advantageously expressed, anchored and exposed at the surface of the membrane of the host cells. Nucleic acid molecules are employed which are able to direct the synthesis of the antigen in the host cell.

They comprise a promoter sequence, a functionally linked secretion signal sequence and a sequence which encodes a membrane anchoring region, which sequences will be adapted by the person skilled in the art.

The immunogen can, in particular, be derived from an RSV surface glycoprotein: F and/or G.

Particularly advantageous results are obtained using fragments of the G protein from RSV subgroups A or B.

The proteins which are derived from the G glycoprotein of RSV subgroup A and subgroup B can be genetically fused or chemically coupled to BB.

The invention relates, therefore, to a complex which is obtained using the sequence which is encompassed between amino acids 130 and 230 of the G protein of RSV, or a sequence exhibiting at least 80% homology with the said sequence of the G protein.

This sequence can be obtained from human or bovine RSV which belongs to subgroups A or B.

The sequence encompassed between amino acids 130 and 230 of the G protein can be subjected to various types of modification for the purpose of modulating its immunogenic activity and its expression by the host system.

The applicant has, in particular, demonstrated that polypeptides are of interest in which:

the Cys amino acid in positions 173 and/or 186 has been replaced by an amino acid which does not form a disulphide bridge, in particular serine, and/or

the amino acids in positions 176 and 182 are capable of forming a covalent bridge other than a disulphide bridge, in particular aspartic acid and ornithine, and/or

the phenylalanine amino acids corresponding to positions 163, 165, 168 and/or 170 of the sequence of the G protein are replaced by a polar amino acid, in particular serine, and/or

the sequence encompassed between the amino acids numbered 162 and 170 is deleted.

Peptides exhibiting one of the sequences ID No: 1 to 73, or a sequence possessing at least 90% homology with one of the sequences ID No: 1 to 73, are thus particularly suitable for implementing the invention.

Other immunogens which are suitable for implementing the novel process include a derivative of the surface protein of hepatitis virus A, B and C, a surface protein of the measles virus, a surface protein of parainfluenza virus 3, in particular a surface glycoprotein such as haemagglutinin, neuraminidase HN and fusion protein F.

The RNA or DNA nucleotide sequences which encode complexes such as those previously defined, and which include elements which enable expression to be targeted in certain specific host cells, are included in the invention. They can be incorporated into a viral or plasmid vector; this vector will be administered to a mammal, in particular within a pharmaceutical composition, in order to enable the complex between the immunogen and the support molecule to be produced in situ.

The invention also relates to the use of a gene fusion or a complex between an immunogen (P) and a support molecule, such as those previously defined, as a medicament. The pharmaceutical compositions containing the gene or the complex together with physiologically acceptable excipients also belong to the invention. They are particularly suitable for preparing a vaccine.

Immunization can be obtained by administering the nucleotide sequence either on its own or through the agency of a viral vector. The host cell, in particular an inactivated bacterium, may also be used. Finally, the complex which is obtained by chemical coupling or is in the form of a fusion protein induces an antibody response which is very powerful compared with that induced by (P) on its own coupled to Freund's adjuvant.

Within the scope of a vaccine against RSV, the applicant has demonstrated the efficacy of the fusion protein BBG2A, where G2A is a 101 amino acid fragment of the G protein of RSV-A (G aa 130-aa 230), seq id No. 1. When used to immunize rodents, BBG2A and BBG2AδC coupled to alum (aluminium hydroxide) confer complete protection against challenge with RSV-A (Long strain).

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

BRIEF DESCRIPTION OF THE DRAWING

In these examples, reference will be made to the following figure:

FIG.

1

: Construction of pVABBG2(A).

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLE 1

Cloning Genes G2A and G2AδC into Expression Vector pVABB308 and Producing Fusion Proteins BBG2A and BBG2AδC in

Escherichia Coli

1) Expression Vector pVABB308

The

E. coli

expression vector pVABB308 (5.4 kbp) encompasses the tryptophan operon promoter (Trp), followed by the gene encoding the human albumin-binding region BB, originating from the G protein of Streptococcus (Nygren et al., J. Mol. Recognit. 1988; 1: 69-74) and a multiple cloning site, mp8, into which various heterologous genes can be inserted (see FIG.

1

). Plasmid pVABB308 contains an ampicillin resistance gene (AMP), a tetracycline resistance gene (Tet) and the

E. coli

origin of replication. Expression of the gene is induced by adding IAA. (indoleacrylic acid) to the

E. coli

culture medium during the exponential growth phase.

2) Cloning the Genes G2A and G2AδC into PVABB308

2.1. BBG2A

The gene encoding RSV-A G (130-230) was obtained by the method of assembling synthetic genes in solid phase (in accordance with Stahl et al., Biotechniques 1992: 14: 424-434) and cloned into expression vector PVABB using the EcoRI and HindIII restriction sites. The resulting vector is termed pVABBG2A (5791 bp). The fusion product BBG2A is purified in two forms from the cytosol of

E. coli

which has been transformed with vector pVABBG2A:

a soluble form, BBG2A (sol), following cell disintegration and centrifugation; the supernatant containing the soluble proteins is loaded directly onto an affinity column. The products are recovered after eluting at acid pH.

an insoluble form, BBG2A (insoluble), which is obtained after renaturing, in an oxidizing medium, the inclusion bodies which have been dissolved in a chaotropic agent (guanidine HCl) (31, 93), and is then subjected to affinity purification.

2.2. BBG2AδC

The two cysteine residues (173 and 186) are replaced by serines (Ser). When the genes are being assembled, the oligonucleotide which encompasses the 2 Cys residues which are encoded by the (TGC) triplet is quite simply replaced by another oligonucleotide in which one of the nucleotides has been changed: (TCC) encoding Ser). We wished deliberately to modify one disulphide bridge in this version in order exclusively to retain the disulphide bridge which is formed by Cys residues (176 and 182) and which is critical for protection (Trudel et al., Virology 1991; 185: 749-757).

We introduced a Met residue between the BB affinity tail and G2A or BB and G2AδC: BB-Met-G2A and BB-Met-G2AδC, thereby rendering it possible to cleave the fusion product chemically using cyanogen bromide (CNBr); the mixture is passed through an HSA-Sepharose affinity column. The cleaved peptide, G2A (G2AδC), is not bound and is therefore recovered in the eluate and then purified by reverse phase HPLC.

3) Fermentation and Purification of Fusion Proteins

Two Erlenmeyer flasks containing 250 ml of TSB (tryptic soy broth, Difco) together with ampicillin (100 μg/ml, Sigma) and tetracycline (8 μg/ml, Sigma) are inoculated with

E. coli

RV308 strains which are transformed with plasmids pVABBG2A and pVABBG2AδC, respectively. The flasks are incubated, with shaking, at T

0

=32° C. for 16 hours. 200 ml of this culture are inoculated into a fermenter (CHEAP CF3000, ALFA LAVAL) which contains 2 litres of culture medium. The medium contains (g/l)=glycerol, 5; ammonium sulphate, 2.6; potassium dihydrogen phosphate, 3; dipotassium hydrogen phosphate, 2; sodium citrate, 0.5; yeast extract, 1; ampicillin, 0.1; tetracycline, 0.008; thiamine, 0.07; magnesium sulphate, 1, and 1 ml of a trace element solution/l and 0.65 ml of a vitamin solution/l. The parameters which are monitored during the fermentation are: pH, shaking, temperature, oxygenation rate and supply of carbon sources (glycerol or glucose). The pH is maintained at 7.3. The temperature is kept at 32° C. Growth is controlled by supplying glycerol at a constant rate in order to keep the tension signal of dissolved oxygen at 30%. When the turbidity of the culture (measured at 580 nm) reaches a value of 80 (after about 27 hours of culture), production of the proteins is induced by adding indoleacrylic acid (IAA) to a final concentration of 25 mg/l. Three hours after the induction, the cells are harvested by centrifugation. The biomass yields which are obtained are of the order of 150 g/l of culture.

A fraction of 30 g of moist biomass is resuspended in 70 ml of TST solution (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 0.05% Tween 20 and 0.05 mM EDTA). These cells are disintegrated by sonication (Vibracell 72401, Sonics & Materials). After the cell lysate has been centrifuged, the supernatant is filtered (1.2 μm) and diluted in 500 ml of TST. The fusion proteins thus obtained in soluble form are purified on an affinity column: HSA-Sepharose (human serum albumin) in accordance with the protocol described by Stahl et al. (J. Immunol. Methods, 1989; 124: 43-52).

After it has been centrifuged, the insoluble lysate is washed once with a buffer (50 mM Tris-HCl, pH 8.5; 5 mM MgCl

2

). After the wash, the pellet is solubilized in 30 ml of 7 M guanidine hydrochloride, 25 mM Tris-HCl (pH 8.5), 10 mM dithiothreitol (DTT), with the mixture then being incubated at 37° C. for 2 hours. The solubilized proteins are added to a renaturation buffer (25 mM Tris-HCl (pH 8.5); 150 mM NaCl; 0.05% Tween 20). The concentration of the guanidine hydrochloride is adjusted to a final concentration of 0.5 M in the renaturation buffer before adding the solubilized fusion proteins. The mixture is incubated at room temperature for 16 hours with gentle stirring. Following centrifugation, the soluble fusion products in the supernatant are purified on an HSA-Sepharose column. The purified fusion proteins are analysed on an SDS-PAGE gel (12%) under reducing conditions using a MINI PROTEAN II SYSTEM (BIORAD) appliance. The proteins are visualized with Coomassie brilliant blue R250.

EXAMPLE 2

Carrier Effect of the BB Polypeptide and Immunogenicity of BBG2AδC

1. Immunization Protocol

C57B1/6 mice (5 per batch) were given 2 subcutaneous injections of the equivalent of 10 μg of G2AδC in the presence of Freund's adjuvants on D0 (complete adjuvant) and D14 (incomplete adjuvant). On D21, the sera were tested individually by ELISA for the production of G2AδC-specific antibodies. The antibody titre is determined as being the inverse of the serum dilution which gives twice the absorbance of the serum of the animal prior to immunization. The results which are presented are the arithmetic mean of the anti-G2AδC antibody titres which were obtained for each of the batches.

RESULTS TABLE

ANTIGEN

Mean titre of anti-G2AδC antibody

1) G2AδC + FA

180

2) BBG2AδC + FA

92800

3) G2AδC + BB + FA

1200

2. Results

The above table demonstrates that G2A6c is a weak immunogen even in the presence of Freund's adjuvant. The BB protein has a poor adjuvant ability since, when it has been added to G2AδC, the anti-G2AδC antibody titre is only increased by 1 log. On the other hand, fusing BB to G2AδC increases the anti-G2AδC antibody production by about 3 logs.

We may conclude, therefore, that BB is an excellent carrier protein for G2AδC and that the fusion protein BBG2AδC is very immunogenic.

EXAMPLE 3

Study of the Protection Induced by Fusion Proteins BBG2A and BBG2AδC in Rodents

a) Study Protocols

Female BALB/c mice and cotton rats (Sigmodon hispidus) (IFFA-CREDO), which are animal models for RSV infection, are employed in the immunization experiments.

