A chimeric protein formed of a small peptide antigen inserted into an exposed surface region of the B subunit of heat-labile enterotoxin of E. coli useful as an immunogenic vaccine composition.

FIELD OF THE INVENTION 
This invention relates to useful immunogenic molecules formed of the beta 
subunit of heat-labile enterotoxin (LTB) and an antigenic peptide antigen. 
More particularly, an antigenic peptide is genetically inserted into an 
exposed loop region of LTB, resulting in the production of a three 
dimensional molecule having the inserted antigen exposed on its surface. 
BACKGROUND OF THE INVENTION 
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 abcesses, skin lesions, and hypersensitivity. These factors are 
unacceptable for the production of a commercially useful vaccine. 
Chimeric molecules formed of large carrier proteins with attached peptide 
epitopes have been suggested as useful vaccines for small peptide 
antigens. However, added peptides extending from a three-dimensional 
protein are generally susceptible to proteolytic degradation. Insertion of 
an antigenic peptide into an interior portion of a carrier protein may 
avoid degradation problems, but disruption of the carrier protein's native 
sequence can alter the carrier's three dimensional structure and thus its 
function, including its ability to act as an efficient immunogen. 
The non-toxic beta subunit of cholera toxin (CTB) and the related B subunit 
of heat-labile enterotoxin from E. coli (LTB) are powerful immunogens that 
have been suggested for use as carriers of foreign epitopes. In studies 
testing the activity of CTB, antigenic peptides have been genetically 
fused to either the N- or C-terminus and tested for activity. These 
constructs were generally susceptible to rapid proteolytic degradation of 
the terminally fused peptide. (European Patent Application No: 89312713.4, 
published Jun. 13, 1990.) In other studies, a CTB-peptide molecule having 
a 10 amino acid peptide from the HIV-1 gp120 envelope protein substituted 
for eight amino acids in CTB at positions 56-63 was shown to be resistant 
from proteolytic degradation as compared with an N-tenninal CTB-peptide 
product. However, only a detectable response to the substituted gp120 
epitope was obtained, and only in some, not all animal hosts. (Backstrom, 
et al., 1994, Gene 149:211-217.) 
It is therefore highly desirable to develop an efficient and commercially 
useful process for producing immunogenic molecules containing antigenic 
peptide epitopes for use as vaccines, where the immunogenic molecule 
permits good recognition of the epitope as antigenic without high 
susceptibility to proteolytic degradation and produces a good immune 
response against the inserted antigen when administered to a host animal 
in the absence of adjuvant. 
SUMMARY OF THE INVENTION 
Chimeric molecules comprising the B subunit of heat-labile enterotoxin 
(LTB) and an inserted antigenic peptide have been found to display the 
antigenic epitope in an exposed surface of the LTB molecule without 
disruption of LTB folding and pentameric assembly and to provide 
immunogenic molecules useful in generating an immune response against the 
inserted small antigen. The LTB protein is also referred to as etxB (for 
enterotoxin B). This protein is encoded by the etxcB gene. 
Specific regions of the nucleic acid sequence encoding LTB have been 
identified as suitable antigen-insertion positions. A nucleic acid 
construct is produced having a nucleic acid sequence encoding the antigen 
inserted into the nucleic acid sequence encoding LTB. The insertion is 
made such that the expressed LTB-antigen fusion protein will include the 
inserted antigen in an external, exposed loop position. For example, when 
the antigen's sequence is inserted at approximately nucleotide 237 of etxB 
without loss of any LTB sequences, the expressed fusion protein displays 
the antigen on an exposed surface of the folded LTB molecule. At 
nucleotide 237, the etxB sequence contains a unique Sma I restriction 
site. In a preferred embodiment of the invention, the antigenic peptide is 
inserted at the unique Sma I site. 
In a most preferred embodiment of the invention, the antigenic fragment is 
a sequence of the .alpha.C subunit of inhibin, the fertility-modulating 
protein. For example, preferred antigens include bINH.alpha.C.sup.1-14 and 
bINH.alpha.C.sup.1-26 containing the first fourteen and first 26 
N-terminal amino acids of the bovine inhibin alpha-C subunit, 
respectively. The antigenic sequence is inserted into the LTB molecule by 
inserting the gene encoding the inhibin fragment into etxB at the unique 
Sma I restriction site. 
Since LTB is a pentameric molecule containing multiple exposed surfaces, 
the LTB:antigen fusion proteins, when used as vaccines, present multiple 
antigens for antibody development.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In the preferred embodiments of the invention, an immunogenic carrier 
molecule, the non-toxic B subunit of the E. coli heat-labile ei-terotoxin 
(LTB), is modified to include an inserted antigenic peptide. The inserted 
antigenic peptide is positioned in an exposed site of the LTB molecule, 
e.g., in an external surface of one of the molecule's loops, resulting in 
the presentation of the antigenic epitope on an exposed surface of the 
three dimensional chimeric molecule. When used as a vaccine, the chimeric 
antigen-LTB molecule is effective in eliciting in antibody response 
against the antigenic peptide in host animals. Vaccination of a host 
animal with the chimeric antigen-LTB results in the development of 
specific anti-antigen antibodies in the animal, preferably in the absence 
of added adjuvant. 
LTB 
Heat-labile enterotoxin from E. coli is a bacterial protein toxin having an 
AB.sub.5 multimer structure. The B pentamer serves a membraine-binding 
function and the A subunit is needed for enzymatic activity. Structurally, 
the B subunits are arranged in a donut shape as a highly stable pentamer. 
The donut shape is formed from five of the identical B subunit monomers 
arranged symmetrically around a central 5-fold axis with a pore in the 
middle. For review of the LTB structure and assembly into pentameric form, 
see Sixma, et al., 1993, J.Mol. Biol. 230:890-918, "Refined structure of 
Escherichia coli heat-labile enterotoxin, a close relative of cholera 
toxin". The crystal structure and three dimensional coordinates of the B 
subunit are known (Sixma, et al., Nature 351:371-377, 1991). 
In the B.sub.5 pentamer, the identical B subunits interact with themselves, 
but also have loops that are exposed on the surface of the pentamer. Each 
subunit takes part in approximately 30 inter-subunit hydrogen bonds and 
six salt bridges with its two neighbors. Although a large portion of the B 
subunit's surface area is buried inside the structure of the AB.sub.5 or 
B.sub.5 complexes, several loop structures are exposed on the surface of 
the subunit, and/or on the surface of the associated pentameric complex, 
as shown by X-ray crystallography. The loops are parts of the secondary 
structure of the B subunit, which includes a small N-terminal helix, two 
three-stranded anti-parallel sheets, and a long alpha-helix. Loops in the 
LTB subunit provide connections between strands and are believed to 
provide shape to the molecule's binding cavities 
Analysis of the LTB protein's structure [Seq. ID NO:2] by, for example, 
interactive computer graphic modeling, identified several domains of the 
pentamer appropriate for display of inserted epitopes. For example, 
appropriate insert regions include external loops formed at amino acid 
positions 10-15; 22-26; 30-37; 41-47; 50-61; 77-82; and 88-94. The 
sequence of the etxB gene, which encodes each of the identical B subunit 
proteins, is known [Seq. ID NO:1](Yamamoto, et al., 1984, J. Biol. Chem., 
259:5037-5044) and the coding sequences for each of the loops can be 
determined. The nucleotide residues encoding the loops include positions 
109-121; 203-218; 229-260; 310-323, and 343-359. One preferred loop 
sequence includes nucleic acid residues 229-260, and contains a unique 
restriction site, SmaI at nucleotide 237. Unique restriction sites can 
also be engineered into other exposed LTB loops by recombinant DNA 
technology, permitting ease of insertion of a desired antigen. For 
example, an external .alpha.-helix encoded by etxB nucleic acid residues 
92-110 can be engineered to contain a unique Bgl II site at nucleotide 97, 
by replacing etxB nucleotide 100 with thymine. 
