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Document A04 
Appl. No. 09/848,616 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCT 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 6 : 

C12N 15/62, 15/16, 15/36, C07K 14/575, 
14/02 



A2 



(11) International Publication Number: WO 99/57289 

(43) International Publication Date: 1 1 November 1999 (U .1 1.99) 



(21) International Application Number: PCT/US99/08238 

(22) International Filing Date: 14 April 1999 (14.04.99) 



(30) Priority Data: 

09/072,323 



4 May 1998 (04.05.98) 



US 



(71) Applicant: MICHIGAN STATE UNIVERSITY [US/US]; 238 
Administration Building, East Lansing, MI 48824-1046 
(US). 

(71) (72) Applicants and Inventors: PUMPENS, Paul [LV/LV]; 

Baznicas 27/29-21, LV-1010 Riga (LV). BORISOVA, 
Galina [LV/LVj; Merkela 2-6, LV-1050 Riga (LV). 
SKRASTINA, Dace [LV/LV]; Dammes 34-10, LV-1069 
Riga (LV). MEZULE, Guna [LV/LV]; Skujenes 10-1, 
LV-1055 Riga (LV). 

(72) Inventor: IRELAND, James, J.; 2805 Brentwood Avenue, East 

Lansing, MI 48823 (US). 

(74) Agent: DAIGNAULT, Ronald, A.; Merchant, Gould, Smith, 
Edell, Welter & Schmidt, P.A., 3100 Norwest Center, 90 
South Seventh Street, Minneapolis, MN 55402-4131 (US). 



(81) Designated States: AE, AL, AM, AT, AT (Utility model), AU, 
AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, CZ 
(Utility model), DE, DE (Utility model), DK, DK (Utility 
model), EE, EE (Utility model), ES, FI, FI (Utility model), 
GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, 
KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG ( 
MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, 
SG, SI, SK, SK (Utility model), SL, TJ, TM, TR, TT, UA, 
UG, UZ, VN, YU, ZA, ZW, ARIPO patent (GH, GM, KE, 
LS, MW, SD, SL, SZ, UG, ZW), Eurasian patent (AM, AZ, 
BY, KG, KZ, MD, RU, TJ, TM), European patent (AT, BE, 
CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, 
NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, 
GN, GW, ML, MR, NE, SN, TD, TG). 



Published 

Without international search report and to be republished 
upon receipt of that report 



(54) Title: INHIBIN-HBc FUSION PROTEIN 



(57) Abstract 



A fusion protein formed of an antigenic inhibin peptide inserted into hepatitis B capsid protein is expressed from a chimeric gene 
forming an effective immunogen which induces anti-inhibin antibodies when administered to a host animal. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT. 



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Armenia 


FI 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


SZ 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


GE 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


BG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


uz 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan 


NE 


Niger 


VN 


Viet Nam 


CG 


Congo 


KE 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzstan 


NO 


Norway 


ZW 


Zimbabwe 


CI 


Cdte d' I voire 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Portugal 






cu 


Cuba 


KZ 


Kazakstan 


RO 


Romania 






cz 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Germany 


LI 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







WO 99/57289 



Inhibin-HBc Fusion Protein 



PCT/US99/08238 



Field of the Invention: 

This invention relates to useful immunogenic molecules formed of an 
antigenic inhibin peptide and hepatitis B capsid protein. More particularly, an 
antigenic inhibin peptide is genetically inserted into the hepatitis B capsid protein, 
resulting in the production of a fusion protein which induces the production of anti- 
inhibin antibodies when administered to host animals, even in the absence of 
adjuvant. 

Background of the Invention: 

The inhibin protein family includes dimeric glycoproteins produced 
by the gonads which act in an endocrine fashion to suppress secretion of follicle 
stimulating hormone (FSH) from the pituitary gland. Since FSH is the major 
hormone involved in stimulation of ovulation and sperm production, inhibin-induced 
suppression of FSH diminishes the rates of ovulation and sperm production. 
Inhibins are therefore natural suppressers of the reproductive process. 

Active immunization of farm animals against inhibin increases the 
rates of ovulation and sperm production, demonstrating that inhibin-based 
immunogens are important agents to enhance fertility in farm animals, including 
swine, bovine, ovine, and equine animals. See, for example: Brown, et al., 
J.Reprod.Fertility 90:199-205, 1990; King, et al., J Animal Science 71:975-982, 
1993; Morris, et al., J.ReprodFertility 97:255-261, 1993; McCue, et al., 
Theriogenology 38:823-831, 1992; Voglmayer, et al., BioLReprod. 42:81-86, 1990; 
Martin, et al., Biol.Reprod. 45:73-77, 1991. 

The immunization of gilts with a small peptide fragment of the 
bovine inhibin a c 126 subunit chemically conjugated to human alpha globulin (HAG) 
and mixed with Freund's adjuvant resulted in a minor increase in FSH during the 
follicular phase and a decrease in FSH during the luteal phase, without effect on 
serum concentrations of estradiol, progesterone, or luteinizing hormone (King, et al., 
J. Animal Science 71 :975-982, 1993). Despite the relatively minor increase in FSH 
during the follicular phase and decrease during the early luteal phase, immunized 



WO 99/57289 PCT/US99/08238 

2 

gilts had a 39% greater ovulation rate as compared with controls. In addition, 
lifetime proliferacy of the immunized gilts was enhanced. 

While the above described merits of using the antigenic inhibin 
peptide to neutralize inhibins and enhance fertility are known, a commercially useful 
vaccine is not available. 