The animal groups are given 1, 2 or 3 doses of 200 μg, 20 μg, 2 μg or 0.2 μg of RSV-A candidate vaccine in 20% aluminium hydroxide (Al(OH)

3

) (v/v) at 2-week intervals. The mice are immunized by the intraperitoneal (i.p.) route while the cotton rats are immunized with intramuscular (i.m.) injections. The control groups are given 10

5

TCID

50

of RSV-A or PBS-A (PBS without Ca

2+

or Mg

2+

) in 20% aluminium hydroxide (v/v).

Three to four weeks after the last immunization, the animals are challenged by the intranasal (i.n.) route with approximately 10

5

TCID

50

RSV-A. They are sacrificed 5 days later following intracardiac blood puncture. The presence of the virus in their lungs is determined in accordance with Trudel et al. (Virology 1991; 185; 749-757).

The different products which are tested are BBG2A, BBG2AδC and BB alone.

TABLE 3.1

Protection results in rodents

Mice

Cotton rats

Complete

Complete

Antigens

Protection*

protection

0

Protection

protection

BBG2A

41/41+

38/41

22/22

22/22

BBG2AδC

32/34

27/34

8/13

7/13

BB

0/20

0/20

0/3

0/3

RSV-A

28/28

28/28

17/17

17/17

PBS-A

0/29

0/29

0/21

0/21

*Protection = a reduction of virus in the lungs of ≧ log

10

2 as compared with the mean virus titre in the lungs of mice immunized with PBS-A.

0

Complete protection = no virus detected in the lungs.

+X/Y where X = number of animals protected or completely protected; Y = number of animals tested

TABLE 3.2

Details of protection in the mouse

3 doses of antigen

2 doses of antigen

1 dose of antigen

BBG2A

BBG2δC

BBG2A

BBG2δC

BBG2A

BBG2δC

200 μg/dose

Protection*

9/9+

9/9

4/4

4/4

4/4

2/4

Complete protection°

9/9

8/9

4/4

3/4

3/4

1/4

20 μg/dose

Protection*

4/4

4/4

3/3

NT

NT

NT

Complete protection°

4/4

4/4

2/3

NT

NT

NT

2 μg/dose

Protection*

4/4

4/4

2/2

NT

NT

NT

Complete protection°

3/4

3/4

2/2

NT

NT

NT

0.2 μg/dose

Protection*

4/4

4/4

NT

NT

NT

NT

Complete protection°

4/4

3/4

NT

NT

NT

NT

*Protection = a reduction of virus in the lungs of ≧ log

10

2 as compared with the mean virus titre in the lungs of mice immunized with PBS-A.

°Complete protection = no virus detected in the lungs.

+ X/Y where X = number of mice protected or completely protected;

Y = number of mice tested

NT = Not tested

TABLE 3.3

Results of the immunological tests in mice

Neutralizing anti-

bodies (mean

Antigens

ELISA(mean LOG

10

)

titre/25 μl)

BBG2A

5.09 (28)

≧512 (15

BBG2AδC

3.71 (29)

≧256 (12)

RSV-A

5.32 (21)

≧512 (12)

( ) = number of animals tested

c) Discussion

The experimental protection results are presented in Tables 3.1. and 3.2. Each molecule was tested in at least 2 independent experiments. The results clearly demonstrate that BBG2A protects rodents against a pulmonary infection with RSV-A independently of the immunization protocol which was employed. Under our experimental conditions, a single injection of 200 μg, 2 injections of 2 μg, or 3 injections of only 0.2 μg of BBG2A are sufficient to protect the mice against infection (Table 3.2). Virus was detected in a third animal of the same group but at the limit of detection. These results suggest that BBG2A displays a potential and an efficacy which are very comparable to those of RSV-A in the immunized control animals and to those of the RSV-A subunit candidate vaccines which are described in the literature.

BBG2AδC was also effective in mice, protecting 32 animals out of 34 aginst pulmonary infection. Two doses of 200 μg were found to be effective, just as were 3 injections of 0.2 μg. Thus, in these immunization protocols consisting of several injections, BBG2AδC was shown to be comparable in its activity and efficacy in mice to the already described RSV-A sub unit candidate vaccines.

The results of the immunological tests of the humoral and cellular response in BALB/c mice are presented in Table 3.3. In general, the mean titres of anti-RSV-A-specific antibodies which are obtained by the ELISA technique are considered to be one of the parameters reflecting the protective activity of the candidate vaccines. The sera of the mice immunized with RSV-A consistently demonstrated elevated anti-RSV-A antibody titres. The virus was never detected in the lungs of these animals. The mice immunized with BBG2A exhibited mean titres of anti-RSV-A antibodies which were similarly elevated and were always protected when challenged with RSV-A.

The mean anti-RSV-A antibody titres induced with BBG2AδC were lower than those obtained with the molecules mentioned above. Furthermore, the animals which were immunized with this molecule exhibited slightly reduced protection. Even if the sera of some animals which were immunized with BBG2AδC exhibited very low titres of anti-RSV-A-specific antibodies (data not shown), certain of these animals were nevertheless completely protected when challenged with RSV-A.

The protection studies demonstrate the protective efficacy of the anti-RSV-A subunit candidate vaccines. Two molecules, BBG2A and BBG2AδC, were found to be very effective in two rodent models for RSV-A infection during challenge with the homologous virus.

EXAMPLE 4

Immunogenic and Protective Efficacy of BBG2AδC as Compared with G2AδC in the BALB/c Mouse

Materials and Methods

Groups of 4 BALB/c mice, which were seronegative with regard to RSV-A, were immunized twice at an interval of 2 weeks, by intraperitoneal (i.p.) injections, with 5.1, 0.51 and 0.051 nM of BBG2AδC and G2AδC. The latter molecule is derived by chemically cleaving BBG2AδC with cyanogen bromide. A group of 3 mice was immunized twice at an interval of 2 weeks with PBS buffer in order to serve as negative controls. Alhydrogel (Al(OH)

3

) (20% v/v) (Superfos BioSector, Denmark) was employed as adjuvant for all the immunizations. A blood puncture was carried out 2 weeks after the last immunization in order to determine the ELISA titres against G2AδC. The mice were challenged with RSV-A (10

5

TCID

50

) at 3 weeks after the last immunization. They were sacrificed 5 days later and subjected to cardiac puncture in order to titrate the post-challenge anti-RSV-A antibodies, and the lungs were removed in order to titrate the pulmonary RSV-A.

Results

See Table 4.

The anti-G2AδC ELISA results indicate that BBG2AδC is always more immunogenic than G2AδC whatever dose (0.051-5.1 nM) is administered. Especially at 0.051 nM, BBG2AδC induces a mean anti-G2AδC titre of log

10

3.27 whereas the same concentration of G2AδC does not induce any detectable anti-G2AδC antibodies. The same applies as far as the anti-RSV-A ELISAs are concerned; 4 mice out of 4 which were immunized with 5.1 or 0.51 nM of BBG2AδC were seropositive, with the mean titres being log

10

2.67 and 2.78, respectively. However, two mice out of 4 which were immunized with 5.1 nM of G2AδC were seropositive, with one being at the limit of detection of the assay and one having a mean titre of log

10

≦2.19. The mice immunized with 0.51 or 0.051 nM of G2AδC did not show evidence of anti-RSV-A antibodies.

The lungs of all the mice which were immunized with 5.1 or 0.51 nM of BBG2AδC were protected against a challenge with the homologous virus. Except for one mouse which was immunized with 0.51 nM of BBG2AδC, which only exhibited virus at the limit of detection of the method, the presence of pulmonary virus was not demonstrated in any of the animals. Following immunization with 0.051 nM of BBG2AδC, 3 mice out of 4 were protected, with 2 of the mice having no evidence of pulmonary virus. In the fourth mouse, the pulmonary virus was diminished by the order of log

10

1.16 as compared with the mean titre of the controls immunized with PBS-A.

The lungs of three mice out of 4 which were immunized with 5.1 nM of G2AδC were protected against a challenge with RSV-A. The pulmonary virus in the fourth mouse was diminished by the order of log

10

1.75 as compared with the mean titre of the controls immunized with PBS-A buffer. In only one of the protected mice was pulmonary virus not detected. The same results are observed after immunizing with 0.51 nM of G2AδC, apart from one unprotected mouse which did not exhibit any significant diminution in pulmonary virus as compared with the controls immunized with PBS-A buffer. The lower respiratory tracts of the mice immunized with 0.051 nM of G2AδC were not protected against a challenge with the homologous virus.

Conclusions

The results indicate that, in accordance with the conditions of this study, BBG2AδC is of the order of 10 to 100 times as effective as G2AδC in inducing immune responses which protect the lungs against a challenge with RSV-A.

TABLE 4

Comparative efficacy of the immunogenicity and protection induced in BALB/c mice which are

immunized with BBG2AδC or G2AδC.

Concentration of

ELISA titre (log

10

)

% protected

log

10

TCID

50

RSV-A/g

immunogen (nM)

vs G2AδC

vs RSV-A

animals

of lung

Immunized with =

BBG2AδC

G2AδC

BBG2AδC

G2AδC

BBG2AδC

G2AδC

BBG2δC

G2AδC

5.1

5.06 ± 0.27

4.70 ± 0.46

2.67 ± 0.83

≦2.19 ± 0.48

100

25

<1.53 ± 0.12

≦1.80 ± 0.35

0.51

4.46 ± 0.46

3.86 ± 0.59

2.78 ± 0.60

<1.95 ± 0.00

75

25

≦1.47 ± 0.04

≦1.97 ± 0.99

0.051

3.27 ± 1.53

<1.95 ± 0.0

≦2.19 ± 0.48

<1.95 ± 0.00

50

0

≦1.93 ± 0.67

4.08 ± 0.48

PBS-A

—

1.95 ± 0.00

0

4.03 ± 0.29

TABLE 5

Protective efficacy of the vaccine candidates in BALB/c mice against a challenge

with RSV-A.

ELISA titres (log

10

)

Log

10

TCID

50

RSV-A

P.Im*vs

P.Im vs

P.Ch•vs

Product

/g of lung

antigen

RSV-A

RSV-A

20 μg BBG7a

<1.45 ± 0.00

6.25 ± 0.00

3.38 ± 0.00

3.38 ± 0.00

20 μg BBG200a

<1.45 ± 0.00

6.41 ± 0.28

4.66 ± 0.28

4.66 ± 0.28

20 μg BBG198a

<1.45 ± 0.00

6.09 ± 0.28

4.66 ± 0.28

4.58 ± 0.35

20 μg BBG196a

<1.45 ± 0.00

5.93 ± 0.28

4.34 ± 0.00

4.18 ± 0.28

20 μg BBG194a

<1.45 ± 0.00

5.77 ± 0.00

4.34 ± 0.48

4.34 ± 0.48

20 μg BBG192a

<1.45 ± 0.00

5.77 ± 0.00

3.54 ± 0.28

3.86 ± 0.00

PBS-A

3.74 ± 0.29

—

2.03 ± 0.20

1.95 ± 0.00

RSV-A

<1.45 ± 0.00

—

4.82 ± 0.00

4.82 ± 0.00

*P.Im. = ELISA results post-immunization but before challenge.