Using these unique restriction sites, foreign niucleic acid sequences 
encoding small peptide antigens from a desired protein may be inserted 
into a the etxB to form a nucleic acid construct. The small peptide 
antigen can be a fragment of a larger protein. The foreign sequences 
encoding a desired antigenic sequence can be inserted so that the reading 
frame encoding LTB is not disrupted and the peptide antigen is expressed 
within the LTB sequence. The fusion proteins containing antigenic peptide 
sequence and the LTB sequence are immunoreactive with antibodies that 
recognize or bind to the inserted peptide or to an epitope of a protein 
containing the sequence of the inserted peptide. The immunogenic fusion 
proteins, or chimeric LTB molecules, can be expressed by methods known for 
expressing proteins in host cells. On expression of the chimeric LTB 
molecule, the inserted peptide antigen is expressed on the surface of the 
molecule and presented for immune response in a host animal. The 
multimeric structure of LTB allows presentation of multiple antigens on 
the multiple exposed surfaces of a single pentamer. 
Antigens 
Antigenic peptides useful in the present invention are generally short 
amino acid sequences, e.g., approximately 8-30 amino acids, and preferably 
10-25 amino acids in length. The peptide is preferably known to be unique 
to a specific protein of choice, and represents an epitope that is able to 
induce a desired immune response against the protein target. For example, 
the antigenic peptide may be known to produce a desired antigenic response 
when used in another carrier protein/adjuvant system such as 
co-administration with Fruend's Adjuvant or other immunogen. 
Alternatively, the peptide antigen may be a portion of a known protein 
having a particularly unique amino acid sequence distinguishing it from 
other 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. 
Antigenic peptides may be inserted into the LTB molecule by recombinant DNA 
methods. For example, a synthetic nucleic acid sequence or vector 
containing the desired nucleic acid sequence to be inserted into etxB is 
specifically designed to include restriction endonuclease sites matched to 
a specified endonuclease-cut etxB sequence. Where the etxB contains a 
single, unique restriction endonuclease site, the antigen's nucleic acid 
sequence preferably is engineered to include matched restriction sites at 
both ends of the sequence, so that it can be inserted into the etxB 
sequence without removal of any etxB nucleotides. Care is taken to match 
the antigenic nucleic acid sequence to be inserted with the reading frame 
of the etxB sequence so that normal expression of the encoded LTB and the 
encoded antigen is achieved. 
Insertion of the antigen's nucleic acid sequence and expression of the 
antigenic peptide does not interfere with normal expression of LTB 
monomers or with folding of the molecule. Preferably, insertion of the 
antigenic peptide does not interfere with the association of the LTB 
monomers into pentameric form. Most preferably, the inserted antigenic 
peptide does not interfere with LTB 's three dimensional structure, and 
permits presentation and recognition of the inserted antigen on an exposed 
surface of the three-dimensional pentameric form of the molecule. 
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., 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-hybridzing nucleic acid sequences. These limitations are generally 
known and can be recognized by review of the amino acid sequence to be 
inserted. 
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 LTB-antigen in E. coli, the 
peptide sequence may be back translated into the nucleotide sequence using 
the codon frequency found in E. coli proteins, as determined by the GCG 
computer program (Devereaux, et al., 1984, Nucleic Acids Res. 12:387-3905) 
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 condon 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 hornone (FSH) from the 
anterior pituitary gland. A vaccine against inhibin can decrease available 
inhibin, with a resulting increase in levels of follicle stimulating 
hormone, and enhanced fertility. Enhanced fertility can be due to enhanced 
production of sperm or ova. Immunization of animals with bovine 
inhibin-.alpha.C subunit has demonstrated the usefulness of inhibinbased 
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 most preferred embodiment and exemplary of the invention, the nucleic 
acid sequence encoding the first 14 N-terminal residues of the antigenic 
inhibin .alpha..sub.c subunit (.alpha.C.sup.1-14) is inserted into the 
unique SmaI restriction site of etxB. Alternatively, the nucleic acid 
sequence encoding the first 26 N-terminal residues (.alpha.C.sup.1-26) is 
inserted. The chimeric gene is subcloned into a broad-host-range 
expression vector. The inserted antigen is expressed on the surface of the 
LTB molecule, such that when the expressed chimeric protein vaccine is 
injected into host animals, an anti-inhibin response is induced in the 
animals, reducing inhibin and thereby enhancing fertility in treated 
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 LTB-antigen and express the fusion 
protein. Suitable vectors for use in Vibrio include pJF 118, as described 
in Furst, et al, 1986, Gene 48:119-131. 
Methods of Administration 
LTB is a known immunogen. The immunogen of the invention, formed of the 
intact LTB protein and an inserted antigenic peptide, are administered 
according to the methods known as effective for the immunogenic 
administration of proteins such as LTB. 
Administration methods include injection 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 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. 
Examples 1 
Analysis of the 3-D Structure of LTB 
The B subunit of the E. Coli heat-labile enterotoxin (LTB) is a multimeric 
protein composed of five identical polypeptides of about 11 kDa each. The 
three-dimensional crystal structure of LTB was analyzed using interactive 
computer graphic modeling for potential exposed, antigenic regions 
appropriate for the display of inserted epitopes. Specifically, the 
structure was analyzed to identify potential sites for insertion of small 
peptides for purposes of producing potentially antigenic molecules or 
vaccines. 
The protein's structure was examiner with the Biosym modelings program 
(Biosym Technologies, 1985, Scranton Road, San Diego, Calif.). Domains of 
the LTB pentamer that are exposed on the surface of the molecule were 
identified by examination of stereo images. The domains encoding external 
loops useful for insertion of antigens were selected using the sequence 
analysis program GCG (Devereaux, et al., 1984, Nucleic Acids Res. 
12:387-3905). Possible insertion sites were identified as loops in the 
three-dimensional structure that could potentially tolerate additional 
amino acids. These loops were also exposed on the surface of the molecule, 
indicating potential antigen presentation. Aligning the amino acid 
sequence and then nucleic acid sequence with the identified structural 
site, potentially useful insertion sites were found. 
A potential insertion site is positioned approximately at nucleotide 237, 
and contains a unique Smal endonuclease restriction site in the native 
sequence. A second potential insertion site is positioned at approximately 
nucleotide 97. While this second site does not contain a unique 
restriction site in the native sequence, one nucleotide was modified by 
site-directed mutagenesis to make this site available for direction 
insertion of foreign oligonucleotides. By replacing adenine at position 
100 with thymine, a unique Bgl II restriction site was created at 
nucleotide 97. In the modified protein encoded by the mutant etxB, Glu-7 
is replaced by Asp as shown below: 
______________________________________ 
Wild Type etxB 
(Seq. ID NO:3) 
GCTCCTCAGTCTATTACAGAACTATGTTCGGAATATCAC 
80 +---------+---------+---------+-------- 118 
1 AlaProGlnSerIleThrGluLeuCysSerGluTyrHis 13 
(Seq. ID NO:4) 
Mutant E7D0etxB 
(Seq. ID NO:5) 
GCTCCTCAGTCTATTACAGATCTATGTTCGGAATATCAC 
80 +---------+---------+---------+-------- 118 
1 AlaProGlnSerIleThrAspLeuCysSerGluTyrHis 13 
(Seq. ID NO:6) 
______________________________________ 
A third potential insertion site was created at nucleotide 176 by 
substitution of adenine nucleotides at positions 179 and 180 with cytosine 
and thymine nucleotides, respectively. This mutation created a unique Stul 
site at nucleotides 176-180. This mutation also created a substitution of 
the Lys-34 by Leu residue. The resulting mutant LTB subunit was expressed 
and formed pentamers as did the wild Type LTB 
______________________________________ 
Wild Type etxB 
(Seq. ID NO:7) 
ATGGCAGGCAAAAGAGAAATGGTTATCATTA 
170 +---------+---------+---------+ 200 
31 MetAlaGlyLysArgGluMetValIleIle 40 
(Seq. ID NO:8) 
Mutant K43L-etxB 
(Seq. ID NO:9) 
ATGGCAGGCCTAAGAGAAATGGTTATCATTA 
170 +---------+---------+---------+ 200 
31 MetAlaGlyLeuArgGluMetValIleIle 40 
(Seq. ID NO:10) 
______________________________________ 
To create these sequences, wild type LTB was constructed as described in 
(Sandkvist, et al., 1987, J. BacterioL. 169:4570-4576). Site-directed 
mutagenesis was performed by the method of Eckstein (Sayers, et al., 1988, 
Nucleic Acids Res. 16:791-802). The sequences of the mutant genes obtained 
were verified by nucleotide sequence determination by di-deoxy sequencing 
procedures (Sanger, et al., 1977, Proc. Nat'l. Acad. Sci. USA, 
74:5463-5467). 