The development of vaccines based on small antigenic epitopes is 
hampered by the inability of the small antigen to elicit a good immune response in a 
host animal. The use of carrier immunogens provides some assistance in the 
immune response, but often decreases the specific activity and yield of the response 
against the desired antigen. Methods for conjugation of antigens to carrier agents 
are costly, and generally utilize hazardous chemicals. Covalent coupling of antigen 
to a carrier protein is inherently variable, resulting in an antigen with an imprecise 
structure, compromising vaccine potency. The use of adjuvants also tends to 
decrease the yield of specific antibodies and can be harmful to the animal host, 
causing abscesses, skin lesions, and hypersensitivity. These factors are unacceptable 
for the production of a commercially useful vaccine. 

These disadvantages are overcome in the present invention by using 
recombinantly produced fusion proteins as immunogens, whose structure is well 
defined. Synthesis of the inventive immunogens does not require hazardous 
chemical treatments, and the molecules are herein demonstrated to induce a desired 
anti-inhibin immune response, even in the absence of additional adjuvants. 



Summary of the Invention 

It has now been found that an inhibin:hepatitis B capsid protein 
fusion (Inh:HBc) provides a useful, defined, easily produced, immunogenic 
molecule which, upon administration to host animals, induces a fertility-enhancing, 
anti-inhibin immune response, even in the absence of additional adjuvant. The 
Inh:HBc fusion protein is produced by inserting a first nucleic acid sequence 
encoding an antigenic inhibin peptide into a second nucleic acid sequence encoding 
hepatitis B capsid protein and expressing a fusion protein a cellular host. When 
administered to host animals, particularly to farm animals, the Inh:HBc fusion 
protein induces an anti-inhibin immune response The anti-inhibin response results 



WO 99/57289 PCT/US99/08238 

3 

in enhanced fertility, measured, for example, as an increase in ovulation rates in 
immunized animals, and preferably, as an increase in lifetime proliferation. 

In a preferred embodiment of the invention, a preferred antigenic 
inhibin peptide is an amino acid sequence of the inhibin a c subunit. For example, a 
preferred antigenic peptide of the invention is formed of the first 25 N-terminal 
amino acids of the inhibin alpha-C subunit ( bINHa c N25 ). 

It was unexpectedly discovered that inhibin antigenic peptide inserted 
at position 78 of the HBc protein results in a preferred fusion protein inducing useful 
antibody titer in host animals with good anti-inhibin specificity. In this construct, 
the inhibin antigenic peptide is inserted in place of a major immunological region of 
HBc Ag. 

Brief Description of the Figures 

Figure 1 A is a photograph of a silver stained gel showing production 

ofInh:HBc-78. 

Figure IB is a photograph of an immunoblot of Inh:HBc-78 probed 
with anti-inhibin antibody. 

Figure 1C is a photograph of a silver stained gel showing production 
ofInh:HBc-144. 

Figure ID is a photograph of an immunoblot of Inh:HBc-144 probed 
with anti-inhibin antibody. 

Figure 2 is a electron micrograph showing capsid formation of 

Inh:HBc-78. 

Figure 3 is a graph showing anti-inhibin antibody titers in mice 
immunized with Inh:HBc-78 with Fruend's; Inh:HBc, Inh:HBc-78 without 
Fruend's; Inh:HBc-144 with Fruend's; and Inh:HBc-144 without Fruend's. 

Figure 4 is a graph showing anti-native inhibin antibody titers in mice 
immunized with Inh:HBc-78 and Inh:HBc-144 with and without Fruend's. 

Figure 5 is a graph showing anti-inhibin titers in gilts immunized 
with Inh:HBc-144 and Inh:HBc-78. 



WO 99/57289 PCT/US99/08238 

4 

Detailed Description of the Invention 

In the preferred embodiments of the invention, an immunogenic 
carrier molecule, the hepatitis B capsid protein (HBc), is modified to include an 
inserted antigenic inhibin peptide. The inserted inhibin peptide is positioned such 
that it does not preclude correct assembly of the HBc protein into core particles and 
is recognized as antigenic in host systems. As shown below, the inhibin antigenic 
peptide is preferentially inserted at position 78 of the HBc protein. 

When used as a vaccine, the Ihn:HBc fusion protein is effective in 
eliciting an antibody response against the antigenic peptide in host animals, 
particularly in farm animals. Vaccination of a host animal with the Inh:HBc fusion 
protein results in the development of specific anti-inhibin antibodies in the animal, 
in the presence or absence of added adjuvant. Vaccination and induction of anti- 
inhibin antibodies results in enhanced fertility. 

Antigenic Inhibin Peptides 

Antigenic inhibin peptides useful in the present invention are 
generally short amino acid sequences, e.g., less than about 100 amino acids, 
preferably about 8-30 amino acids, and more preferably 10-25 amino acids in length. 
The peptide is preferably known to represent an epitope that is able to induce an 
immune response against inhibin, such as the first portion of the inhibin a c chain, 
amino acids 1-30. For example, the antigenic peptide may be known to produce a 
desired antigenic response when used in another carrier protein such as HAG or 
when used with an adjuvant system such as co-administration with Fruend's 
Adjuvant or other immunogen. Alternatively, the peptide antigen may be a portion 
of a known inhibin protein having a particularly unique amino acid sequence 
distinguishing it from other inhibin proteins. These and other techniques for 
identifying and screening potential antigenic peptides useful in vaccine development 
are generally known. See, for example, Scott, et al., 1990, Science 249:386-390. 

Preferred inhibin antigenic peptides include portions of the ct c chain, 
e.g., 10-25 amino acids selected from known antigenic portions of the molecule. 
Most preferred is a sequence of 10-25 amino acids of the first 30 amino acids of the 
a c chain. 