•P.Ch. = ELISA results in sera removed by cardiac puncture at the time of sacrifice.

EXAMPLE 5

Protective Efficacy of the Vaccine Candidates in Balb/c Mice Against a Challenge with RSV-A.

Materials and Methods

Groups of 3 mice were immunized twice at an interval of 2 weeks with 20 μg of the following products:

BBG7A, BBG200A, BBG198A, BBG196A, BBG194A and BBG192A

Two groups of 6 and 4 mice were immunized twice at an interval of 2 weeks with PBS-A and RSV-A (10

5

TCID

50

), respectively, as controls. Alhydrogel (Al(OH)

3

) (20% v/v) was employed as adjuvant for each immunization. All the animals were sampled from the eye before the first immunization in order to verify their seronegativity with respect to RSV-A. All were seronegative or had titres at the detection limit of the ELISA assay. Two weeks after the second immunization, they were sampled from the eye in order to confirm their seroconversion with respect to the antigens and RSV-A. Three weeks after the last immunization, the mice were challenged by the intranasal route with 10

5

TCID

50

of RSV-A. The mice were sacrificed 5 days after the challenge: they were subjected to cardiac puncture; the lungs were removed in order to titre the virus in the lower respiratory tracts. The post-challenge sera were tested by ELISA against the viral antigens.

Results

See Table 5.

The mice which were immunized with BBG200A, BBG198A, BBG196A, BBG194A, BBG192A and BBG7A were protected against a challenge with RSV-A without any evidence of virus in the lungs. All the products induced high mean antibody titres against the immunization antigen (log

10

5.77-6.41) and RSV-A (log

10

3.38-4.66).

These results are in agreement with those obtained from mice which were immunized with RSV-A.

Conclusions

The above molecules are very immunogenic and induce immune responses which are able to protect the lungs of the BALB/c mouse against a challenge with RSV-A. They therefore constitute potential vaccine candidates against RSV-A.

EXAMPLE 6

Protective Efficacy of BB-G4A in the Balb/c Mouse Against a Challenge with RSV-A.

Materials and Methods

Two groups of 3 mice were immunized twice at an interval of 2 weeks with 20 μg of BB-G4A or TT-G4A. The molecules are obtained by chemically coupling the peptide G4A (residues 172-187) onto the carrier proteins (either BB or TT). Two groups of 6 and 4 mice were immunized twice at an interval of 2 weeks with PBS-A and RSV-A (10

5

TCID

50

), respectively, as controls. Alhydrogel (Al(OH)

3

) (20% v/v) was employed as adjuvant for each immunization. All the animals were sampled from the eye before the first immunization in order to verify their seronegativity with regard to RSV-A. All were seronegative or had titres at the detection limit of the ELISA assay. Two weeks after the 2nd immunization, they were sampled from the eye in order to confirm their seroconversion with respect to the antigens and RSV-A. Three weeks after the last immunization, the mice were challenged by the intranasal route with 10

5

TCID

50

of RSV-A. The mice were sacrificed 5 days after the challenge: they were subjected to a cardiac puncture; the lungs were removed in order to titrate the virus in the lower respiratory tracts. The post-challenge sera were tested by ELISA against the viral antigens.

Results

BB-G4A, a protein which is obtained by coupling the peptide G4A to BB, protected the mice without any evidence of pulmonary virus. TT-G4A, a protein which is obtained by coupling the peptide G4A to TT, was less effective than BB-G4A as regards protecting the lungs; 2 mice out of 3 were protected, in this case, in one of which there was no evidence of pulmonary virus. The diminution in the virus count in the non-protected mouse was of the order of log

10

1.52 as compared with the controls immunized with PBS-A. The carrier:peptide ratios for BB-G4A and TT-G4A are ˜1:7 and ˜1:21, respectively. These results indicate, therefore, that BB is a better carrier for G4A than is TT.

The 2 products introduced high titres of antibodies against the immunization antigen (log

10

5.77 and 6.73, respectively, for the anti-BB-G4A and anti-TT-G4A sera post-immunization). By contrast, the animals which were immunized with these candidate vaccines had very low anti-RSV-A titres (logl

10

2.11±0.28 ard 2.42±0.48, respectively, for the anti-BB-G4A and anti-TT-G4A sera post-immunization).

Conclusions

BB-G4A is able to protect the mice against a challenge with RSV-A without any evidence of pulmonary virus. It therefore confirms its potential as an anti-RSV-A vaccine. The results also indicate that BB is a better carrier for G4A than is TT.

TABLE 6

Protective efficacy of BB-G4A in the BALB/c mouse against a challenge with RSV-A.

ELISA titres (log

10

)

Log

10

TCID

50

RSV-A

P.Im* vs

P.Im vs

P.Ch• vs

Product

/g of lung

antigen

RSV-A

RSV-A

20 μg BB-G4A

<1.45 ± 0.00

5.77 ± 0.00

2.11 ± 0.28

1.95 ± 0.00

20 μg TT-G4A

≦1.78 ± 0.38

6.41 ± 0.28

2.43 ± 0.48

2.27 ± 0.55

PBS-A

3.74 ± 0.29

—

2.03 ± 0.20

1.95 ± 0.00

RSV-A

<1.45 ± 0.00

—

4.82 ± 0.00

4.82 ± 0.00

*P.Im. = ELISA results post-immunization but before challenge.

•P.Ch. = ELISA results in the sera removed by cardiac puncture at the time of sacrifice.

EXAMPLE 7

Cross Protection of the Lungs of BALB/c Mice which Have Been Immunized with BBG2A by the Intraperitoneal Route with Respect to a Heterologous Challenge with RSV-B (Strain 8/60).

Materials and Methods

BALB/c mice were immunized twice or three times at 2-week intervals with 20 μg of BBG2A by means of intraperitoneal injection. Another group of mice were immunized in the same way with PBS-A as controls. Alhydrogel (Al(OH)

3

) (20% v/v) was employed as adjuvant for each immunization. A blood sampling was carried out before the first immunization in order to verify their seronegativity with regard to RSV-A. Three weeks after the last immunization, the mice were challenged intranasally with 10

5

TCID

50

of RSV-A or with 10

5

TCID

50

of RSV-B. The mice were sacrificed 5 days after the challenge: they were subjected to cardiac puncture; the lungs were removed in order to titrate the virus in the lower respiratory tracts. The post-challenge sera were tested by ELISA against the viral antigens.

Results

All the mice were seronegative for RSV-A at the beginning of the study. The first group, 11 mice out of 11, which were immunized with 20 μg of BBG2A, were protected with respect to a challenge with RSV-A. The second group, 11 mice out of 11, were also protected with respect to a heterologous challenge with RSV-B (Table 7).

Conclusions

The immunization of BALB/c mice with the BBG2A antigen confers protection not only against RSV-A but also with respect to a challenge with RSV-B. Antigen BBG2A therefore induces cross-protection with respect to a heterologous challenge.

TABLE 7

Cross-protection of the lungs of BALB/c mice which are immunized intraperitoneally

with BBG2A.

Challenge with RSV-a

Challenge with RSV-B

Log

10

Number of

Number of

TCID

50

a

/g of

immunized

Log

10

TCID

50

/g

immunized

lung

% protection

b

animals

of lung

% protection

animals

20 μg BBG2A

<1.45

c

± 0.00

100

11

1.68 ± 0.36

100

11

PBS-A

4.08 ± 0.60

0

4

4.25 ± 0.27

0

5

TCID

50

a

= tissue culture infectious dose 50

% protection

b

= a reduction in virus in the lungs of ≧log

10

1.8 as compared with the mean virus titre in the lungs of mice immunized with PBS-A.

<1.45

c

= limit of virus detection in this assay.

EXAMPLE 8

Study of the Priming Effect BB on Immun with BBG2A

BALB/c mice are sensitized to the BB protein are then given an injection of BBG2A. The anti-G2A titres obtained in these animals are compared with thot obtained in mice which are given two injections of BBG2A.

Material and Methods

BALB/C mice (N=5/batch) are immunized subcutaneously as described below:

D0

D14

batch 1

0.1

ml PBS

0.1

ml PBS

batch 2

20

μg BBG2A + FCA

20

μg BBG2A + FIA

batch 3

100

μg BB + FCA

20

μg BBG2A + FIA

FCA: Freund's complete adjuvant; FIA: Freund's incomplete adjuvant

The blood of the animals is sampled on D7 and D21 and the serum titres of anti-G2A IgM and IgG are determined individually by ELISA.

Results

Table of anti-G2A IgG titres

D7

D21

BATCH 2

BATCH 1

BATCH 2

BATCH 3

M1

2

2

3.81

3.51

M2

2

2

3.81

4.11

M3

2

2

3.81

4.41

M4

2

2

4.41

3.51

M5

2

2

3.81

4.71

m ± σ

2

2

3.93 ± 0.27

4.05 ± 0.54

In summary, the table of anti-G2A IgG titres at D7 and D21 is as follows:

D0

D7

D14

D21

batch 1

0.1

ml PBS

—

0.1

ml PBS

2

batch 2

20

μg BBG2A + FCA

2

20

μg BBG2A +

4.93 ±

FIA

0.27

batch 3

100

μg BB + FCA

—

20

μg BBG2A +

4.05 ±

FIA

0.54

BATCH 2: 2 Injections of BBG2A

Anti-G2A IgG is not detected at one week after the first injection of 20 μg of BBG2A. On the other hand, there is a strong production of anti-G2A IgG, approximately 4 log

10

, at one week after the second injection of BBG2A.

BATCH 3: Injection No. 1=BB, Injection No. 2=BBG2A

After the mice have been sensitized with 100 μg of BB, one injection of 20 μg of BBG2A is sufficient to induce an anti-G2A IgG titre of 4 log

10

, which is a titre which is similar to that obtained with 2 injections of 20 μg of BBG2A.

Conclusion

These results demonstrate that BB induces the production of Th memory cells which supplied the help required to the G2A-specific B cells at the time of the primary immunization with BBG2A, resulting in a secondary response of the IgG type. Thus, naive B cells can therefore be stimulated to produce anti-G2A antibodies.

BB thus supplies the T-cell help which is appropriate for producing antibodies directed against G2A; in this respect, it behaves as a carrier protein.