Example 2 
Production of a LTB-Inhibin Fusion Polypeptide 
A nucleic acid sequence encoding an immuno-dominant epitope of inhibin, was 
inserted into the nucleic acid sequence encoding LTB (etxB) at its unique 
Smal site. To accomplish the insertion of the sequence, the N-terminal 
portion of the .alpha.C-subunit was back-translated into the nucleotide 
sequence using the codon frequencies found in E. coli proteins,using the 
GCG program as described above (Devereaux, et al., 1984, Nucleic Acids 
Res. 12:387-3905). Complementary oligonucleotides of this back-translated 
sequence were chemically synthesized (MSU Macromolecular Structure 
Facility, Biochem. Dept.) and inserted into etxB in a manner that ensured 
the continuation of the reading frame after insertion at the SmaI 
restriction site, as shown in the sequences in the table below. 
The pair of complementary oligonucleotides (Seq. ID No:11) encoding 
bINH.alpha.C.sup.1-14 (Seq. ID No:12) was inserted into the pMMB68 vector 
(Sandkvist, et al., 1987, J Bacteriol. 169:4570-4576), a broad host-range 
vector, containing the etxB sequence under the control of the inducible 
isopropyl-.beta.-D thiogalactopyranoside (IPTG) tac promoter (Sandkvist, 
et al., 1987, J: Bacteriol. 169:4570-4576), digested with SmaI, as shown 
below. The sequences of the insert are shown in bold type, the nucleotide 
numbers of the etxB sequences are indicated, and extra nucleotides 
introduced to maintain the proper reading frame, are underlined. The 
encoded amino acid sequence is also shown. 
__________________________________________________________________________ 
230 240 
GTCGAAGTCCCTGGATCCACCCCGCCGCTGCCGTGGCCGTGGTCCCCGGCTGCTCTGGGCAGTCAA 
(Seq. ID No:11) 
ValGluValProGlySerThrProProLeuProTrpProTrpSerProAlaAlaLeuGlySerGln 
(Seq. ID No:12) 
__________________________________________________________________________ 
Recombinant plasmids containing the inhibin subunit sequence were 
introduced into E. coli cells (CB1360, GIBCO BRL, Life Technologies) by 
standard calcium phosphate transformnation methods. A diagram showing the 
constructed plasmid (pMMB552) is shown in FIG. 1. 
Colonies of transformed E. Coli were screened for inhibin and LTB 
expression by immunoblotting. Colonies were grown on parallel 
nitrocellulose membrane filters (Schleicher & Schuell), placed on nutrient 
agar (LB) plates containing ampicillin and IPTG as inducer. 
A first filter was blotted with anti-inhibin antibody, and a second filter 
was blotted with anti-LTB antibodies. Whole cell inhibin immunoblot assays 
using mink anti-bINH.alpha.C.sup.1-26 gly.tyr antiserum (Ireland, et al., 
1994, Biol. Reprod. 50:1265-1276) or anti-pentaneric LTB monoclonal 
antibody 118-8 (Sandkvist, et al., 1987, J Bacteriol., 169:4570-4576) were 
performed separately on each of the two parallel membranes. Colonies 
exhibiting both inhibin and LTB immunoreactivity were selected for further 
DNA sequence screening. DNA from these colonies was isolated by standard 
methods, cloned into the sequencing vector, M13mp19 and sequenced. The 
sequence analysis of both DNA strands was done by automated fluorescent 
sequencing (MSU-DOE-PRL Plant Biochemistry Facility) using ABI catalyst 
800 Taq cycle sequencing and the ABI 373A sequencer for the analysis of 
products. One of the many colonies containing the expected 
etxB:bINH.alpha..sub.C.sup.1-14 DNA sequence wasidentified as harboring 
pMMB552 and was retained for continued analysis and development. 
To determine if the fusion etxB-inhibin protein exhibited the same 
pentameric subunit structure as wild type B-subunit, protein produced by 
E. coli cells containing pMMB522 was run on SDS-polyacrylamide gels 
without denaturation (no boiling of samples) and with denaturation 
(boiling for 5 minutes). 
The electrophoretic mobility of the non-denatured fusion protein was about 
the same as that of the native LTB. After denaturation, the pentameric 
form dissociated into monomeric subunits that ran slightly slower than the 
wild type-B monomers, reflecting their larger size. 
Example 3 
Expression and Secretion of LTB-Inhibin Fusion Polypeptide in Cellular Host 
Systems 
The LTB-Inhibin fusion construct expressing the LTB-inhibin fusion protein, 
prepared as described for Example 2, was introduced into Vibrio cholerae 
TRH (Sandkvist, et al., 1987, J: Bacteriol. 169:4570-4576) and Vibrio 
anguilarum H3 (Crosa, 1980, Infec. Immunity, 27:897-902) by conjugative 
mobilization under conditions sufficient to produce chimeric protein at 
concentrations greater than 6 mg/I. Conjugation was achieved by making a 
mixed suspension of the donor strain (E. coli CB1360 harboring pMMB552), 
the helper strain, HB101pRK2013:kanamycin resistant (Km.sup.R), and the 
recipient strains (E anguillarium Rif.sup.R or V cholerae TRHY 7000: 
polymyxin resistant, Pmx.sup.R)at a 1:1:1 ratio in LB broth in a sterile 
1.5 ml eppendorf tube. The suspension mixture was plated on LB medium is a 
discrete droplet and incubated overnight at 37.degree. C. 
The overnight bacterial growth was scraped off the plate and suspended in 
sterile 0.9% saline solution, then replated on a Rif, A.sub.p selective 
plate. Selected colonies were individually streaked on fresh LB media 
(1.5% W/V Bacto-agar plates containing Luria Broth (LB, Difco, Detroit, 
Mich.) supplemented with 1.5% NaCl and 100 .mu.g/ml amnpicillin (Ap, 
Sigma, St. Louis, Mo.). Plates were incubated overnight at 30.degree. C. A 
single colony was picked from the plate, grown in 20 ml LB, and inoculated 
into one liter LB supplemented with 1.5% NaCl and 100 .mu.g/ml Ap for 
culture to an absorbance at 650 nm of 0.02-0.05. At A.sub.650 =0.02, IPTG 
was added to a final concentration of 1.0 mM to induce transcription of 
etxB. Cultures were harvested 6 or 48 hours after IPTG addition, and 
medium was saved for analysis and protein purification. 
A one ml sample of the cell culture was separated by centrifugation. The 
separated cells were resuspended in 1.0 ml PBS and broken by sonication 
with two ten second pulses. The amount of LTB pentamers present in the 
growth medium and in the lysed cells was determined by GMI ELISA using the 
methods described in Svennerholm and Holmgren, 1978, Curr. Microbiol., 
1:19-27. Briefly, pentameric, but not monomeric LTB binds to 
galactosyl-N-acetogalactosamninyl-(N-acetylneuraminyl)galactosylglucosylce 
ramide (GM1). To determine if the expressed fusion protein, 
etxB:bINH.alpha.C.sup.1-14, retained the LTB pentameric structure, the 
produced protein was tested for its ability to bind ELISA plates coated 
with GM1 ganglioside (Sigma). Wild type LTB was used as control. The 
amount of LTB in each sample was estimated from the ED.sub.50 of the E. 
coli enterotoxin standard curve. The percent LTB in the media was 
calculated as the amount in the media/the amount in media plus 
cells.times.100%. 