WO 99/57289 PCT/US99/08238 

5 

Antigenic peptides are inserted into the HBc molecule by 
recombinant DNA methods. For example, a synthetic nucleic acid sequence or 
vector containing a nucleic acid sequence encoding a desired inhibin antigenic 
peptide to be inserted into HBc is specifically designed to include restriction 
endonuclease sites matched to a specified endonuclease-cut nucleic acid sequence 
encoding HBc. Where a desirable HBc insertion site contains a single, unique 
restriction endonuclease site, the inhibin antigen's nucleic acid sequence is 
preferably engineered to include matched restriction sites at both ends of the 
sequence. In this manner, the sequence encoding the inhibin antigen is inserted into 
the HBc sequence without removal of any HBc-encoding nucleotides. Care is taken 
to match the antigenic inhibin-encoding nucleic acid sequence to be inserted with the 
reading frame of the HBc sequence so that normal expression of the encoded HBc 
with the encoded inhibin antigen is achieved. 

For HBc, specific display vectors containing cloning sites specifically 
engineered into the HBc nucleic acid sequence have been constructed. In these 
vectors, expression of the HBc gene is under the control of a tandem stretch of 
strong E. coli trp promoters. Vector pCT31, prepared as described in Borisova, et 
al., 1988, Proc. USSR Acad Sci. 298:1474-1478 and Borisova, et al., 1989, FEES 
Lett. 259:121-124 (containing a truncated form of HBc with amino acids 145-183 
removed), is designed for insertion of antigens at amino acid position 144, and 
allows in-frame insertion and translation termination in all three possible frames. 
Vector p2- 19, prepared as described in Borisova, et al., 1996, Intervirology 39:16- 
22, is designed for insertion of antigens at amino acid position 78, and allows for 
blunt-ended insertion of the antigenic fragment in frame. 

It is contemplated that the compositions and methods of the invention 
may be limited by the antigenic peptide's amino acid chain length (e.g., less than 
100 amino acids, and preferably no greater than about 30 amino acids), net charge of 
the inserted amino acid sequence (e.g, less than about 50% highly charged amino 
acid residues), potentially cross-linking residues, or a density of potentially self- 
hybridizing nucleic acid sequences. These limitations are generally known and can 
be recognized by review of the amino acid sequence to be inserted. 



WO 99/57289 PCT/US99/08238 

6 

It is generally known that a nucleic acid sequence may be modified 
for enhanced expression in a particular host cell by modifying the codons of the 
nucleic acid sequence to those more preferred in the specific host cell. Thus, for 
example, to express the Inh:HBc fusion protein in E. coli, the peptide sequence is 
back translated into the nucleotide sequence using the codon frequency found in E. 
coli proteins, as determined, for example, by the GCG computer program 
(Devereaux, et al., 1984, Nucleic Acids Res. 12:387-3905) and modified as 
suggested by E. coli codon frequencies. 

It is generally understood that protein expression in a given host cell 
may be enhanced by modification of one or more nucleotides in the coding sequence 
to reduce the number of unique or rare codons. In a preferred embodiment of the 
invention, the nucleic acid sequence contains one or more codons modified 
according to the codon frequency preferences for a particular cellular host. 

Inhibin Vaccine 

Inhibin is a glycoprotein produced by the gonads that selectively 
suppresses the secretion of follicle stimulating hormone (FSH) from the anterior 
pituitary gland. Vaccination against inhibin decreases available inhibin, with a 
resulting increase in levels of follicle stimulating hormone (FSH), and enhanced 
fertility. Enhanced fertility may be due to enhanced production of sperm or ova, to 
increased rates of ovulation or spermatogenesis, or to increased lifetime proliferacy 
in animals, for example. 

Immunization of animals with bovine inhibin - a c subunit has 
demonstrated the usefulness of inhibin-based antigens as fertility-enhancing 
vaccines. However, to date, a practical commercial vaccine has not been produced, 
at least in part due to the limitations of chemical synthesis, conjugation, and 
adjuvant toxicity discussed above. 

In a preferred embodiment and exemplary of the invention, the 
nucleic acid sequence encoding the first 25 N-terminal residues of the antigenic 
inhibin a c subunit (a c 125 ) is inserted into nucleic acid sequence encoding HBc such 
that the expressed fusion protein will include the inhibin antigenic peptide inserted at 
HBc amino acid position 78 (Inh:HBc-78). Multiple copies of the antigen may be 



WO 99/57289 PCT/US99/08238 

7 

inserted, e.g., at more than one site in the HBc molecule, and preferably at two or 
more sites, where at least one inhibin antigen insertion site is at position 78. 

The chimeric gene encoding the Inh:HBc fusion protein is subcloned 
into an expression vector, preferably a broad-host-range expression vector. The 
inserted antigen is expressed with expression of the HBc molecule, such that when 
the expressed fusion protein is administered to host animals, an anti-inhibin immune 
response is produced in the animals, reducing endogenous inhibin and thereby 
enhancing fertility in treated animals. 

Hepatitis B Capsid Protein 

The hepatitis B capsid protein (HBc) is an immunogenic carrier 
protein having several advantages over other potential carrier molecules (Nassal, et 
al., Trends in Microbiology 1:221-228, 1993). These advantages include high level 
production and correct assembly into core particles in the absence of virus in a wide 
variety of eukaryotic and prokaryotic expression systems. Because recombinant 
HBc molecules expressed in E.coli self-assemble into particles containing 180 or 
240 subunits, an inserted inhibin antigen is present in 1 80 or 240 copies per particle. 

A hepatitis B capsid fusion protein including an antigenic inhibin 
peptide, capable of inducing the production of anti-inhibin antibodies in a host 
animal, preferably in the absence of adjuvant, would be very useful, particularly for 
enhancing the fertility of farm animals. 