303 base pairs

nucleotide

single

linear

cDNA

CDS

1..303

1
ACC GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG CCG AGC AAA 48
Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gln Thr Gln Pro Ser Lys
1 5 10 15
CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG AAC AAC 96
Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro Pro Asn Lys Pro Asn Asn
20 25 30
GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG CCG TGC AGC ATC TGC AGC 144
Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys Ser
35 40 45
AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA CGT ATC CCG AAC AAA AAA 192
Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro Asn Lys Lys
50 55 60
CCG GGC AAA AAA ACC ACG ACC AAA CCG ACC AAA AAA CCG ACC TTC AAA 240
Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys
65 70 75 80
ACC ACC AAA AAA GAT CAT AAA CCG CAG ACC ACC AAA CCG AAA GAA GTG 288
Thr Thr Lys Lys Asp His Lys Pro Gln Thr Thr Lys Pro Lys Glu Val
85 90 95
CCG ACC ACC AAA CCG 303
Pro Thr Thr Lys Pro
100

303 base pairs

nucleotide

single

linear

cDNA

CDS

1..303

2
ACC GCG CAG ACC AAA GGC CGT ATC ACC ACC AGC ACC CAG ACC AAC AAA 48
Thr Ala Gln Thr Lys Gly Arg Ile Thr Thr Ser Thr Gln Thr Asn Lys
1 5 10 15
CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG CCG AAA AAA CCG AAA GAT 96
Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro Pro Lys Lys Pro Lys Asp
20 25 30
GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG CCC TGC AGC ATC TGC GGC 144
Asp Tyr His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys Gly
35 40 45
AAC AAC CAG CTG TGC AAA AGC ATC TGC AAA ACC ATC CCG AGC AAC AAA 192
Asn Asn Gln Leu Cys Lys Ser Ile Cys Lys Thr Ile Pro Ser Asn Lys
50 55 60
CCG AAA AAG AAA CCG ACC ATC AAA CCG ACC AAC AAA CCG ACC ACC AAA 240
Pro Lys Lys Lys Pro Thr Ile Lys Pro Thr Asn Lys Pro Thr Thr Lys
65 70 75 80
ACC ACC AAC AAA CGT GAT CCG AAA ACC CCG GCG AAA ATG CCG AAG AAG 288
Thr Thr Asn Lys Arg Asp Pro Lys Thr Pro Ala Lys Met Pro Lys Lys
85 90 95
GAA ATC ATC ACC AAC 303
Glu Ile Ile Thr Asn
100

303 base pairs

nucleotide

single

linear

cDNA

CDS

1..303

3
ACC GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG CCG AGC AAA 48
Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gln Thr Gln Pro Ser Lys
1 5 10 15
CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG AAC AAC 96
Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro Pro Asn Lys Pro Asn Asn
20 25 30
GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG CCG AGC AGC ATC TGC AGC 144
Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Ser Ser Ile Cys Ser
35 40 45
AAC AAC CCG ACC TGC TGG GCG ATC AGC AAA CGT ATC CCG AAC AAA AAA 192
Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys Arg Ile Pro Asn Lys Lys
50 55 60
CCG GGC AAA AAA ACC ACG ACC AAA CCG ACC AAA AAA CCG ACC TTC AAA 240
Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys
65 70 75 80
ACC ACC AAA AAA GAT CAT AAA CCG CAG ACC ACC AAA CCG AAA GAA GTG 288
Thr Thr Lys Lys Asp His Lys Pro Gln Thr Thr Lys Pro Lys Glu Val
85 90 95
CCG ACC ACC AAA CCG 303
Pro Thr Thr Lys Pro
100

303 base pairs

nucleotide

single

linear

cDNA

CDS

1..303

4
ACC GCG CAG ACC AAA GGC CGT ATC ACC ACC AGC ACC CAG ACC AAC AAA 48
Thr Ala Gln Thr Lys Gly Arg Ile Thr Thr Ser Thr Gln Thr Asn Lys
1 5 10 15
CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG CCG AAA AAA CCG AAA GAT 96
Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro Pro Lys Lys Pro Lys Asp
20 25 30
GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG CCC AGC AGC ATC TGC GGC 144
Asp Tyr His Phe Glu Val Phe Asn Phe Val Pro Ser Ser Ile Cys Gly
35 40 45
AAC AAC CAG CTG TGC AAA AGC ATC AGC AAA ACC ATC CCG AGC AAC AAA 192
Asn Asn Gln Leu Cys Lys Ser Ile Ser Lys Thr Ile Pro Ser Asn Lys
50 55 60
CCG AAA AAG AAA CCG ACC ATC AAA CCG ACC AAC AAA CCG ACC ACC AAA 240
Pro Lys Lys Lys Pro Thr Ile Lys Pro Thr Asn Lys Pro Thr Thr Lys
65 70 75 80
ACC ACC AAC AAA CGT GAT CCG AAA ACC CCG GCG AAA ATG CCG AAG AAG 288
Thr Thr Asn Lys Arg Asp Pro Lys Thr Pro Ala Lys Met Pro Lys Lys
85 90 95
GAA ATC ATC ACC AAC 303
Glu Ile Ile Thr Asn
100

42 base pairs

nucleotide

single

linear

cDNA

CDS

1..42

5
AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA 42
Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys
1 5 10

42 base pairs

nucleotide

single

linear

cDNA

CDS

1..42

6
AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC TGC AAA 42
Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Cys Lys
1 5 10

42 base pairs

nucleotide

single

linear

cDNA

CDS

1..42

7
AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC AGC AAA 42
Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys
1 5 10

42 base pairs

nucleotide

single

linear

cDNA

CDS

1..42

8
AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC AGC AAA 42
Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Ser Lys
1 5 10

14 amino acids

amino acid

single

linear

Peptide

Modified-site

/Xaa means Orn

9
Ser Ile Asp Ser Asn Asn Pro Thr Xaa Trp Ala Ile Cys Lys
1 5 10

14 amino acids

amino acid

single

linear

Peptide

Modified-site

/Xaa means Orn

10
Ser Ile Asp Gly Asn Asn Gln Leu Xaa Lys Ser Ile Cys Lys
1 5 10

14 amino acids

amino acid

single

linear

Peptide

Modified-site

/Xaa means Orn

11
Ser Ile Asp Ser Asn Asn Pro Thr Xaa Trp Ala Ile Ser Lys
1 5 10

14 amino acids

amino acid

single

linear

Peptide

Modified-site

/Xaa means Orn

12
Ser Ile Asp Gly Asn Asn Gln Leu Xaa Lys Ser Ile Ser Lys
1 5 10

48 base pairs

nucleotide

single

linear

cDNA

CDS

1..48

13
AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG 48
Ser Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro Pro Asn Lys Pro
1 5 10 15

303 base pairs

nucleotide

single

linear

cDNA

CDS

1..303

14
ACC GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG CCG AGC AAA 48
Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gln Thr Gln Pro Ser Lys
1 5 10 15
CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG AAC AAC 96
Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro Pro Asn Lys Pro Asn Asn
20 25 30
GAT TCC CAT TCC GAA GTG TCC AAC TCC GTG CCG AGC AGC ATC TGC AGC 144
Asp Ser His Ser Glu Val Ser Asn Ser Val Pro Ser Ser Ile Cys Ser
35 40 45
AAC AAC CCG ACC TGC TGG GCG ATC AGC AAA CGT ATC CCG AAC AAA AAA 192
Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys Arg Ile Pro Asn Lys Lys
50 55 60
CCG GGC AAA AAA ACC ACG ACC AAA CCG ACC AAA AAA CCG ACC TTC AAA 240
Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys
65 70 75 80
ACC ACC AAA AAA GAT CAT AAA CCG CAG ACC ACC AAA CCG AAA GAA GTG 288
Thr Thr Lys Lys Asp His Lys Pro Gln Thr Thr Lys Pro Lys Glu Val
85 90 95
CCG ACC ACC AAA CCG 303
Pro Thr Thr Lys Pro
100

51 base pairs

nucleotide

single

linear

cDNA

CDS

1..51

15
GTG CCG TGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC TGC 48
Val Pro Cys Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys
1 5 10 15
AAA 51
Lys

51 base pairs

nucleotide

single

linear

cDNA

CDS

1..51

16
GTG CCG AGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC AGC 48
Val Pro Ser Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Ser
1 5 10 15
AAA 51
Lys

51 base pairs

nucleotide

single

linear

cDNA

CDS

1..51

17
GTG CCC TGC AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC TGC 48
Val Pro Cys Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Cys
1 5 10 15
AAA 51
Lys

51 base pairs

nucleotide

single

linear

cDNA

CDS

1..51

18
GTG CCC AGC AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC AGC 48
Val Pro Ser Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Ser
1 5 10 15
AAA 51
Lys

17 amino acids

amino acid

single

linear

Peptide

Modified-site

/Xaa means Orn

Modified-site

/Xaa means Orn

19
Val Pro Asp Ser Ile Asp Ser Asn Asn Pro Thr Xaa Trp Ala Ile Xaa
1 5 10 15
Lys

17 amino acids

amino acid

single

linear

Peptide

Modified-site

/Xaa means Orn

20
Val Pro Ser Ser Ile Asp Ser Asn Asn Pro Thr Xaa Trp Ala Ile Ser
1 5 10 15
Lys

17 amino acids

amino acid

single

linear

Peptide

Modified-site

/Xaa means Orn

Modified-site

/Xaa means Orn

21
Val Pro Asp Ser Ile Asp Gly Asn Asn Gln Leu Xaa Lys Ser Ile Xaa
1 5 10 15
Lys

17 amino acids

amino acid

single

linear

Peptide

Modified-site

/Xaa means Orn

22
Val Pro Ser Ser Ile Asp Gly Asn Asn Gln Leu Xaa Lys Ser Ile Ser
1 5 10 15
Lys

183 base pairs

nucleotide

single

linear

cDNA

CDS

1..183

23
CAG ACC CAG CCG AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG 48
Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro
1 5 10 15
CCG AAC AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG 96
Pro Asn Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val
20 25 30
CCG TGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA 144
Pro Cys Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys
35 40 45
CGT ATC CCG AAC AAA AAA CCG GGC AAA AAA ACC ACG ACC 183
Arg Ile Pro Asn Lys Lys Pro Gly Lys Lys Thr Thr Thr
50 55 60

177 base pairs

nucleotide

single

linear

cDNA

CDS

1..177

24
CAG ACC CAG CCG AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG 48
Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro
1 5 10 15
CCG AAC AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG 96
Pro Asn Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val
20 25 30
CCG TGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA 144
Pro Cys Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys
35 40 45
CGT ATC CCG AAC AAA AAA CCG GGC AAA AAA ACC 177
Arg Ile Pro Asn Lys Lys Pro Gly Lys Lys Thr
50 55

171 base pairs

nucleotide

single

linear

cDNA

CDS

1..171

25
CAG ACC CAG CCG AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG 48
Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro
1 5 10 15
CCG AAC AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG 96
Pro Asn Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val
20 25 30
CCG TGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA 144
Pro Cys Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys
35 40 45
CGT ATC CCG AAC AAA AAA CCG GGC AAA 171
Arg Ile Pro Asn Lys Lys Pro Gly Lys
50 55

165 base pairs

nucleotide

single

linear

cDNA

CDS

1..165

26
CAG ACC CAG CCG AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG 48
Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro
1 5 10 15
CCG AAC AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG 96
Pro Asn Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val
20 25 30
CCG TGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA 144
Pro Cys Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys
35 40 45
CGT ATC CCG AAC AAA AAA CCG 165
Arg Ile Pro Asn Lys Lys Pro
50 55