TABLE 1 
______________________________________ 
% EtxB 
Bacterial EtxB (.mu.g) Pentamers 
Strains Cells Media in Media 
______________________________________ 
EtxB:bINH.alpha..sub.c.sup.1-14 
V. cholerae 148.8 16.0 9.0 
V. anguillarum 
92.2 91.6 50.0 
EtxB 
V. cholerae 0.0 57.0 100.0 
V. anguillarum 
4.6 70.0 94.0 
______________________________________ 
As shown in Table 1 above, both host cells V. anguillarum and V. cholerae 
produced the recombinant fusion protein. Although the total amount of 
fusion protein produced in each host was similar, more fusion protein was 
detected in the medium of V. anguillarum as compared with V. cholerae. 
Fusion protein accumulated in the medium of 1 liter cultures was 
precipitated with ammonium sulfate (65% saturation) according to the 
methods described in Amin et.al., 1993, Biochem. Soc. Trans., 21:213S. The 
precipitate was recovered by centrifugation (10,000.times.g, Beckman L-80 
ultracentrifuge) for 25 minutes, was redissolved in PBS containing 5% 
glycerol, and ws dialized against the same buffer at 40.degree. C. 
An ion exchange column (CL 6B DEAE-Sepharose, 9.5.times.1.6 cm, Pharinacia, 
Piscataway, N.J.) equilibrated with PBS containing 5% glycerol, was used 
to further purify the fusion protein. An increasing NaCl gradient (137 to 
600 mM in PBS-glycerol) was used to elute fractions. An aliquot of each 
collected fraction was diluted 1:1000 in PBS-Tween containing 0.1% BSA and 
assayed by GM1 ELISA as described above to detect pentameric fusion 
protein. Fractions with the highest amount of the fusion protein were 
pooled, and the protein mass estimated from spectrophotometer readings 
using the Warburg equation: A.sub.280 =1 for 0.1% protein solution. LTB 
specific activity was estimated as the amount of GM1-reactivity as 
determined by the GM1 assay per total protein estimated by 
spectrophotometer readings. 
The fusion protein was eluted as a single peak between 300 and 430 mM of 
the NaCl gradient. An average of 84.9+/-21.5 mg of the fusion protein was 
isolated (67+/-11% recovery) with an LTB specific activity of 0.84+/-0.02 
per liter. 
Protein eluted from the ion exchange column was assessed for purity and 
molecular weight by SDS-PAGE. Pentameric or heat-disrupted (100.degree. 
C.) monomeric samples (1.2 to 3.6 .mu.g) of LTB or the fusion protein, 
LTB:bINH.alpha..sub.C.sup.1-14 were separated in one dimensional 12% 
SDS-PAGE in a mini-gel apparatus following the manufacturer's instructions 
(Mini-Protean II, BioRad). Separated proteins were visualized by Coomassie 
blue staining and compared with molecular weight markers for estimation of 
size. 
A single major band for each of the pentamneric LTB (39.5.+-.1 kDa) and 
monomeric LTB (8.9.+-.0.4 kDa) was observed in the stained gels. The major 
band for each of the pentameric LTB:bINH.alpha..sub.C.sup.1-14 (41.6.+-.2 
kDa) and monomeric LTB:bINH.alpha..sub.C.sup.1-14 (10.1.+-.0.2 kDa) were 
larger than the LTB bands, according to the expected size of the insert. 
The pentameric or heat-disrupted monomeric fusion protein samples were 
further analyzed for inhibin and LTB specificity by immunoblotting 
techniques. Briefly, duplicate samples were electrophoresed in 12% 
SDS-PAGE and electroeluted onto nitrocellulose membranes, as described 
above. After blocking with 0.01% Blotto (Food Club, Skokie, Ill.) in TBS 
for 2 hours, the membrane was washed in Tween-TBS (0.05% Tween-20), and 
cut to present two replicate membranes for antibody binding. 
One of the membranes was incubated with mink 
anti-bINH.alpha..sub.C.sub.1-26 gly.tyr antiserum (1:1000 in TTBS) 
overnight at room temperature, as described in Ireland et.al., 1994, Biol. 
Reprod., 50:1265-1276. After washing in TTBS (5, 10-minute washes), the 
membrane was further incubated in 20 ml .sup.125 
I-bINH.alpha..sub.C.sup.1-26 gly.tyr (1.times.10.sup.6 cpm/ml in TTBS with 
1% gelatin), for competition. The membrane was washed and placed on Xray 
film (Kodak X-OMAT AR) with a Cronex intensifying screen and exposed for 
ten days at -80.degree. C. 
The second membrane was incubated with a monoclonal anti-LTB antibody 
(118-8, 1:100 dilution) as described in Sandvikst et.al., 1987, J 
Bacteriol., 169:4570-4576. Incubation with second antibody goat anti-mouse 
peroxidase conjugate (Vector) diluted 1:5000 in TTBS was followed by 
visualization in 0.2% (w/v) 4-chloro-1-napthol (Sigma) in PBS containing 
20% methanol and 0.01% H.sub.2 O.sub.2. 
As shown in FIG. 2, the immunoblot assays confirmed 
LTB:bINH.alpha..sub.C.sup.1-14 fusion protein was produced having dual 
inhibin and LTB immunoreactivity. Anti-LTB antibody recognized both wild 
type LTB and the fusion protein in both monomeric and pentameric forms. 
Anti-bINH.alpha..sub.C.sup.1-26 gly.tyr antiserum recognized only the 
monomeric and pentameric forms of the fusion protein, and did not bind the 
LTB alone. Molecular weight determinations for the immunoreactive proteins 
were similar for the monomeric (11 kDa, open arrow) and pentameric (45 
kDa, shaded arrow) forms as compared with the size estimates obtained from 
Coomassie blue stained gels. 
Inhibin Radioimmunoassay (RIA) 
Inhibin immunoactivity in duplicate samples of the fusion protein (0.5-64 
.mu.g) in both monomeric and pentameric forms, and in control samples, 
including bINH.alpha..sub.C.sup.1-26 gly.tyr peptide standards (0.039 
ng-10 ng), pentameric and monomeric LTB (0.625 .mu.g-80.mu.g), was 
determined by radioimmunoassay using mink anti-bINH.alpha..sub.C.sup.1-26 
gly.tyr antiserum diluted 1:40,000 in RIA buffer (0.01M NaH.sub.x 
PO.sub.4, 0.1M NaCl, 0.025M EDTA, 0.1% NaN.sub.3, 0.1% Triton X-100, 
0.1%BSA). .sup.125 I-bINH.alpha..sub.C.sup.1-26 gly.tyr (20,000 cpm/tube) 
was used as tracer. The RIA conditions were as described in Ireland, et 
al., 1992 Biol. Reprod., 47:746-50; and Good, et al., 1995, Biol. Reprod., 
53:1478-1488. Briefly, 200 .mu.l of protein sample, 200 .mu.l mink 
anti-bINH.alpha..sub.C.sup.1-26 gly.tyr antiserum and 100 .mu.l 
125-bINH.alpha..sub.c.sup.1-26 gly.tyr tracer were sequentially added into 
test tubes and incubated for 16-18 hours at 4.degree. C. The antibody: 
.sup.125 I-bINH.alpha..sub.c.sup.1-26 gly.tyr complex was incubated for 2 
hours with 100 .mu.l Staphylococcus protein A (Staph A; Boehringer 
Mannheim) diluted 1:50 in RIA buffer at room temperature, followed by the 
addition of 2 ml/tube PBS (pH 7.4 with 0.025 M EDTA) and centrifugation 
for 30 minutes at 2,200.times.g at 4.degree. C. (Beckman GPR centrifuge) 
to sediment the inhibin-antibody complex. Tubes were decanted and 
radioactivity in the Staph A:antibody:.sup.125 
I-bINH.alpha..sub.c.sup.1-26 gly.tyr complex determined using a MACC 
Micromedic .gamma.-counter. Inhibin immunoactivity was plotted as percent 
.sup.125 I-bINH.alpha..sub.c.sup.1-26 gly.tyr tracer bound. 