Cellular Hosts 

Many known cellular host systems are suitable for expression of the 
chimeric genes of the invention. For example, non-pathogenic strains such as Vibrio 
and including Vibrio anguillarium are transfected with suitable vectors containing 
the gene encoding Inh:HBc and express the fusion protein. Suitable vectors for use 
in Vibrio include pJF 1 1 8, as described in Furst, et al, 1 986, Gene 48: 1 1 9-1 3 1 . 

Additional host systems useful in the invention include E. coli, useful 
for example with the HBc display vectors described more fully in the examples 
below, as well as other known host and vector systems. 



WO 99/57289 PCT/US99/08238 

8 

HBc is a known immunogen. The fusion protein immunogen of the 
invention, formed of the intact HBc protein and an inserted antigenic inhibin 
peptide, is administered to farm animals such as sheep, pigs, horses, cows, and the 
like, according to the methods known as effective for the immunogenic 
administration of HBc and other protein immunogens. 

Administration methods include injection, e.g., intramuscular, 
subcutaneous, or intraperitoneal injection and nasal administration of protein 
compositions to induce effective antibody titers. In a preferred embodiment, the 
fusion protein of the invention is expressed in edible plants or animals for oral 
ingestion. This oral delivery method has been described for immunogenic delivery 
of the immunogen LTB. See, for example, Mason, et al., 1995, TIBTECH 13:388- 
392, describing oral immunization against LTB via ingestion of transgenic potato 
tubers expressing LTB antigen. 

Examples 

The present invention may be better understood with reference to the 
following examples. These examples are intended to be representative of specific 
embodiments of the invention, and are not intended as limiting the scope of the 
invention. 

Example 1 
Production of In:HBc Fusion Protein 

HBc Display Vectors 

Hepatitis B capsid antigen display vectors containing the HBc gene 
under the control of a tandem string of strong E.coli trp promoters (Borisova, et al., 
1988, Proc. USSR AcadSci. 298:1474-1478; Borisova, et al., FEBS Lett. 259:121- 
124, 1989; and Borisova, et al., 1996, Intervirology 39:16-22) were used to insert the 
bovine inhibin a c N25 (blNH-CLc 1 ~ 25 ) antigenic epitope into HBc at amino acid positions 
144 and 78. Vector pCT31 was used for insertions into position 144, and allowed 
in-frame insertion and translation termination in all three possible frames to produce 
Inh:HBc-144. Vector p2-19 was used for insertions into position 78, allowing for 
blunt-ended insertion of the antigenic fragment in frame to produce Inh:HBc-78. 



WO 99/57289 PCT/US99/08238 

9 

Inhibin Antigenic Peptide 

The Inhibin antigenic peptide was prepared by synthesizing the 
following oligonucleotides: 

5'-TCT ACC CCG CCG CTG CCG TGG CCG TGG TCC CCG 
GCT GCT CTG CTG CAG CGT CCG CCG GAA GAA CCG 
GCT GCT CCG-3' [SEQ. ID. NO: 1] ; and 

3' -CGG AGC AGC CGG TTC TTC CGG CGG ACG CTG CAG 
CAG AGC AGC CGG GGA CCA CGG CCA CGG CAG CGG 
CGG CCT AGA-5' [SEQ. ID. NO: 2] . 

Each oligonucleotide was phosphorylated by T4 polynucleotide 
kinase, and the pair of oligonucleotides was annealed to form a double-strand. The 
double-stranded nucleic acid sequence was then cloned as a blunt-ended fragment 
into the HBc display vectors to form chimeric genes encoding the InhrHBc fusion 
proteins. 

Cloning 

Ampicillin-resistant plasmid pCT3 1 carrying the C-terminally 
truncated (having amino acids 145-183 removed) HBc gene was used as a vector. 
For insertion at amino acid 144, the vector was cleaved with Smal (1MB Fermentas, 
Vilnius, Lithuania) at position 1741, purified from agarose gel by the DEAE paper 
method, and used for ligation with the inhibin antigenic double-stranded nucleic acid 
sequence described above as phosphorylated or dephosphorylated by bacterial 
alkaline phosphatase (1MB Fermentas). Ligation was performed in 20 mM TRIS pH 
7.6, 0.5 mM MgCl 2 , 5mM dithiothreitol (in 10 |il) with 100 ng vector and fragment, 
1 Weiss unit of T4 DNA ligase, at 4°C for 12 hours. The molar ratio of 
oligonucleotide: vector was either 5:1 or 100:1. 

For insertion at amino acid 78, ampicillin-resistant plasmid p2-19 
carrying the HBc gene with a poly linker inserted at position 78 was also used as a 
vector. Polylinker encodes an immunomarker sequence "DPAFR" recognized by 
monoclonal anti-preSl antibody MAI 8/7 (W.H.Gerlich, Giessen). The p2-19 vector 
was cleaved simultaneously by Eco 721 (position 1531) and Eco 1051 (position 



WO 99/57289 PCT/US99/08238 

10 

1 552) (IBM Fermentas), purified from agarose gel by DEAE paper method, 
dephosphorylated by bacterial alkaline phosphatase, and used for ligation with the 
double-stranded antigenic inhibin peptide. The oligonucleotidervector molar ratio 
was 100:1. 

First selection of plasmids containing the antigenic inhibin insert was 
by Pst\ cleavage. The vector contains a unique Pst\ cleavage site at position 4742. 
Clones containing the Pst\ site in the insert (presenting two fragments) were 
subjected to further screening by immunoblotting and sequencing. 

Purified fusion protein 

E. coli strain RR1 (F-, hsd S20 (r b -, m b -), recA+, ara-14, proA2, 
lacYl, galK2, rpsL20 (Sm r ), xyl-5, mlt-l, supE44, X-) was used for transformation 
with the chimeric Inh:HBc vectors, resulting in a transformation efficiency of 10 9 
clones/jag of supercoiled DNA. Recombinant fusion protein was purified from cells 
by cell lysis followed by Sepharose CL4B column chromatography. Production 
yield for the fusion protein Inh:HBc-144 was higher than for Inh:HBc-78, generating 
5 mg of purified intact product per liter of E. coli cells. 