159 base pairs

nucleotide

single

linear

cDNA

CDS

1..159

27
CAG ACC CAG CCG AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG 48
Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro
1 5 10 15
CCG AAC AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG 96
Pro Asn Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val
20 25 30
CCG TGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA 144
Pro Cys Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys
35 40 45
CGT ATC CCG AAC AAA 159
Arg Ile Pro Asn Lys
50

153 base pairs

nucleotide

single

linear

cDNA

CDS

1..153

28
CAG ACC CAG CCG AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG 48
Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro
1 5 10 15
CCG AAC AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG 96
Pro Asn Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val
20 25 30
CCG TGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA 144
Pro Cys Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys
35 40 45
CGT ATC CCG 153
Arg Ile Pro
50

99 base pairs

nucleotide

single

linear

cDNA

CDS

1..99

29
AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG CCG TGC 48
Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Cys
1 5 10 15
AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA CGT ATC 96
Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile
20 25 30
CCG 99
Pro

183 base pairs

nucleotide

single

linear

cDNA

CDS

1..183

30
CAG ACC CAG CCG AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG 48
Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro
1 5 10 15
CCG AAC AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG 96
Pro Asn Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val
20 25 30
CCG AGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC AGC AAA 144
Pro Ser Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys
35 40 45
CGT ATC CCG AAC AAA AAA CCG GGC AAA AAA ACC ACG ACC 183
Arg Ile Pro Asn Lys Lys Pro Gly Lys Lys Thr Thr Thr
50 55 60

177 base pairs

nucleotide

single

linear

cDNA

CDS

1..177

31
CAG ACC CAG CCG AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG 48
Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro
1 5 10 15
CCG AAC AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG 96
Pro Asn Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val
20 25 30
CCG AGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC AGC AAA 144
Pro Ser Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys
35 40 45
CGT ATC CCG AAC AAA AAA CCG GGC AAA AAA ACC 177
Arg Ile Pro Asn Lys Lys Pro Gly Lys Lys Thr
50 55

171 base pairs

nucleotide

single

linear

cDNA

CDS

1..171

32
CAG ACC CAG CCG AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG 48
Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro
1 5 10 15
CCG AAC AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG 96
Pro Asn Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val
20 25 30
CCG AGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC AGC AAA 144
Pro Ser Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys
35 40 45
CGT ATC CCG AAC AAA AAA CCG GGC AAA 171
Arg Ile Pro Asn Lys Lys Pro Gly Lys
50 55

165 base pairs

nucleotide

single

linear

cDNA

CDS

1..165

33
CAG ACC CAG CCG AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG 48
Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro
1 5 10 15
CCG AAC AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG 96
Pro Asn Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val
20 25 30
CCG AGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC AGC AAA 144
Pro Ser Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys
35 40 45
CGT ATC CCG AAC AAA AAA CCG 165
Arg Ile Pro Asn Lys Lys Pro
50 55

159 base pairs

nucleotide

single

linear

cDNA

CDS

1..159

34
CAG ACC CAG CCG AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG 48
Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro
1 5 10 15
CCG AAC AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG 96
Pro Asn Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val
20 25 30
CCG AGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC AGC AAA 144
Pro Ser Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys
35 40 45
CGT ATC CCG AAC AAA 159
Arg Ile Pro Asn Lys
50

153 base pairs

nucleotide

single

linear

cDNA

CDS

1..153

35
CAG ACC CAG CCG AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG 48
Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro
1 5 10 15
CCG AAC AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG 96
Pro Asn Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val
20 25 30
CCG AGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC AGC AAA 144
Pro Ser Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys
35 40 45
CGT ATC CCG 153
Arg Ile Pro
50

99 base pairs

nucleotide

single

linear

cDNA

CDS

1..99

36
AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG CCG AGC 48
Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Ser
1 5 10 15
AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC AGC AAA CGT ATC 96
Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys Arg Ile
20 25 30
CCG 99
Pro

183 base pairs

nucleotide

single

linear

cDNA

CDS

1..183

37
AGC ACC CAG ACC AAC AAA CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG 48
Ser Thr Gln Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro
1 5 10 15
CCG AAA AAA CCG AAA GAT GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG 96
Pro Lys Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn Phe Val
20 25 30
CCC TGC AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC TGC AAA 144
Pro Cys Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Cys Lys
35 40 45
ACC ATC CCG AGC AAC AAA CCG AAA AAG AAA CCG ACC ATC 183
Thr Ile Pro Ser Asn Lys Pro Lys Lys Lys Pro Thr Ile
50 55 60

177 base pairs

nucleotide

single

linear

cDNA

CDS

1..177

38
AGC ACC CAG ACC AAC AAA CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG 48
Ser Thr Gln Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro
1 5 10 15
CCG AAA AAA CCG AAA GAT GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG 96
Pro Lys Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn Phe Val
20 25 30
CCC TGC AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC TGC AAA 144
Pro Cys Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Cys Lys
35 40 45
ACC ATC CCG AGC AAC AAA CCG AAA AAG AAA CCG 177
Thr Ile Pro Ser Asn Lys Pro Lys Lys Lys Pro
50 55

171 base pairs

nucleotide

single

linear

cDNA

CDS

1..171

39
AGC ACC CAG ACC AAC AAA CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG 48
Ser Thr Gln Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro
1 5 10 15
CCG AAA AAA CCG AAA GAT GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG 96
Pro Lys Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn Phe Val
20 25 30
CCC TGC AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC TGC AAA 144
Pro Cys Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Cys Lys
35 40 45
ACC ATC CCG AGC AAC AAA CCG AAA AAG 171
Thr Ile Pro Ser Asn Lys Pro Lys Lys
50 55

165 base pairs

nucleotide

single

linear

cDNA

CDS

1..165

40
AGC ACC CAG ACC AAC AAA CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG 48
Ser Thr Gln Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro
1 5 10 15
CCG AAA AAA CCG AAA GAT GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG 96
Pro Lys Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn Phe Val
20 25 30
CCC TGC AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC TGC AAA 144
Pro Cys Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Cys Lys
35 40 45
ACC ATC CCG AGC AAC AAA CCG 165
Thr Ile Pro Ser Asn Lys Pro
50 55

159 base pairs

nucleotide

single

linear

cDNA

CDS

1..159

41
AGC ACC CAG ACC AAC AAA CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG 48
Ser Thr Gln Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro
1 5 10 15
CCG AAA AAA CCG AAA GAT GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG 96
Pro Lys Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn Phe Val
20 25 30
CCC TGC AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC TGC AAA 144
Pro Cys Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Cys Lys
35 40 45
ACC ATC CCG AGC AAC 159
Thr Ile Pro Ser Asn
50

153 base pairs

nucleotide

single

linear

cDNA

CDS

1..153

42
AGC ACC CAG ACC AAC AAA CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG 48
Ser Thr Gln Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro
1 5 10 15
CCG AAA AAA CCG AAA GAT GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG 96
Pro Lys Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn Phe Val
20 25 30
CCC TGC AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC TGC AAA 144
Pro Cys Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Cys Lys
35 40 45
ACC ATC CCG 153
Thr Ile Pro
50

99 base pairs

nucleotide

single

linear

cDNA

CDS

1..99

43
AAA CCG AAA GAT GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG CCC TGC 48
Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn Phe Val Pro Cys
1 5 10 15
AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC TGC AAA ACC ATC 96
Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Cys Lys Thr Ile
20 25 30
CCG 99
Pro

183 base pairs

nucleotide

single

linear

cDNA

CDS

1..183

44
AGC ACC CAG ACC AAC AAA CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG 48
Ser Thr Gln Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro
1 5 10 15
CCG AAA AAA CCG AAA GAT GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG 96
Pro Lys Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn Phe Val
20 25 30
CCC AGC AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC AGC AAA 144
Pro Ser Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Ser Lys
35 40 45
ACC ATC CCG AGC AAC AAA CCG AAA AAG AAA CCG ACC ATC 183
Thr Ile Pro Ser Asn Lys Pro Lys Lys Lys Pro Thr Ile
50 55 60

177 base pairs

nucleotide

single

linear

cDNA

CDS

1..177

45
AGC ACC CAG ACC AAC AAA CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG 48
Ser Thr Gln Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro
1 5 10 15
CCG AAA AAA CCG AAA GAT GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG 96
Pro Lys Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn Phe Val
20 25 30
CCC AGC AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC AGC AAA 144
Pro Ser Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Ser Lys
35 40 45
ACC ATC CCG AGC AAC AAA CCG AAA AAG AAA CCG 177
Thr Ile Pro Ser Asn Lys Pro Lys Lys Lys Pro
50 55

171 base pairs

nucleotide

single

linear

cDNA

CDS

1..171

46
AGC ACC CAG ACC AAC AAA CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG 48
Ser Thr Gln Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro
1 5 10 15
CCG AAA AAA CCG AAA GAT GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG 96
Pro Lys Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn Phe Val
20 25 30
CCC AGC AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC AGC AAA 144
Pro Ser Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Ser Lys
35 40 45
ACC ATC CCG AGC AAC AAA CCG AAA AAG 171
Thr Ile Pro Ser Asn Lys Pro Lys Lys
50 55

165 base pairs

nucleotide

single

linear

cDNA

CDS

1..165

47
AGC ACC CAG ACC AAC AAA CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG 48
Ser Thr Gln Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro
1 5 10 15
CCG AAA AAA CCG AAA GAT GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG 96
Pro Lys Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn Phe Val
20 25 30
CCC AGC AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC AGC AAA 144
Pro Ser Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Ser Lys
35 40 45
ACC ATC CCG AGC AAC AAA CCG 165
Thr Ile Pro Ser Asn Lys Pro
50 55

159 base pairs

nucleotide

single

linear

cDNA

CDS

1..159

48
AGC ACC CAG ACC AAC AAA CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG 48
Ser Thr Gln Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro
1 5 10 15
CCG AAA AAA CCG AAA GAT GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG 96
Pro Lys Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn Phe Val
20 25 30
CCC AGC AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC AGC AAA 144
Pro Ser Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Ser Lys
35 40 45
ACC ATC CCG AGC AAC 159
Thr Ile Pro Ser Asn
50

153 base pairs

nucleotide

single

linear

cDNA

CDS

1..153

49
AGC ACC CAG ACC AAC AAA CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG 48
Ser Thr Gln Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro
1 5 10 15
CCG AAA AAA CCG AAA GAT GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG 96
Pro Lys Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn Phe Val
20 25 30
CCC AGC AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC AGC AAA 144
Pro Ser Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Ser Lys
35 40 45
ACC ATC CCG 153
Thr Ile Pro
50

99 base pairs

nucleotide

single

linear

cDNA

CDS

1..99

50
AAA CCG AAA GAT GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG CCC AGC 48
Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn Phe Val Pro Ser
1 5 10 15
AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC AGC AAA ACC ATC 96
Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Ser Lys Thr Ile
20 25 30
CCG 99
Pro