As shown in FIG. 3, the pentameric fusion protein, 
LTB:bINH.alpha..sub.c.sup.1-14 reacted with the anti-inhibin antiserum 
parallel to the reaction of bINH.alpha..sub.c.sup.1-26 gly.tyr peptide. In 
a separate RIA, monomeric fusion protein did not react with the antiserum 
(FIG. 4). 
Example 4 
Passive Immunization of Mice and Rabbits with 
Anti-EtxB:bINH.alpha..sub.c.sup.1-14 Antiserum 
Use of the LTB:bINH.alpha..sub.c.sup.1-14 fusion protein as a fertility 
vaccine requires that LTB:bINH.alpha..sub.c.sup.1-14 stimulates production 
of serum inhibin antibodies when injected into animals, ideally in the 
absence of adjuvant. As shown above in Example 3, pentameric form of 
LTB:bINH.alpha..sub.c.sup.1-14 fusion protein cross-reacted in a parallel 
fashion with a synthetic bINH.alpha..sub.c.sup.1-26 gly.tyr. peptide 
during radioimmunoassay (RIA) indicating that bINH.alpha..sub.c.sup.1-14 
peptide is on the hydrophilic surface of the intact, non-denatured 
molecule. These RIA data imply that the bINH.alpha..sub.c.sup.1-14 peptide 
portion of EtxB:bINH.alpha..sub.c.sup.1-14 is immunogenic, especially 
since immunogenicity is closely correlated with hydrophilicity (Sagar, et 
al., 1989 J. Pept. Protein Res. 33:452-456). In addition, 
bINH.alpha..sub.c.sup.1-4 peptide was inserted between amino acid 53 and 
54 in EtxB a region of LTB known to be highly immunodominant (Jacob, et 
al., 1984 EMBO J. 3:2889-2893, and 1985 EMBOJ. 4:3339-3343). 
Animals 
Rabbits and mice were maintained by the University Laboratory Animal 
Resources (ULAR, Michigan State University) in their animal care 
facilities for the duration of the experiments. All animals were 
maintained on a 12L: 12D cycle with food and water provided ad libitum. 
Treatments 
I. Active Immunization 
Adult female New Zealand White rabbits of 6-7 kg body weight were purchased 
from ULAR and housed 1/cage. Purified LTB:bINH.alpha..sub.c.sup.1-14 
fusion protein produced in Vibrio anguillarum as described in Example 3, 
was mixed with or without Freund's adjuvant and used to actively immunize 
rabbits. For antigen preparation in Freund's adjuvant, 3 ml 
LTB:bINH.alpha..sub.c.sup.1-14 in phosphate buffered saline (PBS-0.01M 
phosphate buffer, pH 7.4 with 0.15M NaCl) was mixed with an equal volume 
of Freund's complete (primary injection) or incomplete (boosters) 
adjuvants (Calbiochem, La Jolla, Calif.) to give a final sample 
concentration of 100 (primary) or 50 (boosters) .mu.g 
LTB:bINH.alpha..sub.c.sup.1-14 per ml. Antigen and adjuvant were mixed in 
12.times.75 mm glass test-tubes and emulsified using a 10 ml syringe with 
an 18 gauge needle. Mixing was done by suction and expulsion of the 
antigen and adjuvant through the needle until a stable emulsion was 
obtained. The emulsion was considered stable when a droplet of emulsion on 
the surface of the water in a beaker did not disperse when the beaker was 
shaken. For antigen preparation without Freund's adjuvant, 
LTB:bINH.alpha..sub.c.sup.1-14 fusion protein was diluted in PBS to a 
concentration of 100 (primary) or 10 (boosters) .mu.g 
LTB:bINH.alpha..sub.c.sup.1-14 per 0.5 ml (2 ml total volume) and 
sterilized by expulsion through a 2.2 .mu.m Millex GV sterile filter 
(Millipore, Bedford, Mass.) attached to a 5 ml syringe into a 15 ml 
sterile polypropylene tube. 
All rabbits were bled from the marginal ear vein before immunization to 
recover preimmune serum which was used as the negative control. For the 
adjuvant group (n=3), the primary dose (volume=1 ml) of 
LTD:bINH.alpha..sub.c.sup.1-14 was given s.c. in the nape of the neck or 
in the back at 10 sites (0.1 ml per site) followed by five s.c. boosters 
at 2-week intervals. For the group immunized without Freund's adjuvant 
(n=3), the primary dose of LTB:bINH.alpha..sub.c.sup.1-14 (volume=0.5 ml) 
was injected i.v. into the marginal ear vein followed by five boosters at 
2-week intervals. LTB:BINH injections were performed while restraining 
rabbits in a towel (ULAR recommendations). To collect blood, rabbits were 
anesthetized with sodium phenobarbitone (Nembutal, Sigma, St. Louis, Mo.) 
and blood was removed from a marginal ear vein (5-15 ml) or the heart 
(100-150 ml) using a 20 or 50 ml syringe with a 20 gauge needle. Serum 
samples were collected 14 days after each booster for a total of five 
bleeds per rabbit. Blood samples were incubated at room temperature for 2 
hours and then at 4.degree. C. overnight. Serum was separated from clotted 
blood cells by centrifugation at 1000.times.g for 30 minutes at 4.degree. 
C. then stored at -20.degree. C. until assayed by ELISA. Serum from three 
rabbits with the highest inhibin antibody titer was pooled and used to 
passively immunize mice. 
II. Passive Immunization 
Twenty prepubertal male BALB/c mice (25 days old) averaging 12.2.+-.1.4 
grams body weight were purchased from Harland Sprague Dawley (Wilmington, 
Mass.) and housed 5/cage. Rabbit anti-LTB:bINH.alpha..sub.c.sup.1-14 
antiserum generated, as described above, was used to passively immunize 
mice, and preimmune serum was used as control. Crude 
anti-LTB:bINH.alpha..sub.c.sup.1-14 antiserum or preimmune serum was 
filter-sterilized through a 2.2 .mu.m Millex GV sterile filter attached to 
a 5 ml syringe and collected into 15 ml sterile polypropylene tubes. 
Mice were divided into four groups (5 mice/group). The first two groups 
were given one 0.5 ml i.p. injection of either rabbit 
anti-LTB:bINH.alpha..sub.c.sup.1-14 antiserum or preimmune control serum, 
whereas the other two groups received two, 0.5 ml i.p. injections of 
either anti-LTB:bINH.alpha..sub.c.sup.1-14 or preimmune serum spaced 12 
hours apart. After mice were anesthetized with Metofane (Methoxyflurane; 
Pitman-Moore, Mundelein, Ill.), blood was collected via heart puncture 12 
hours after the last injection in each group, serum processed, as 
described above in Active Immunization, and inhibin antibody titer and 
concentrations of FSH and LH in serum determined, as described below. 
Antibody Titer Determination by ELISA 
I. Anti-LTB Antibody Titer 
A modification of the GM1 ELISA, described in Svennerholm and Holmgren, 
1978, supra, was used to determine anti-LTB antibody titer in serum from 
actively immunized rabbits (5 bleeds/rabbit). Probind microliter plates 
(Falcon, Lincoln Park, N.J.) were coated overnight at room temperature 
with 0.2 .mu.g/well 
galactosyl-N-acetogalactosaminyl-(N-acetylneuraminyl)-galactosyl 
glucosylceramide (GM1) in PBS. After washing wells three times with PBS 
containing 0.05% Tween-20 (PBS-T), non-specific binding sites were blocked 
by adding 1% BSA (Sigma) in PBS-T to each microwell and incubating for 2 
hours at room temperature. Plates were washed and incubated with 100 
ng/well partially purified pentameric LTB for 1 hour at room temperature. 