Immunoblot Screening 

Transformed E. coli cells expressing the fusion protein were selected 
by immunoblotting and sequencing. For selection by immunoblotting, E. coli strain 
K802 cells harboring the appropriate plasmids were grown to saturation overnight in 
M9 synthetic medium supplemented with 2%g/l Casamino acids (Difco). Bacteria 
were pelleted, suspended in SDS-gel electrophoresis sample buffer containing 2% 
SDS and 2% 2-mercaptoethanol, and lysed by heating at 100°C for five minutes. 
Proteins were separated by PAGE in a slab gel (150X150X0.75 mm) apparatus. 

Western blotting was conducted as described by Towbin et al., PNAS 
USA,76:4350-4354, 1979. Aliquots (4ug) of the fusion proteins In3-69 and In4-56, 
as well as control vector HBc were subjected to 15% SDS-PAGE under non- 
reducing conditions. Each gel was stained for protein by the silver stain method 
described, for example, in Ohsawa, et al., Anal BiochemA35:409-4l5 9 1983. 



WO 99/57289 PCT/US99/08238 

11 

Parallel gels containing lOOng aliquots of the fusion protein and 
control HBc were run under both non-reducing and reducing conditions. Separated 
protein was electrophoretically transferred to Immobilon P membrane (Millipore) , 
and processed for immunoblot assay with mink anti-bovine c^ 1 26 gly.tyr antiserum 
as described in Good, et al., BiolReprod. 53:1478-1488, 1995. The protein- 
transferred membranes were incubated with the anti-inhibin antibody at a dilution of 
1 : 1000 in TTBS overnight at room temperature, as described in Ireland, et al., 
BiolReprod. 50:1265-1276, 1994. 

After washing in TTBS (five 10-minute washes), the membranes 
were further incubated in 20 ml of ^I-blNHa. 1 ' 26 gly.tyr (1 X 10 6 cpm/ml in TTBS 
with 1 % gelatin) for competition. The membrane was washed and placed on X-ray 
film (Kodak X-OMAT AR) with a Cronex intensifying screen and exposed for ten 
days at -80°C. 

As show in Figures 1 A-D, the silver-stained gels as compared with 
the immunoblots demonstrated the purity of the fusion protein preparations. The 
immunoblots further demonstrated immunoreactivity of the fusion proteins with 
anti-inhibin antibody. 

DNA sequencing of positive clones 

The primary structure of the DNA insert in the positive clones was 
determined by DNA sequencing, using standard methods for Sanger's dideoxy 
sequencing. Two different clones were selected for sequence analysis, the first 
having the inhibin epitope inserted at amino acid position 144 (Inh:HBc-144) and 
the second inserted at amino acid position 78 (Inh:HBc-78). The following 
sequences were identified, confirming the insertion of the antigenic inhibin peptide 
sequence shown below surrounded by HBc-poiy linker sequences (in bold): 

In position 144, clone In3-38, encoding Inh:HBc-144: 



GGG 


ccc 


TCT 


ACC 


CCG 


CCG 


CTG 


CCG TGG 


CCG 


TGG 


TCC 


Gly 


Pro 


Ser 


Thr 


Pro 


Pro 


Leu 


Pro Trp 


pro 


Trp 


Ser 


CCG 


GCT 


GCT 


CTG 


CTG 


CAG 


CGT 


CCG CCG 


GAA 


GAA 


CCG 


Pro 


Ala 


Ala 


Leu 


Leu 


Gin 


Arg 


Pro Pro 


Glu 


Glu 


Pro 


GCT 


GCT 


CCG 


GGG 


TAA 


[SEQ. ID. 


.NO: 3] 









WO 99/57289 



Ala Ala Pro Gly 



12 

[SEQ.ID.N0:4] 



PCT/US99/08238 



In position 78, clone In4-56, encoding Inh:HBc-78: 

GAT CAC TCT ACC CCG CCG CTG CCG TGG CCG TGG TCC 
Asp His Ser Thr Pro Pro Leu Pro Trp pro Trp Ser 

CCG GCT GCT CTG CTG CAG CGT CCG CCG GAA GAA CCG 
Pro Ala Ala Leu Leu Gin Arg Pro Pro Glu Glu Pro 

GCT GCT CCG GTA GAT [ SEQ . ID . NO : 5 ] 
Ala Ala Pro Val Asp [SEQ. ID. NO: 6] 

Self-Assembly 

The Inh:HBc fusion protein self-assembled and remained intact after 
insertion of the antigenic inhibin peptide into HBc, as shown by double radial 
immunodiffusion against capsid specific antibodies, using the method of 
Ouchterlony, 1965, In: Immunochemie. 15th Colloquium of the Gesellschaft fur 
Physiologische Chemie, Springer, Berlin, Heidelberg, New York, 1979, pages 15- 
35. Gel filtration on Separose CL4B, and electron microscopy of negatively stained 
InhrHBc fusion protein capsid preparations were also used to confirm correct 
assembly. 

Natural human and hyperimmune rabbit anti-HBc antibodies were 
used as the particulate HBc-specific polyclonal antibodies for double radial 
immunidiffusion test according to Ouchterlony, supra. Hyperimmune anti-HBc 
antibodies were generated by immunization of rabbits with purified recombinant 
HBc (Mezule, BMC, Riga). 