303 base pairs

nucleotide

single

linear

cDNA

CDS

1..303

51
CAA AAC AGA AAA ATC AAA GGT CAA TCA ACA CTA CCA GCC ACA AGA AAA 48
Gln Asn Arg Lys Ile Lys Gly Gln Ser Thr Leu Pro Ala Thr Arg Lys
1 5 10 15
CCA CCA ATT AAT CCA TCA GGA AGC ATC CCA CCA GAA AAC CAT CAA GAC 96
Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro Pro Glu Asn His Gln Asp
20 25 30
CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT CCC TGC AGT ACA TGT GAA 144
His Asn Asn Phe Gln Thr Leu Pro Tyr Val Pro Cys Ser Thr Cys Glu
35 40 45
GGT AAT CTT GCA TGC TTA TCA CTC TGC CAT ATT GAG ACG GAA AGA GCA 192
Gly Asn Leu Ala Cys Leu Ser Leu Cys His Ile Glu Thr Glu Arg Ala
50 55 60
CCA AGC AGA GCA CCA ACA ATC ACC CTC AAA AAG ACA CCA AAA CCA AAA 240
Pro Ser Arg Ala Pro Thr Ile Thr Leu Lys Lys Thr Pro Lys Pro Lys
65 70 75 80
ACC ACA AAA AAG CCA ACC AAG ACA ACA ATC CAT CAC AGA ACC AGC CCA 288
Thr Thr Lys Lys Pro Thr Lys Thr Thr Ile His His Arg Thr Ser Pro
85 90 95
GAA ACC AAA CTG CAA 303
Glu Thr Lys Leu Gln
100

303 base pairs

nucleotide

single

linear

cDNA

CDS

1..303

52
CAA AAC AGA AAA ATC AAA GGT CAA TCA ACA CTA CCA GCC ACA AGA AAA 48
Gln Asn Arg Lys Ile Lys Gly Gln Ser Thr Leu Pro Ala Thr Arg Lys
1 5 10 15
CCA CCA ATT AAT CCA TCA GGA AGC ATC CCA CCA GAA AAC CAT CAA GAC 96
Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro Pro Glu Asn His Gln Asp
20 25 30
CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT CCC AGC AGT ACA TGT GAA 144
His Asn Asn Phe Gln Thr Leu Pro Tyr Val Pro Ser Ser Thr Cys Glu
35 40 45
GGT AAT CTT GCA TGC TTA TCA CTC AGC CAT ATT GAG ACG GAA AGA GCA 192
Gly Asn Leu Ala Cys Leu Ser Leu Ser His Ile Glu Thr Glu Arg Ala
50 55 60
CCA AGC AGA GCA CCA ACA ATC ACC CTC AAA AAG ACA CCA AAA CCA AAA 240
Pro Ser Arg Ala Pro Thr Ile Thr Leu Lys Lys Thr Pro Lys Pro Lys
65 70 75 80
ACC ACA AAA AAG CCA ACC AAG ACA ACA ATC CAT CAC AGA ACC AGC CCA 288
Thr Thr Lys Lys Pro Thr Lys Thr Thr Ile His His Arg Thr Ser Pro
85 90 95
GAA ACC AAA CTG CAA 303
Glu Thr Lys Leu Gln
100

183 base pairs

nucleotide

single

linear

cDNA

CDS

1..183

53
CTA CCA GCC ACA AGA AAA CCA CCA ATT AAT CCA TCA GGA AGC ATC CCA 48
Leu Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro
1 5 10 15
CCA GAA AAC CAT CAA GAC CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT 96
Pro Glu Asn His Gln Asp His Asn Asn Phe Gln Thr Leu Pro Tyr Val
20 25 30
CCC TGC AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC TGC CAT 144
Pro Cys Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Cys His
35 40 45
ATT GAG ACG GAA AGA GCA CCA AGC AGA GCA CCA ACA ATC 183
Ile Glu Thr Glu Arg Ala Pro Ser Arg Ala Pro Thr Ile
50 55 60

177 base pairs

nucleotide

single

linear

cDNA

CDS

1..177

54
CTA CCA GCC ACA AGA AAA CCA CCA ATT AAT CCA TCA GGA AGC ATC CCA 48
Leu Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro
1 5 10 15
CCA GAA AAC CAT CAA GAC CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT 96
Pro Glu Asn His Gln Asp His Asn Asn Phe Gln Thr Leu Pro Tyr Val
20 25 30
CCC TGC AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC TGC CAT 144
Pro Cys Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Cys His
35 40 45
ATT GAG ACG GAA AGA GCA CCA AGC AGA GCA CCA 177
Ile Glu Thr Glu Arg Ala Pro Ser Arg Ala Pro
50 55

171 base pairs

nucleotide

single

linear

cDNA

CDS

1..171

55
CTA CCA GCC ACA AGA AAA CCA CCA ATT AAT CCA TCA GGA AGC ATC CCA 48
Leu Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro
1 5 10 15
CCA GAA AAC CAT CAA GAC CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT 96
Pro Glu Asn His Gln Asp His Asn Asn Phe Gln Thr Leu Pro Tyr Val
20 25 30
CCC TGC AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC TGC CAT 144
Pro Cys Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Cys His
35 40 45
ATT GAG ACG GAA AGA GCA CCA AGC AGA 171
Ile Glu Thr Glu Arg Ala Pro Ser Arg
50 55

165 base pairs

nucleotide

single

linear

cDNA

CDS

1..165

56
CTA CCA GCC ACA AGA AAA CCA CCA ATT AAT CCA TCA GGA AGC ATC CCA 48
Leu Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro
1 5 10 15
CCA GAA AAC CAT CAA GAC CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT 96
Pro Glu Asn His Gln Asp His Asn Asn Phe Gln Thr Leu Pro Tyr Val
20 25 30
CCC TGC AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC TGC CAT 144
Pro Cys Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Cys His
35 40 45
ATT GAG ACG GAA AGA GCA CCA 165
Ile Glu Thr Glu Arg Ala Pro
50 55

159 base pairs

nucleotide

single

linear

cDNA

CDS

1..159

57
CTA CCA GCC ACA AGA AAA CCA CCA ATT AAT CCA TCA GGA AGC ATC CCA 48
Leu Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro
1 5 10 15
CCA GAA AAC CAT CAA GAC CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT 96
Pro Glu Asn His Gln Asp His Asn Asn Phe Gln Thr Leu Pro Tyr Val
20 25 30
CCC TGC AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC TGC CAT 144
Pro Cys Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Cys His
35 40 45
ATT GAG ACG GAA AGA 159
Ile Glu Thr Glu Arg
50

153 base pairs

nucleotide

single

linear

cDNA

CDS

1..153

58
CTA CCA GCC ACA AGA AAA CCA CCA ATT AAT CCA TCA GGA AGC ATC CCA 48
Leu Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro
1 5 10 15
CCA GAA AAC CAT CAA GAC CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT 96
Pro Glu Asn His Gln Asp His Asn Asn Phe Gln Thr Leu Pro Tyr Val
20 25 30
CCC TGC AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC TGC CAT 144
Pro Cys Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Cys His
35 40 45
ATT GAG ACG 153
Ile Glu Thr
50

99 base pairs

nucleotide

single

linear

cDNA

CDS

1..99

59
AAC CAT CAA GAC CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT CCC TGC 48
Asn His Gln Asp His Asn Asn Phe Gln Thr Leu Pro Tyr Val Pro Cys
1 5 10 15
AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC TGC CAT ATT GAG 96
Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Cys His Ile Glu
20 25 30
ACG 99
Thr

183 base pairs

nucleotide

single

linear

cDNA

CDS

1..183

60
CTA CCA GCC ACA AGA AAA CCA CCA ATT AAT CCA TCA GGA AGC ATC CCA 48
Leu Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro
1 5 10 15
CCA GAA AAC CAT CAA GAC CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT 96
Pro Glu Asn His Gln Asp His Asn Asn Phe Gln Thr Leu Pro Tyr Val
20 25 30
CCC AGC AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC AGC CAT 144
Pro Ser Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Ser His
35 40 45
ATT GAG ACG GAA AGA GCA CCA AGC AGA GCA CCA ACA ATC 183
Ile Glu Thr Glu Arg Ala Pro Ser Arg Ala Pro Thr Ile
50 55 60

177 base pairs

nucleotide

single

linear

cDNA

CDS

1..177

61
CTA CCA GCC ACA AGA AAA CCA CCA ATT AAT CCA TCA GGA AGC ATC CCA 48
Leu Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro
1 5 10 15
CCA GAA AAC CAT CAA GAC CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT 96
Pro Glu Asn His Gln Asp His Asn Asn Phe Gln Thr Leu Pro Tyr Val
20 25 30
CCC AGC AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC AGC CAT 144
Pro Ser Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Ser His
35 40 45
ATT GAG ACG GAA AGA GCA CCA AGC AGA GCA CCA 177
Ile Glu Thr Glu Arg Ala Pro Ser Arg Ala Pro
50 55

171 base pairs

nucleotide

single

linear

cDNA

CDS

1..171

62
CTA CCA GCC ACA AGA AAA CCA CCA ATT AAT CCA TCA GGA AGC ATC CCA 48
Leu Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro
1 5 10 15
CCA GAA AAC CAT CAA GAC CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT 96
Pro Glu Asn His Gln Asp His Asn Asn Phe Gln Thr Leu Pro Tyr Val
20 25 30
CCC AGC AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC AGC CAT 144
Pro Ser Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Ser His
35 40 45
ATT GAG ACG GAA AGA GCA CCA AGC AGA 171
Ile Glu Thr Glu Arg Ala Pro Ser Arg
50 55

165 base pairs

nucleotide

single

linear

cDNA

CDS

1..165

63
CTA CCA GCC ACA AGA AAA CCA CCA ATT AAT CCA TCA GGA AGC ATC CCA 48
Leu Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro
1 5 10 15
CCA GAA AAC CAT CAA GAC CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT 96
Pro Glu Asn His Gln Asp His Asn Asn Phe Gln Thr Leu Pro Tyr Val
20 25 30
CCC AGC AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC AGC CAT 144
Pro Ser Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Ser His
35 40 45
ATT GAG ACG GAA AGA GCA CCA 165
Ile Glu Thr Glu Arg Ala Pro
50 55

159 base pairs

nucleotide

single

linear

cDNA

CDS

1..159

64
CTA CCA GCC ACA AGA AAA CCA CCA ATT AAT CCA TCA GGA AGC ATC CCA 48
Leu Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro
1 5 10 15
CCA GAA AAC CAT CAA GAC CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT 96
Pro Glu Asn His Gln Asp His Asn Asn Phe Gln Thr Leu Pro Tyr Val
20 25 30
CCC AGC AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC AGC CAT 144
Pro Ser Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Ser His
35 40 45
ATT GAG ACG GAA AGA 159
Ile Glu Thr Glu Arg
50