After another wash, microwells were incubated with serum from actively 
immunized rabbits diluted 1:5000 in PBS-T containing 0.1% BSA (PBS-T-B) 
for 1 hour at room temperature. After washing with PBS-T, horseradish 
peroxidase-labeled goat anti-rabbit IgG diluted 1:5000 (Vector, 
Burlingame, Calif.) in PBS-T-B was added to each microwell and incubated 
for 1 hour at room temperature. Color was developed using 
ortho-phenylenediamine (OPD) in 0.1M citrate buffer, pH 4.5, containing 
0.01% H.sub.2 O.sub.2. After 10 minutes, color development was terminated 
by adding 3M phosphoric acid. Titer of anti-LTB antibodies in serum was 
defined as absorbance at 490 nm (A.sub.490, Bio-Rad microplate reader, 
Model 3550). 
II. Inhibin Antibody Titer 
A modified ELISA method, as described in Groome and O'Brien, 1993 J. Immun. 
Methods 165:167-176, was used to estimate titer of anti-inhibin antibodies 
in serum. Xenobind microliter plates (Xenopore Inc., Hawthrone, N.J.) were 
covalently coated overnight at room temperature with 1 .mu.g/well 
bINH.alpha..sub.c.sup.1-26 gly.tyr. peptide or 1 .mu.g/well partially 
purified bovine inhibin ppbINH, prepared as described in Good, et al., 
1995, Biol. Reprod., 53:1478-1488, in PBS as recommended by the 
manufacturer. After washing wells three times using PBS-T, non-specific 
binding sites were blocked by incubating wells with 3% gelatin (Bio-Rad) 
in PBS-T for 2 hours at room temperature. Plates were washed and the 
coated microwells incubated for 2 hours with serum from actively immunized 
rabbits or passively immunized mice diluted 1:100 in PBS-T-B. After 
washing with PBS-T, horseradish peroxidase labeled goat anti-rabbit IgG 
(Vector, Burlingame, Calif.) diluted 1:5000 in PBS-T-B was added to each 
microwell and incubated for I hour at room temperature. Microwells were 
thoroughly washed, color was developed using OPD-H.sub.2 O.sub.2, and 
titer of inhibin antibodies in serum was determined, as described in LTB 
antibody titer. 
FSH and LH RIA 
I. Iodination and Validation 
Concentrations of FSH and LH in serum from passively immunized mice were 
determined in duplicate samples using rat FSH and LH reagents kindly 
supplied by the National Institute of Diabetes and Digestive and Kidney 
Diseases (NIDDK). The chloramine-T method described in Hunter and 
Greenwood, 1962, Nature 194:495-496, was used to radiolabel 5 .mu.g of 
rFSH (NIDDK-rFSH-I-8) or rLH (NIDDK-rLH-I-9) dissolved in 20 .mu.l PBS in 
a Nalgene cryovial (Nalge, Rochester, N.Y.). Iodinated rFSH and rLH was 
stored at 4.degree. C. for use as tracer in each RIA. 
The ability of radioiodinated hormone to bind antiserum was determined in 
duplicate by incubating 50 .mu.l antiserum (NIDDK-anti-rFSH-S-11 or 
NIDDK-anti-rLH-S-11) diluted 1:1000 to 1:640,000 in assay buffer (0.0095 M 
Na.sub.2 HPO.sub.4, 0.014 M NaH.sub.2 PO.sub.4, 0.15 M NaCl, 0.01 M EDTA, 
0.1% NaN.sub.3, 0.5% Chicken Egg Albumin, pH 7.2) with 100 .mu.l assay 
buffer and 50 .mu.l .sup.125 IrFSH or .sup.125 IrLH tracer diluted to 
12,000 cpm/tube with assay buffer. Tubes were incubated in the 
aforementioned buffers for 18 hours at room temperature followed by: 1) 
precipitation of the bound antibody by incubating tubes with 50 .mu./tube 
of Staphylococcus protein A (Staph A; Boehringer Mannheim) diluted 1:100 
in PBS-EDTA (PBS, pH 7.4, 0.025 M EDTA) for 1.5 hours at room temperature; 
2) addition of 2 ml/tube PBS-EDTA to wash tubes; and 3) immediate 
centrifugation for 30 minutes at 2,200.times.g at 4.degree. C. (Beckman 
GPR centrifuge) to sediment the Staph A:antibody:tracer complex. The tubes 
were decanted and radioactivity in the dried pellet determined in a MACC 
Micromedic .gamma.-counter. Percent binding of tracer to antibody was 
calculated as: 
EQU % binding=(average cpm for each dilution).times.100 average total cpm 
An antiserum dilution of 1:100,000 resulted in 20% binding of tracer to LH 
or FSH antibody, thus the 1:100,000 dilution was used in RIA of rFSH or 
rLH in serum (data not shown). 
II. RIA 
Mouse FSH and LH cross-react with the NIDDK rat gonadotropin antibodies 
(Bearner, et al., 1972 Endocrinology 90:823-827; Kovacic and Parlow, 1972 
Endocrinology 91 :910-915). In the present study, several dilutions of 
BALB/c mouse serum were used to confirm parallelism of mouse serum to the 
standard curve produced by NIDDK-rat-FSH-RP-2 or NIDDK-rat-LH-RP-3 
reference preparations. The standard FSH and LH assay (Parkening, et al., 
1980) was miniaturized to reduce the total incubation volume from 600 
.mu.l to 200 .mu.l. Duplicate mouse serum samples (5 to 50 .mu.l) diluted 
to 100 .mu.l in assay buffer were incubated with 50 .mu.l antiserum 
(1:100,000 in assay buffer, NIDDK-anti-rFSH-S-11 or NIDDK-anti-rLH-S-11) 
at room temperature for 18 hours. The following day, 50 .mu.l tracer was 
added at 12,000 cpm/tube and the mixture further incubated at room 
temperature for 24 hours. This second incubation was followed by 
precipitation with Staph A as described in lodination and Validation. 
Tubes were decanted and the radioactivity in each dried pellet determined 
in a MACC Micromedic .mu.-counter. FSH values were expressed in terms of 
the rat FSH-NIDDK-RP-2 reference standard, whereas LH values were 
expressed in terms of the rat LH-NIDDK-RP-3 reference standard. Samples 
were analyzed in a single assay for each hormone. rFSH and rLH assay 
sensitivities were 0.625 and 0.156 ng/ml and intra-assay coefficients of 
variation (cv) were 6.3 and 1.6%, respectively. The cross reaction of FSH 
with LH and of LH with FSH was &lt;2% (per NIDDK guidelines). 
Statistics 
Results were subjected to ANOVA. Whether significant (P&lt;0.05) differences 
existed between means was determined by Student's t-test. 
RESULTS 
Active Immunization of Rabbits 
Antibodies were generated to both LTB and bINH.alpha..sub.c.sup.1-14 
components of the LTB:bINH.alpha..sub.c.sup.1-14 fusion protein in the two 
groups of rabbits injected with LTB:bINH.alpha..sub.c.sup.1-14 mixed with 
or without Freund's adjuvant. Specifically, both LTB and inhibin antibody 
titers reached a peak after the first booster and stayed elevated for the 
duration of the experiment, although a transient decrease (P&lt;0.05) in the 
inhibin antibody titer after booster 3 was observed (FIG. 5). In addition, 
rabbits immunized with LTB:bINH.alpha..sub.c.sup.1-14 in Freund's adjuvant 
had anti-LTB antibody titers twice (P&lt;0.05) as high as those immunized 
without Freund's adjuvant. However, anti-inhibin antibody titers were 
similar between rabbits injected with LTB:bINH.alpha..sub.c.sup.1-14 mixed 
with or without Freund's adjuvant. 
As shown in FIG. 5, preimmune serum did not bind to LTB and 
bINH.alpha..sub.c.sup.1-26 gly.tyr. peptide in ELISA. Antibodies generated 
against the LTB:bINH.alpha..sub.c.sup.1-14 fusion protein mixed with or 
without Freund's adjuvant bound to native inhibin but not to preimmune 
control serum. 