Ouchterlony's test for lysozyme lysates of the fusion proteins 
demonstrated their full capability to self-assemble. Ouchterlony's assay employs the 
basic principles of double radial immunodiffusion in 0.8% agarose gel of antibodies 
(center) and antigens at the step 1 :2 dilutions (radial) after 24 hours at 4°C. Titers of 
antibody are recognized as the last precipitation - line-forming dilution. These 
studies demonstrated the two fusion protein clones, Inh:HBc-144 (In3-38) and 
Inh:HBc-78 (In4-56) were able to form capsids (see Figure 2). 

Purification of Fusion Protein 



WO 99/57289 PCT/US99/08238 

13 

Inh:HBc recombinants 144 (In3-38) and 78 (In4-56) were expressed 
in E. coli strain K802 harboring the appropriate plasmid encoding a recombinant 
gene under the control of a tandem string of strong bacterial trp promoters. Bacteria 
were grown overnight on a rotary shaker at 37°C in 500 ml flasks containing 200 ml 
of M9 minimal medium supplemented with 1% Casamino acids (Difco) and 0.2% 
glucose. An optical density (650 nm) of 4-5 for one ml of the suspension was 
generally reached. Cells were pelleted and lysed with a 30 minute incubation on ice 
in lysis buffer containing 50 mM TRIS-HC1 (ph 8.0), 5 mM EDTA, 100 ug/ml 
PMSF, 2 mg/ml lysozyme and then frozen and thawed three times. After freeze- 
thaw, lOmM MgCl 2 and 20 ug/ml DNase were added. After low speed 
centrifiigation, proteins were precipitated from the supernatant with ammonium 
sulphate at 30% saturation for 12 hours at 4°C. Pellets were resuspended in standard 
PBS buffer containing 0.1% Triton X- 100 (30-40 mg/ml total protein). A volume of 
5 ml of protein solution was loaded on a Sepharose CL4B column (2.5 x 85 cm) and 
eluted with PBS buffer without Triton X-100. Fractions containing caspids 
(detected by double radial immunodiffusion test against human polyclonal anti-HBc 
antibodies) were pooled and concentrated by ammonium sulphate precipitation at 
50% saturation. Pellets were resuspended in TRIS-saline buffer (10 mM TRIS-HC1 
(pH 7.5), 150 mM NaCl) to a final concentration of about 10-15 mg/ml total protein, 
dialyzed overnight against 1000 volumes of the same buffer and stored at -70°C. 
Quality of capsid preparations was checked by electron microscopy (V.Ose, BMC, 
Riga). See Figure 2. 

Immunodiffusion, silver stained PAGE, and immunoblots with anti- 
HBc monoclonal 14E1 1 antibodies of SDS and lysozyme lysates of Inh:HBc-144 
and Inh:HBc-78 cells were used to follow the purification process. On silver 
staining and immunoblot of gels run with the fusion protein, the inhibin fusion 
protein preparation was shown to be relatively pure. The immunoblot data 
demonstrated that only the fusion protein, and not the HBc vector reacted with anti- 
cs 1 " 25 antibodies. (See Figures 1 A-D). 



WO 99/57289 PCT/US99/08238 

14 

Example 2 

Immunization of Mice with Inh:HBc Fusion Protein 

A total of 45 BALB/C mice, 5 per treatment group, were immunized 
subcutaneously with 20 \ig of the Inh:HBc fusion proteins 144 (In3-96) and 78 (In4- 
56), or with the HBc control, both in the presence and absence of Fruend's complete 
adjuvant (0.1ml). One group of mice was untreated. The primary immunization was 
followed by four boosters spaced two weeks apart. Two weeks after boosts 1,2, and 
3, all mice were bled. A volume of 20jul of blood was placed in PBS (1:10 dilution) 
containing heparin to prevent clotting. Each boost was given immediately following 
each bleed. Mice were sacrificed 2 weeks after boost 4, and trunk blood was 
collected. 

The blood samples were analyzed for anti-inhibin antibody activity 
by ELISA. Microtiter plates (Xenobind, Xenopore, Inc.) were coated with 1 jig/well 
of bovine inhibin a c 1-26 peptide, and a solid phase, non-radiometric ELISA protocol 
was used to estimate titer. Absorbance at 490 nm (A 490 ) was measured using a 
microplate reader (BioRad Model 35500). Titer was defined as the serum/plasma 
dilution giving an A490 four times the average value for untreated controls. 

The A 490 for blood samples of HBc-treated mice were not different 
than the untreated control samples (data not shown). As shown in Figure 3, 
significant antibody titers against the a c 126 inhibin fragment were elicited in all mice 
immunized with either Inh:HBc-144, Inh:HBc-78, with Fruend's adjuvant (+FR), 
and importantly, in the absence of the adjuvant. Mice immunized with Inh:HBc-78 
plus Fruend's adjuvant demonstrated the highest titer. The values shown in the 
figure represent the mean ±SEM of pooled mouse titers for Boost 1, but individual 
mouse titers for Boosts 2-4 are shown. 

The immunogenic blood samples were further tested for their ability 
to recognize native inhibin. Microtiter test plates were coated with l|ig of a partially 
purified preparation of bovine inhibin, prepared as described in Good, et al., Biology 
Reprod. 53:1478-1488, 1995. The native inhibin preparation contains nine different 
molecular variants of bovine inhibin dimers and a subunits. Titer was determined 
for mouse serum collected after Boost 4. The A 490 values for HBc-treated mice did 
not differ from the untreated control (data not shown). Figure 4 shows A 490 values as 



WO 99/57289 PCT/US99/08238 

15 

mean ± SEM of individual mouse titers for each treatment group after Boost 4. The 
results demonstrate that mice immunized with any of the HBc- Inhibin fusion 
proteins, with or without adjuvant, produced antibodies capable of reacting with 
native inhibin. The Inh:HBc-78 (In4-56) fusion protein produced the highest 
antibody titers, with or without added adjuvant. 