153 base pairs

nucleotide

single

linear

cDNA

CDS

1..153

65
CTA CCA GCC ACA AGA AAA CCA CCA ATT AAT CCA TCA GGA AGC ATC CCA 48
Leu Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro
1 5 10 15
CCA GAA AAC CAT CAA GAC CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT 96
Pro Glu Asn His Gln Asp His Asn Asn Phe Gln Thr Leu Pro Tyr Val
20 25 30
CCC AGC AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC AGC CAT 144
Pro Ser Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Ser His
35 40 45
ATT GAG ACG 153
Ile Glu Thr
50

99 base pairs

nucleotide

single

linear

cDNA

CDS

1..99

66
AAC CAT CAA GAC CAC AAC AAC TTC CAA ACA CTC CCC TAT GTT CCC AGC 48
Asn His Gln Asp His Asn Asn Phe Gln Thr Leu Pro Tyr Val Pro Ser
1 5 10 15
AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC AGC CAT ATT GAG 96
Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Ser His Ile Glu
20 25 30
ACG 99
Thr

51 base pairs

nucleotide

single

linear

cDNA

CDS

1..51

67
GTT CCC TGC AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC TGC 48
Val Pro Cys Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Cys
1 5 10 15
CAT 51
His

51 base pairs

nucleotide

single

linear

cDNA

CDS

1..51

68
GTT CCC AGC AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC AGC 48
Val Pro Ser Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Ser
1 5 10 15
CAT 51
His

17 amino acids

amino acid

single

linear

Peptide

Modified-site

/Xaa means Orn

Modified-site

/Xaa means Orn

69
Val Pro Asp Ser Thr Asp Glu Gly Asn Leu Ala Xaa Leu Ser Leu Xaa
1 5 10 15
His

17 amino acids

amino acid

single

linear

Peptide

Modified-site

/Xaa means Orn

70
Val Pro Ser Ser Thr Asp Glu Gly Asn Leu Ala Xaa Leu Ser Leu Ser
1 5 10 15
His

42 base pairs

nucleotide

single

linear

cDNA

CDS

1..42

71
AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC TGC CAT 42
Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Cys His
1 5 10

42 base pairs

nucleotide

single

linear

cDNA

CDS

1..42

72
AGT ACA TGT GAA GGT AAT CTT GCA TGC TTA TCA CTC AGC CAT 42
Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Ser His
1 5 10

14 amino acids

amino acid

single

linear

Peptide

Modified-site

/Xaa means Orn

73
Ser Thr Asp Glu Gly Asn Leu Ala Xaa Leu Ser Leu Ser His
1 5 10

657 base pairs

nucleotide

single

linear

cDNA

CDS

1..657

74
AAA TAT GGA GTA AGT GAC TAT TAC AAG AAT CTA ATC AAC AAT GCC AAA 48
Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asn Asn Ala Lys
1 5 10 15
ACT GTT GAA GGC GTA AAA GAC CTT CAA GCA CAA GTT GTT GAA TCA GCG 96
Thr Val Glu Gly Val Lys Asp Leu Gln Ala Gln Val Val Glu Ser Ala
20 25 30
AAG AAA GCG CGT ATT TCA GAA GCA ACA GAT GGC TTA TCT GAT TTC TTG 144
Lys Lys Ala Arg Ile Ser Glu Ala Thr Asp Gly Leu Ser Asp Phe Leu
35 40 45
AAA TCA CAA ACA CCT GCT GAA GAT ACT GTT AAA TCA ATT GAA TTA GCT 192
Lys Ser Gln Thr Pro Ala Glu Asp Thr Val Lys Ser Ile Glu Leu Ala
50 55 60
GAA GCT AAA GTC TTA GCT AAC AGA GAA CTT GAC AAA TAT GGA GTA AGT 240
Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser
65 70 75 80
GAC TAT CAC AAG AAC CTA ATC AAC AAT GCC AAA ACT GTT GAA GGT GTA 288
Asp Tyr His Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val
85 90 95
AAA GAC CTT CAA GCA CAA GTT GTT GAA TCA GCG AAG AAA GCG CGT ATT 336
Lys Asp Leu Gln Ala Gln Val Val Glu Ser Ala Lys Lys Ala Arg Ile
100 105 110
TCA GAA GCA ACA GAT GGC TTA TCT GAT TTC TTG AAA TCA CAA ACA CCT 384
Ser Glu Ala Thr Asp Gly Leu Ser Asp Phe Leu Lys Ser Gln Thr Pro
115 120 125
GCT GAA GAT ACT GTT AAA TCA ATT GAA TTA GCT GAA GCT AAA GTC TTA 432
Ala Glu Asp Thr Val Lys Ser Ile Glu Leu Ala Glu Ala Lys Val Leu
130 135 140
GCT AAC AGA GAA CTT GAC AAA TAT GGA GTA AGT GAC TAT TAC AAG AAC 480
Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn
145 150 155 160
CTA ATC AAC AAT GCC AAA ACT GTT GAA GGT GTA AAA GCA CTG ATA GAT 528
Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile Asp
165 170 175
GAA ATT TTA GCT GCA TTA CCT AAG ACT GAC ACT TAC AAA TTA ATC CTT 576
Glu Ile Leu Ala Ala Leu Pro Lys Thr Asp Thr Tyr Lys Leu Ile Leu
180 185 190
AAT GGT AAA ACA TTG AAA GGC GAA ACA ACT ACT GAA GCT GTT GAT GCT 624
Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala Val Asp Ala
195 200 205
GCT ACT GCA AGA TCT TTC AAT TTC CCT ATC CTC 657
Ala Thr Ala Arg Ser Phe Asn Phe Pro Ile Leu
210 215

324 base pairs

nucleotide

single

linear

cDNA

CDS

1..324

75
AAA TAT GGA GTA AGT GAC TAT CAC AAG AAC CTA ATC AAC AAT GCC AAA 48
Lys Tyr Gly Val Ser Asp Tyr His Lys Asn Leu Ile Asn Asn Ala Lys
1 5 10 15
ACT GTT GAA GGT GTA AAA GAC CTT CAA GCA CAA GTT GTT GAA TCA GCG 96
Thr Val Glu Gly Val Lys Asp Leu Gln Ala Gln Val Val Glu Ser Ala
20 25 30
AAG AAA GCG CGT ATT TCA GAA GCA ACA GAT GGC TTA TCT GAT TTC TTG 144
Lys Lys Ala Arg Ile Ser Glu Ala Thr Asp Gly Leu Ser Asp Phe Leu
35 40 45
AAA TCA CAA ACA CCT GCT GAA GAT ACT GTT AAA TCA ATT GAA TTA GCT 192
Lys Ser Gln Thr Pro Ala Glu Asp Thr Val Lys Ser Ile Glu Leu Ala
50 55 60
GAA GCT AAA GTC TTA GCT AAC AGA GAA CTT GAC AAA TAT GGA GTA AGT 240
Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser
65 70 75 80
GAC TAT TAC AAG AAC CTA ATC AAC AAT GCC AAA ACT GTT GAA GGT GTA 288
Asp Tyr Tyr Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val
85 90 95
AAA GCA CTG ATA GAT GAA ATT TTA GCT GCA TTA CCT 324
Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro
100 105

1050 base pairs

nucleotide

single

linear

cDNA

CDS

1..1050

76
ATG AAA GCA ATT TTC GTA CTG AAT GCG CAA CAC GAT GAA GCC GTA GAC 48
Met Lys Ala Ile Phe Val Leu Asn Ala Gln His Asp Glu Ala Val Asp
1 5 10 15
GCG AAT TTC GAC CAA TTC AAC AAA TAT GGA GTA AGT GAC TAT TAC AAG 96
Ala Asn Phe Asp Gln Phe Asn Lys Tyr Gly Val Ser Asp Tyr Tyr Lys
20 25 30
AAT CTA ATC AAC AAT GCC AAA ACT GTT GAA GGC GTA AAA GAC CTT CAA 144
Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Asp Leu Gln
35 40 45
GCA CAA GTT GTT GAA TCA GCG AAG AAA GCG CGT ATT TCA GAA GCA ACA 192
Ala Gln Val Val Glu Ser Ala Lys Lys Ala Arg Ile Ser Glu Ala Thr
50 55 60
GAT GGC TTA TCT GAT TTC TTG AAA TCA CAA ACA CCT GCT GAA GAT ACT 240
Asp Gly Leu Ser Asp Phe Leu Lys Ser Gln Thr Pro Ala Glu Asp Thr
65 70 75 80
GTT AAA TCA ATT GAA TTA GCT GAA GCT AAA GTC TTA GCT AAC AGA GAA 288
Val Lys Ser Ile Glu Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu
85 90 95
CTT GAC AAA TAT GGA GTA AGT GAC TAT CAC AAG AAC CTA ATC AAC AAT 336
Leu Asp Lys Tyr Gly Val Ser Asp Tyr His Lys Asn Leu Ile Asn Asn
100 105 110
GCC AAA ACT GTT GAA GGT GTA AAA GAC CTT CAA GCA CAA GTT GTT GAA 384
Ala Lys Thr Val Glu Gly Val Lys Asp Leu Gln Ala Gln Val Val Glu
115 120 125
TCA GCG AAG AAA GCG CGT ATT TCA GAA GCA ACA GAT GGC TTA TCT GAT 432
Ser Ala Lys Lys Ala Arg Ile Ser Glu Ala Thr Asp Gly Leu Ser Asp
130 135 140
TTC TTG AAA TCA CAA ACA CCT GCT GAA GAT ACT GTT AAA TCA ATT GAA 480
Phe Leu Lys Ser Gln Thr Pro Ala Glu Asp Thr Val Lys Ser Ile Glu
145 150 155 160
TTA GCT GAA GCT AAA GTC TTA GCT AAC AGA GAA CTT GAC AAA TAT GGA 528
Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly
165 170 175
GTA AGT GAC TAT TAC AAG AAC CTA ATC AAC AAT GCC AAA ACT GTT GAA 576
Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu
180 185 190
GGT GTA AAA GCA CTG ATA GAT GAA ATT TTA GCT GCA TTA CCT AAG ACT 624
Gly Val Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro Lys Thr
195 200 205
GAC ACT TAC AAA TTA ATC CTT AAT GGT AAA ACA TTG AAA GGC GAA ACA 672
Asp Thr Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr
210 215 220
ACT ACT GAA GCT GTT GAT GCT GCT ACT GCA AGA TCT TTC AAT TTC CCT 720
Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Arg Ser Phe Asn Phe Pro
225 230 235 240
ATC CTC GAG AAT TCC ATG ACC GTG AAA ACC AAA AAC ACC ACG ACC ACC 768
Ile Leu Glu Asn Ser Met Thr Val Lys Thr Lys Asn Thr Thr Thr Thr
245 250 255
CAG ACC CAG CCG AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG 816
Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro
260 265 270
CCG AAC AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG 864
Pro Asn Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val
275 280 285
CCG TGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA 912
Pro Cys Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys
290 295 300
CGT ATC CCG AAC AAA AAA CCG GGC AAA AAA ACC ACG ACC AAA CCG ACC 960
Arg Ile Pro Asn Lys Lys Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr
305 310 315 320
AAA AAA CCG ACC TTC AAA ACC ACC AAA AAA GAT CAT AAA CCG CAG ACC 1008
Lys Lys Pro Thr Phe Lys Thr Thr Lys Lys Asp His Lys Pro Gln Thr
325 330 335
ACC AAA CCG AAA GAA GTG CCG ACC ACC AAA CCG GTC GAC TAA 1050
Thr Lys Pro Lys Glu Val Pro Thr Thr Lys Pro Val Asp
340 345