Passive Immunization of Mice 
I. Inhibin Antibody Titer Following Passive Immunization 
The inhibin antibody titer was higher (P&lt;0.05) in mice that received one 
injection of anti-LTB:bINH.alpha..sub.c.sup.1-14 antiserum compared with 
preimmune controls (FIG. 6A). Inhibin antibody titer was also higher 
(P&lt;0.05) in mice that received two injections of 
anti-LTB:bINH.alpha..sub.c.sup.1-14 antiserum than those that received a 
single anti-LTD:bINH.alpha..sub.c.sup.1-14 injection or preimmune controls 
(FIG. 6A). 
II. Serum Concentrations of FSH and LH 
Administration of two injections of anti-LTB:bINH.alpha..sub.c.sup.1-14 
antiserum resulted in a nearly two-fold increase (P&lt;0.05) in plasma 
concentrations of FSH compared with preimmune controls (FIG. 6B). In 
contrast, concentrations of serum LH were similar (P&gt;0.1) for 
anti-LTB:bINH.alpha..sub.c.sup.1-14 treated and preimmune control mice 
(FIG. 6C). 
The results of this study demonstrate that: 
1) Immunization with LTB:bINH.alpha..sub.c.sup.1-14 fusion protein 
stimulated production of anti-bINH.alpha..sub.c.sup.1-14 antibodies when 
injected into rabbits with or without Freund's adjuvant; 
2) Anti-bINH.alpha..sub.c.sup.1-14 antibodies stimulated by immunization 
with LTB:bINH.alpha..sub.c.sup.1-14 bound to native inhibin; and, 
3) Anti-bINH.alpha..sub.c.sup.1-14 antibodies stimulated by immunization 
with LTB:bINH.alpha..sub.c.sup.1-14 effectively neutralized endogenous 
inhibin in host animals. 
Example 5 
Active Immunization of Mice with EtxB:bINH.alpha..sub.c.sup.1-14 
Animals 
Mice were maintained by the University Laboratory Animal Resources (ULAR, 
Michigan State University) in their animal care facilities for the 
duration of the experiment. Male BALB/c mice (25 days old) were purchased 
from Harland Sprague Dawley (Wilmington, Mass.) and housed 5/cage, 
maintained on a 12L:12D cycle, and provided with food and water ad 
libitum. 
Immunization Protocol 
After Metofane anesthesia (Methoxyflurane; Pitman-Moore, Mundelein, Ill.), 
mice were bled by gently inserting a heparinized microhematocrit capillary 
tube (200 .mu.l, Fisher, Pittsburg, Pa.) into the orbital sinus of the 
mouse. After filling the microhematocrit capillary tube with blood, the 
tube was sealed using hemato-seal tube sealing compound (Fisher, 
Pittsburg, Pa.) then centrifuged for 5 minutes at full speed to recover 
preimmune plasma. 
Purified LTB:bINH.alpha..sub.c.sup.1-14 and LTB proteins were used to 
actively immunize mice. Beginning at 26 days of age, male mice (n=5/group) 
were injected subcutaneously (s.c.) over a 10-week period at either 2-or 
4-week intervals with two doses (10 or 40 pg) of 
LTB:bINH.alpha..sub.c.sup.1-14 fusion protein or one dose (40 .mu.g) wild 
type LTB (controls). Each immunogen was injected in its pentameric or 
heat-disrupted monomeric form mixed with or without Freund's adjuvant. A 
group of ten untreated mice served as additional controls. To prepare the 
monomeric form, 500 .mu.l aliquots of LTB:bINH.alpha..sub.c.sup.1-14 or 
LTB were heated in a heating block (100.degree. C. for 15 minutes) then 
left to cool at room temperature for 15 minutes before preparation for 
immunization. Henceforth, sample preparations for pentameric and monomeric 
LTB:bINH.alpha..sub.c.sup.1-14 and EtxB are similar. 
For antigen preparation in Freund's adjuvant, 400 or 800 .mu.l 
LTB:bINH.alpha..sub.c.sup.1-14 or LTB in PBS (0.01M phosphate buffer, pH 
7.4 containing 0.15 M NaCl) was mixed with an equal volume of Freund's 
complete (primary injection) or incomplete (boosters) adjuvant 
(Calbiochem, La Jolla, Calif.) to give a final sample concentration of 10 
or 40 .mu.g LTB:bINH.alpha..sub.c.sup.1-14 or 40 .mu.g LTB per 100 .mu.l 
PBS. Antigen and adjuvant were mixed to give a stable emulsion as 
described for Example 3. For preparation of LTB:bINH.alpha..sub.c.sup.1-14 
(10 or 40 .mu.g/100 .mu.l) or LTB (40 .mu.g/100 .mu.l) without Freund's 
adjuvant, samples were diluted in PBS and sterilized by expulsion through 
a 2.2 .mu.m Millex GV sterile filter (Millipore, Bedford, Mass.) attached 
to a 5 ml syringe into a 15 ml sterile polypropylene tube. 
Mice injected every 2 weeks received a primary injection of 10 or 40 .mu.g 
LTB:bINH.alpha..sub.c.sup.1-14 or 40 .mu.g LTB either in Freund's complete 
adjuvant or PBS followed by four subsequent boosters in Freund's 
incomplete or PBS. Mice injected every 4 weeks received a total of two 
boosters in 8 weeks. Control groups immunized against pentarneric or 
monomeric LTB (40 .mu.g/mouse) were injected using the 2-week injection 
paradigm only. 
At 68 days of age, 6 weeks after the first injection (two boosters for 20 
week and one booster for 4-week interval), mice were anesthetized with 
Metofane, bled from the orbital sinus, and plasma collected as described 
above. Two weeks after the final booster, at 96 days of age, all mice were 
anesthetized with Metofane, body weight taken, and blood collected by 
heart puncture as described for Example 3. Blood samples (500 to 1,500 
.mu.l/mouse) were incubated at room temperature for 2 hours and then at 
4.degree. C. overnight, and serum was separated from clotted blood cells 
by centrifugation at 1000.times.g (Beckman GPR centrifuge) for 30 minutes 
at 4.degree. C. and stored at-20.degree. C. until assayed. After bleeding, 
testis, epididymis, seminal vesicle, heart, kidney, liver and spleen for 
each mouse were weighed. 
Antibody Titer Determination 
I. Pentameric EtxB Antibody Titer 
Probind microliter plates (Falcon, Lincoln Park, N.J.) were coated 
overnight at room temperature with 0.2 .mu.g/well GM1 in PBS followed by 
incubation with 100 ng/well pentameric LTB. After blocking non-specific 
binding sites with 1% bovine serum albumin (BSA) dissolved in PBS-T, the 
ELISA plates were incubated with mouse serum diluted 1:5000 in PBS-T 
containing 0.1% BSA (PBS-T-B, 1 hour), horseradish peroxidase-labeled goat 
anti-rabbit IgG (GAM0HRP, 1 hour; Vector, Burlingame, Calif.) diluted 
1:5000 in PBS-T-B, then ortho-phenylenediamine in 0.1M citrate buffer, pH 
4.5, containing 0.01% H.sub.x O.sub.2 (OPD-H.sub.2 O.sub.2) as described 
in Example 4. Titer=A.sub.490. 
Monomeric EtxB Antibody Titer 
Xenobind microliter plates (Xenopore Inc., Hawthorne, N.J.) were incubated 
with 200 ng/well monomeric LTB in PBS overnight (room temperature) to 
covalently link antigen to ELISA plate wells, as recommended by the 
manufacturer. After blocking non-specific binding sites with 3% gelatin in 
IIBS-T, ELISA plates were incubated with mouse serum diluted 1:5000 in 
PBS-T-B (1 hour), GAM-HRP (1 hour), then OPD-H.sub.2 O.sub.2, as described 
for Example 4. Titer=A.sub.490. 
Inhibin Antibody Titer 
Xenobind microliter plates were incubated with 1 .mu.g/well 
bINH.alpha..sub.c.sup.1-26 gly.tyr. or 1 .mu.g/well partially purified 
bovine inhibin (ppbINH) in PBS overnight (room temperature) to covalently 
link antigen to ELISA plate wells, as recommended by the manufacturer. 