Example 3 

Intraperitoneal immunization of mice with Inh:HBc 

Mice (5 per treatment group) were immunized intraperitoneally with 
the Inh:HBc fusion proteins Inh:HBc-144 and Inh:HBc-78 or with HBc mixed in 
Fruend's adjuvant. A boost was given 24 days after primary injection , and titer was 
determined 7 days after the boost, using the methods described above for Example 2. 

In the analysis of antibody titer, serum was pooled for all mice in a 
treatment group, and 2\ig of the inhibin antigen a c ] 26 was added to each well of 
Maxisorp, NUNC plates. Both controls and treated animals exhibited high titers 
against HBc (data not shown). Importantly, antibodies against the inhibin antigen 
were detected for all mice immunized with Inh:HBc-144 or Inh:HBc-78, but not the 
HBc control (see Table 1). The titers of anti-inhibin antibodies elicited by Inh:HBc- 
78 were 5 times the level elicited by Inh:HBc-144, and with greater specificity for 
the inhibin antigen versus the carrier HBc molecule. 



Table 1 



Immunogen y ; 




Anit-InUibin T$U&lM 


Inh:HBc-144 


1:10,000 


1:3,000 


Inh:HBc-78 


1:500 


1:15,000 


HBc 


1:100,000 





Example 4 

Immunization of Gilts with HBcAg: g J' 2 S Fusion Protein 

Latvian White gilts, 2 gilts per treatment group, were administered a 
single dose of 1 mg (0.8 ml) of Inh:HBc fusion protein, Inh:HBc-144 or Inh:HBc-78, 



WO 99/57289 PCT/US99/08238 

16 

or with HBc control, mixed in 0.8 ml Fruend's Complete Adjuvant. Injections were 
made into multiple sites across each animal's back. Blood samples were collected 
and titers against the antigenic inhibin 1-26 peptide were determined, as described 
above for Example 2, assaying individual animal titers. 

Within ten days after the single injection, the two gilts immunized 
with Inh:HBc-78 had developed anti-inhibin titers of 1:600 and 1:125, which 
increased to 1 :3000 and 1 :600, respectively, by 19 days after the injection, as shown 
in Figure 5. The two gilts immunized with Inh:HBc-144 had titers of 1 : 125 or less, 
and the two gilts immunized with HBcAg had non-detectable titers. Thirty days 
after the primary immunization, the titre determined for each gilt was determined. 
The 30 day data are shown below in Table 2. 



Table 2 



Immunogen 


anti-HBc 


anti-inhibin :. 


Inh:HBc-78 


1:10,000 1:10,000 


1:15,000 1:3,000 


Inh:HBc-144 


1:100,000 1:1,000,000 


1:100 1:125 


HBc 


1:100,000 1:100,000 





These results demonstrate that an HBcAg:ct c 125 fusion protein, 
Inh:HBc-78, is highly immunogenic in gilts, as well as in mice, and the 
immunogenicity is demonstrated in the absence of adjuvant. The data further 
demonstrate that an inhibin antigenic peptide, when inserted at position 78 of HBc, 
induces a useful antibody titre with preferred anti-inhibin specificity. 



WO 99/57289 
WE CLAIM: 



17 



PCT/US99/08238 



1 . A nucleic acid construct comprising: 

a first nucleic acid sequence encoding an inhibin antigenic peptide; 

and 

a second nucleic acid sequence encoding a hepatitis B capsid protein, 
wherein the first sequence is inserted into the second sequence to 

encode a fusion protein having the inhibin antigenic peptide inserted at position 78 

of the hepatitis B capsid protein. 

2. The nucleic acid construct of claim 1, wherein said second nucleic 
acid sequence encoding hepatitis B capsid protein is truncated to remove nucleic 
acid residues 145-183. 

3. The nucleic acid construct of claim 1, wherein the inhibin antigenic 
peptide comprises an antigenic portion of the a c chain. 

4. The nucleic acid construct of claim 1, wherein the inhibin antigenic 
peptide comprises a sequence of 10 or more of amino acids 1-30 of inhibin a c . 

5. The nucleic acid construct of claim 1, wherein the inhibin antigenic 
peptide comprises inhibin-aj" 25 . 

6. A fusion protein comprising: 
hepatitis B capsid protein; and 
an inhibin antigenic peptide, 

wherein the inhibin antigenic peptide is inserted at amino acid 78 of 
the hepatitis B capsid protein. 

7. The fusion protein of claim 6, wherein the inhibin antigenic peptide 
comprises an antigenic portion of the ot c chain. 



WO 99/57289 PCT/US99/08238 

18 

8. The fusion protein of claim 6, wherein the inhibin antigenic peptide 
comprises a sequence of 10 or more of amino acids 1 -30 of inhibin a c . 

9. The fusion protein of claim 6, wherein the inhibin antigenic peptide 
comprises inhibin-c^ 1 " 25 . 

1 0. A method for producing an anti-inhibin immunogen, the method 
comprising the steps of: 

inserting a first nucleic acid sequence encoding an inhibin antigenic peptide 
into a second nucleic acid sequence encoding hepatitis B capsid protein to form a 
fusion construct expressing a fusion protein having the inhibin antigenic peptide 
inserted at position 78 of the hepatitis B capsid protein; 

expressing the fusion protein in a host cell. 

11. The method of claim 10, wherein the inhibin antigenic peptide 
comprises an antigenic portion of the inhibin a c chain. 

12. The method of claim 10, wherein the inhibin antigenic peptide 
comprises a sequence of 10 or more of amino acids 1-30 of inhibin ct c . 