1071 base pairs

nucleotide

single

linear

cDNA

CDS

1..1071

77
ATG AAA GCA ATT TTC GTA CTG AAT GCG CAA CAC GAT GAA GCC GTA GAC 48
Met Lys Ala Ile Phe Val Leu Asn Ala Gln His Asp Glu Ala Val Asp
1 5 10 15
GCG AAT TTC GAC CAA TTC AAC AAA TAT GGA GTA AGT GAC TAT TAC AAG 96
Ala Asn Phe Asp Gln Phe Asn Lys Tyr Gly Val Ser Asp Tyr Tyr Lys
20 25 30
AAT CTA ATC AAC AAT GCC AAA ACT GTT GAA GGC GTA AAA GAC CTT CAA 144
Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Asp Leu Gln
35 40 45
GCA CAA GTT GTT GAA TCA GCG AAG AAA GCG CGT ATT TCA GAA GCA ACA 192
Ala Gln Val Val Glu Ser Ala Lys Lys Ala Arg Ile Ser Glu Ala Thr
50 55 60
GAT GGC TTA TCT GAT TTC TTG AAA TCA CAA ACA CCT GCT GAA GAT ACT 240
Asp Gly Leu Ser Asp Phe Leu Lys Ser Gln Thr Pro Ala Glu Asp Thr
65 70 75 80
GTT AAA TCA ATT GAA TTA GCT GAA GCT AAA GTC TTA GCT AAC AGA GAA 288
Val Lys Ser Ile Glu Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu
85 90 95
CTT GAC AAA TAT GGA GTA AGT GAC TAT CAC AAG AAC CTA ATC AAC AAT 336
Leu Asp Lys Tyr Gly Val Ser Asp Tyr His Lys Asn Leu Ile Asn Asn
100 105 110
GCC AAA ACT GTT GAA GGT GTA AAA GAC CTT CAA GCA CAA GTT GTT GAA 384
Ala Lys Thr Val Glu Gly Val Lys Asp Leu Gln Ala Gln Val Val Glu
115 120 125
TCA GCG AAG AAA GCG CGT ATT TCA GAA GCA ACA GAT GGC TTA TCT GAT 432
Ser Ala Lys Lys Ala Arg Ile Ser Glu Ala Thr Asp Gly Leu Ser Asp
130 135 140
TTC TTG AAA TCA CAA ACA CCT GCT GAA GAT ACT GTT AAA TCA ATT GAA 480
Phe Leu Lys Ser Gln Thr Pro Ala Glu Asp Thr Val Lys Ser Ile Glu
145 150 155 160
TTA GCT GAA GCT AAA GTC TTA GCT AAC AGA GAA CTT GAC AAA TAT GGA 528
Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly
165 170 175
GTA AGT GAC TAT TAC AAG AAC CTA ATC AAC AAT GCC AAA ACT GTT GAA 576
Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu
180 185 190
GGT GTA AAA GCA CTG ATA GAT GAA ATT TTA GCT GCA TTA CCT AAG ACT 624
Gly Val Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro Lys Thr
195 200 205
GAC ACT TAC AAA TTA ATC CTT AAT GGT AAA ACA TTG AAA GGC GAA ACA 672
Asp Thr Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr
210 215 220
ACT ACT GAA GCT GTT GAT GCT GCT ACT GCA AGA TCT TTC AAT TTC CCT 720
Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Arg Ser Phe Asn Phe Pro
225 230 235 240
ATC CTC GAG AAT TCG AGC TCG GTA CCC GGG GAT CCT ATG ACC GTG AAA 768
Ile Leu Glu Asn Ser Ser Ser Val Pro Gly Asp Pro Met Thr Val Lys
245 250 255
ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG CCG AGC AAA CCG ACC ACC 816
Thr Lys Asn Thr Thr Thr Thr Gln Thr Gln Pro Ser Lys Pro Thr Thr
260 265 270
AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG AAC AAC GAT TTC CAT 864
Lys Gln Arg Gln Asn Lys Pro Pro Asn Lys Pro Asn Asn Asp Phe His
275 280 285
TTC GAA GTG TTC AAC TTC GTG CCG AGC AGC ATC TGC AGC AAC AAC CCG 912
Phe Glu Val Phe Asn Phe Val Pro Ser Ser Ile Cys Ser Asn Asn Pro
290 295 300
ACC TGC TGG GCG ATC AGC AAA CGT ATC CCG AAC AAA AAA CCG GGC AAA 960
Thr Cys Trp Ala Ile Ser Lys Arg Ile Pro Asn Lys Lys Pro Gly Lys
305 310 315 320
AAA ACC ACG ACC AAA CCG ACC AAA AAA CCG ACC TTC AAA ACC ACC AAA 1008
Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys Thr Thr Lys
325 330 335
AAA GAT CAT AAA CCG CAG ACC ACC AAA CCG AAA GAA GTG CCG ACC ACC 1056
Lys Asp His Lys Pro Gln Thr Thr Lys Pro Lys Glu Val Pro Thr Thr
340 345 350
AAA CCG GTC GAC TAA 1071
Lys Pro Val Asp
355

726 base pairs

nucleotide

single

linear

cDNA

CDS

1..726

78
ATG AAA GCA ATT TTC GTA CTG AAT GCG CAA CAC GAT GAA GCC GTA GAC 48
Met Lys Ala Ile Phe Val Leu Asn Ala Gln His Asp Glu Ala Val Asp
1 5 10 15
GCG AAT TTC GAC CAA TTC AAC AAA TAT GGA GTA AGT GAC TAT TAC AAG 96
Ala Asn Phe Asp Gln Phe Asn Lys Tyr Gly Val Ser Asp Tyr Tyr Lys
20 25 30
AAT CTA ATC AAC AAT GCC AAA ACT GTT GAA GGC GTA AAA GAC CTT CAA 144
Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Asp Leu Gln
35 40 45
GCA CAA GTT GTT GAA TCA GCG AAG AAA GCG CGT ATT TCA GAA GCA ACA 192
Ala Gln Val Val Glu Ser Ala Lys Lys Ala Arg Ile Ser Glu Ala Thr
50 55 60
GAT GGC TTA TCT GAT TTC TTG AAA TCA CAA ACA CCT GCT GAA GAT ACT 240
Asp Gly Leu Ser Asp Phe Leu Lys Ser Gln Thr Pro Ala Glu Asp Thr
65 70 75 80
GTT AAA TCA ATT GAA TTA GCT GAA GCT AAA GTC TTA GCT AAC AGA GAA 288
Val Lys Ser Ile Glu Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu
85 90 95
CTT GAC AAA TAT GGA GTA AGT GAC TAT CAC AAG AAC CTA ATC AAC AAT 336
Leu Asp Lys Tyr Gly Val Ser Asp Tyr His Lys Asn Leu Ile Asn Asn
100 105 110
GCC AAA ACT GTT GAA GGT GTA AAA GAC CTT CAA GCA CAA GTT GTT GAA 384
Ala Lys Thr Val Glu Gly Val Lys Asp Leu Gln Ala Gln Val Val Glu
115 120 125
TCA GCG AAG AAA GCG CGT ATT TCA GAA GCA ACA GAT GGC TTA TCT GAT 432
Ser Ala Lys Lys Ala Arg Ile Ser Glu Ala Thr Asp Gly Leu Ser Asp
130 135 140
TTC TTG AAA TCA CAA ACA CCT GCT GAA GAT ACT GTT AAA TCA ATT GAA 480
Phe Leu Lys Ser Gln Thr Pro Ala Glu Asp Thr Val Lys Ser Ile Glu
145 150 155 160
TTA GCT GAA GCT AAA GTC TTA GCT AAC AGA GAA CTT GAC AAA TAT GGA 528
Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly
165 170 175
GTA AGT GAC TAT TAC AAG AAC CTA ATC AAC AAT GCC AAA ACT GTT GAA 576
Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu
180 185 190
GGT GTA AAA GCA CTG ATA GAT GAA ATT TTA GCT GCA TTA CCT AAG ACT 624
Gly Val Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro Lys Thr
195 200 205
GAC ACT TAC AAA TTA ATC CTT AAT GGT AAA ACA TTG AAA GGC GAA ACA 672
Asp Thr Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr
210 215 220
ACT ACT GAA GCT GTT GAT GCT GCT ACT GCA AGA TCT TTC AAT TTC CCT 720
Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Arg Ser Phe Asn Phe Pro
225 230 235 240
ATC CTC 726
Ile Leu

Number	Name	Date	Kind
4415491	Girish	Nov 1983	A
5958736	Stahl et al.	Sep 1999	A
6149911	Binz et al.	Nov 2000	A

Number	Date	Country
0 327 522	Aug 1989	EP
WO 9101743	Feb 1991	WO
WO 9116926	Nov 1991	WO
WO 9201471	Feb 1992	WO
WO 9306218	Apr 1993	WO

	Number	Date	Country
Parent	08/836501		US
Child	09/626830		US

Complexes of immunogens derived from RSV surface glycoprotein G covalently coupled to a support molecule

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Disclaimer

Abstract

Description

Claims

Priority Claims (1)

Parent Case Info

US Referenced Citations (3)

Foreign Referenced Citations (5)

Non-Patent Literature Citations (5)

Continuations (1)

Entry
Sjolander et al.; “Bacterial Expression Systems Based on Protein A and Protein G Designed for the Production of Immunogens: Applications to Plasmodium Falciparum Malaria Antigens”. Immunomethods; vol. 2, No. 1, Feb. 1993, pp. 79-92.
Sjolander et al.; “Immunogenicity and Antigenicity in Rabbits of a Repeated Sequence of Plasmodium Falciparum Antigen PF 155/RESA Fused to two Immunoglobulin G-Binding Domains of Staphylococcal Protein A”. Infection and Immunity; vol. 58, No. 4, Apr. 1990, pp. 854-859.
Sjolander et al.; “Plasmodium Falciparum: The Immune Response in Rabbits to the Clustered Asparagine-Rich Protein (CARP) After Immunization in Freund's Adjuvant or Immunostimulating Complexes (ISCOMS)”; Mar. 1993, 76 (2), 134-45. Database Medline; File Server STN Karlsruhe; 93202225.
Nygren et al.; “Analysis and use of the Serum Albumin Binding Domains of Streptococcal Protein G”. Journal of Molecular Recognition; vol. 1, No. 2, Apr. 1988, pp. 69-74.
Berzins et al.; “Immunogencity in Aotus Monkeys of ISCOM Formulated Repeat Sequences from the Plasmodium Falciparum Asexual Blood Stage Antigen PF155/RESA”; 1995, 4(3), 121-33. Database Chemical Abstracts; File Server STN Karlsruhe; Abstract No. 124:84244.