1 3 . The method of claim 1 0, wherein the inhibin antigenic peptide 
comprises inhibin-a c 1-25 . 

14. A method of inducing an anti-inhibin immune response in a host 
animal comprising the steps of: 

administering to a host animal a fusion protein comprising a hepatitis 
B capsid protein having an inhibin antigenic peptide inserted at position 78. 

15. The method of claim 14, wherein the inhibin antigenic peptide 
comprises an antigenic portion of the a c chain. 



WO 99/57289 PCT/US99/08238 

19 

16. The method of claim 14, wherein the inhibin antigenic peptide 
comprises a sequence of 10 or more of amino acids 1-30 of inhibin <x c . 

17. The method of claim 14, wherein the inhibin antigenic peptide 
comprises bovine inhibin-a c N25 . 

18. The method of claim 14, wherein said host animal is swine. 

19. The method of claim 14, wherein said administering is in the absence 
of adjuvant. 



WO 99/57289 



PCT/US99/08238 



FIG. 1A 



Non-Reduced 
kDa Inh:HBc-78 HBc 



37 
29 



Reduced 
Inh:HBc-78 HBc 



22 
21 
17 




FIG. 1B 

Non-Reduced Reduced 
kDa Inh:HBc-78 HBc Iqb:HBc-78 HBc 




1 / 6 



WO 99/57289 



PCT/US99/08238 



FIG. 1C 



Non-Reduced 
kDa Inh:HBc-144 HBc 



Reduced 
lnh:HBc-144 HBc 



45 

27 

21 
<14 



IF 



FIG. 1D 



Non-Reduced 
141)8 Inh:HBc-144 HBc 



Reduced 
Inh:HBc-144 HBc 



WO 99/57289 



PCT/US99/08238 



FIG. 2 




3 / 6 



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PCT/US99/08238 




4 / 6 



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PCT/US99/08238 




5 / 6 



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PCT/US99/08238 




6 / 6 



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PCT/US99/08238 



SEQUENCE LISTING 

(1) GENERAL INFORMATION 
(i) APPLICANT: MICHIGAN STATE UNIVERSITY 

(ii) TITLE OF THE INVENTION: INHIBIN-HBc FUSION PROTEIN 

(iii) NUMBER OF SEQUENCES; 6 

(iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: Merchant, Gould, Smith, Edell, Welter & Schmidt 

(B) STREET: 3100 Norwest Center, 90 South 7th Street 

(C) CITY: Minneapolis 

(D) STATE: MN 

(E) COUNTRY: USA 

(F) ZIP: 55402 

(v) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Diskette 

(B) COMPUTER; IBM Compatible 

(C) OPERATING SYSTEM: DOS 

(D) SOFTWARE: FastSEQ for Windows Version 2,0 

(vi) CURRENT APPLICATION DATA: 

(A) APPLICATION NUMBER; NEW FILING 

(B) FILING DATE: 04-APRIL-1999 

( C ) CLASS I FI CAT I ON : 

(vii) PRIOR APPLICATION DATA: 

(A) APPLICATION NUMBER: 09/072,323 

(B) FILING DATE: 04-MAY-1998 



(viii) ATTORNEY /AGENT INFORMATION: 

(A) NAME: Kettelberger , Denise M 

(B) REGISTRATION NUMBER: 33,924 

(C) REFERENCE/ DOCKET NUMBER: 11526. 2WO01 

(ix) TELECOMMUNICATION INFORMATION: 

(A) TELEPHONE: 612-332-5300 

(B) TELEFAX: 612-332-9081 

(C) TELEX: 



(2) INFORMATION FOR SEQ ID NO:l: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 75 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: Other 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: 

TCTACCCCGC CGCTGCCGTG GCCGTGGTCC CCGGCTGCTC TGCTGCAGCG TCCGCCGGAA 
GAACCGGCTG CTCCG 

(2) INFORMATION FOR SEQ ID NO: 2: 

(i) SEQUENCE CHARACTERISTICS: 



1 



WO 99/57289 



PCT/US99/08238 



(A) LENGTH: 75 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: Other 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 

AGATCCGGCG GCGACGGCAC CGGCACCAGG GGCCGACGAG ACGACGTCGC AGGCGGCCTT 60 
CTTGGCCGAC GAGGC 75 

<2) INFORMATION FOR SEQ ID NO : 3 : 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 8 9 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

( D) TOPOLOGY : linear 

(ii) MOLECULE TYPE: cDNA 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 

GGGCCCTCTA CCCCGCCGCT GCCGTGGCCG TGGTCCGYCC GGCTGCTCTG CTGCAGCGTC 60 
CGCCGGAAGA ACCGGCTGCT CCGGGGTAA 8 9 

(2) INFORMATION FOR SEQ ID NO : 4 : 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 27 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: unknown 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 

Gly Pro Ser Thr Pro Pro Leu Pro Trp Trp Ser Pro Ala Ala Leu Leu 

1 5 10 15 

Gin Arg Pro Pro Glu Glu Pro Ala Ala Pro Gly 
20 25 

(2) INFORMATION FOR SEQ ID NO: 5: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 87 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 

GATCACTCTA CCCCGCCGCT GCCGTGGCCG TGGTCCCCGG CTGCTCTGCT GCAGCGTCCG 60 
CCGGAAGAAC CGGCTGCTCC GGTAGAT 87 

(2) INFORMATION FOR SEQ ID NO: 6: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 28 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: unknown 

(D) TOPOLOGY: linear 



2 



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PCT/US99/08238 



(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 

Asp His Ser Thr Pro Pro Leu Pro Trp Trp Ser Pro Ala Ala Leu Leu 

15 10 15 

Gin Arg Pro Pro Glu Glu Pro Ala Ala Pro Val Asp 
20 25 



18 



3