After blocking non-specific binding sites with 3% gelatin in PBS-T, ELISA 
plates were incubated with mouse plasma or serum diluted 1:100 in PBS-T-B 
(2 hours), GAM-HRP (1 hour), then OPD-H.sub.2 O.sub.2, as described for 
Example 4. Titer=A.sub.490. 
FSH and LH RIA 
Concentrations of FSH or LH in serum or plasma were determined in duplicate 
samples in RIA, as described for Example 4. Samples were analyzed in a 
single assay for each hormone. rFSH and rLH assay sensitivities were 0.625 
and 0.156 ng/ml and intra-assay coefficients of variation (cv) were 6.3 
and 1.6%, respectively. The cross reaction of FSH with LH and LH with FSH 
is &lt;2% (per NIDDK guidelines). 
Testosterone RIA 
Concentrations of testosterone in serum were determined using the 
Coat-A-Count Total Testosterone assay kit from Diagnostic Products (DPC, 
Los Angeles, Calif.), per manufacturer's instruction for a non-extraction 
assay. Briefly, 50 .mu.l of mouse serum was incubated with 1 ml tracer in 
antiserum-coated tubes at 37.degree. C. for 3 hours. The coated tubes were 
decanted, and radioactivity bound to the dried tubes determined in a MACC 
Micromedic .gamma.-counter. All samples were analyzed in a single assay. 
Assay sensitivity was 0.2 ng/ml and intraassay CV was 1.2%. The cross 
reactivities are: estradiol=0.02%; 5.alpha.-dihydrotestosterone=3.4%; and 
other steroids=&lt;1% (per DPC guidelines). 
Statistics 
Results were subjected to ANOVA. Whether significant (P&lt;0.05) differences 
existed between means was determined by Student's t-test. 
Results 
As shown in FIGS. 7 and 8, active immunization of animals with either the 
monomeric or pentameric form of the fusion protein 
LTB:bINH.alpha..sub.c.sup.1-14 with Freund's adjuvant resulted in 
increased anti-inhibin antibody titers as compared with the LTB control, 
and also resulted in increased levels of FSH and LH. Testosterone levels 
were decreased (data not shown). In contrast, active immunization with the 
fusion protein in the absence of adjuvant failed to alter anti-inhibin 
antibody titers or reproductive hormone levels. 
The invention has been described with reference to various specific and 
preferred embodiments and techniques. However, it should be understood 
that many variations and modifications may be made while remaining within 
the spirit and scope of the invention. All publications and patent 
applications in this specification are indicative of the level of ordinary 
skill in the art to which this invention pertains. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 12 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 587 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: Genomic DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (ix) FEATURE: 
(A) NAME/KEY: Coding Se - #quence 
(B) LOCATION: 16...387 
(D) OTHER INFORMATION: 
- (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:1: 
- AATTCGGGAT GAATT ATG AAT AAA GTA AAA TTT TAT - # GTT TTA TTT ACG GCG 
51 
#Met Asn Lys Val Lys Phe Tyr Val Leu Phe T - #hr Ala 
# 10 
#CCT CAG TCT ATT ACA GAA 99AC GGA GCT 
#Pro Gln Ser Ile Thr Glu Ala His Gly Ala 
# 25 
#TAT ACG ATA AAT GAC AAG 147CA CAA ATA 
#Tyr Thr Ile Asn Asp Lys Asn Thr Gln Ile 
# 40 
#AAA AGA GAA ATG GTT ATC 195TG GCA GGC 
#Lys Arg Glu Met Val Ile Ser Met Ala Gly 
# 60 
#GTC GAA GTC CCG GGC AGT 243CA TTT CAG 
#Val Glu Val Pro Gly Ser Ala Thr Phe Gln 
# 75 
#GAA AGG ATG AAG GAC ACA 291AA GCC ATT 
#Glu Arg Met Lys Asp Thr Lys Lys Ala Ile 
# 90 
#ATT GAT AAA TTA TGT GTA 339AG ACC AAA 
#Ile Asp Lys Leu Cys Val Thr Glu Thr Lys 
# 105 
#GCA ATC AGT ATG GAA AAC TA 389CA ATT GCG 
#Ala Ile Ser Met Glu Asn Asn Ser Ile Ala 
# 120 
#TACTTATACT 449GCATGTC TAATGCTAGG AACCTATATA ACAACTACTG 
#CCTTAAACTG 509CTGCATT TGAAAAGGCG GTAGAGGATG CAATACCGAT 
#AACTAAGCTA 569GCTTCCA CTACAGGGAG CTGTTATAGC AAACAGAAAA 
# 587 CTT 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 124 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:2: 
#Thr Ala Leu Leu Ser Serhe Tyr Val Leu Phe 
# 15 
#Thr Glu Leu Cys Ser Glula Pro Gln Ser Ile 
# 30 
#Asp Lys Ile Leu Ser Tyrle Tyr Thr Ile Asn 
# 45 
#Val Ile Ile Thr Phe Lysly Lys Arg Glu Met 
# 60 
#Gly Ser Gln His Ile Aspln Val Glu Val Pro 
# 80 
#Asp Thr Leu Arg Ile Thrle Glu Arg Met Lys 
# 95 
#Cys Val Trp Asn Asn Lysys Ile Asp Lys Leu 
# 110 
#Glu Asnro Asn Ser Ile Ala Ala Ile Ser Met 
# 120 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 39 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: Genomic DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (ix) FEATURE: 
(A) NAME/KEY: Coding Se - #quence 
(B) LOCATION: 1...39 
(D) OTHER INFORMATION: 
- (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:3: 
# 39T CAC GAA CTA TGT TCG 
#Glu Tyr His Ser Ile Thr Glu Leu Cys Ser 
# 10 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 13 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:4: 
#Glu Tyr Hisln Ser Ile Thr Glu Leu Cys Ser 
# 10 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 39 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: Genomic DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (ix) FEATURE: 
(A) NAME/KEY: Coding Se - #quence 
(B) LOCATION: 1...39 
(D) OTHER INFORMATION: 
- (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:5: 
# 39T CAC GAT CTA TGT TCG 
#Glu Tyr His Ser Ile Thr Asp Leu Cys Ser 
# 10 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 13 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:6: 
#Glu Tyr Hisln Ser Ile Thr Asp Leu Cys Ser 
# 10 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 31 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: Genomic DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (ix) FEATURE: 
(A) NAME/KEY: Coding Se - #quence 
(B) LOCATION: 1...31 
(D) OTHER INFORMATION: 
- (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:7: 
# 31 GA GAA ATG GTT ATC ATT 
#Ilt Ala Gly Lys Arg Glu Met Val Ile Ile 
# 10 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 11 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:8: 
#Ileaa Xaa Gly Lys Arg Glu Met Val Ile Ile 
# 10 
- (2) INFORMATION FOR SEQ ID NO:9: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 31 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: Genomic DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (ix) FEATURE: 
(A) NAME/KEY: Coding Se - #quence 
(B) LOCATION: 1...31 
(D) OTHER INFORMATION: 
- (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:9: 
# 31 GA GAA ATG GTT ATC ATT 
#Ilt Ala Gly Leu Arg Glu Met Val Ile Ile 
# 10 
- (2) INFORMATION FOR SEQ ID NO:10: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 11 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:10: 
#Ileet Ala Gly Leu Arg Glu Met Val Ile Ile 
# 10 
- (2) INFORMATION FOR SEQ ID NO:11: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 66 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: Genomic DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (ix) FEATURE: 
(A) NAME/KEY: Coding Se - #quence 
(B) LOCATION: 1...31 
(D) OTHER INFORMATION: 
- (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:11: 
#C CGTGGCCGTG GTCCCCGGCT GC 53CG CCG CTG 
#Pro Glu Val Pro Gly Ser Thr Pro Pro Leu 
# 10 
# 66 
- (2) INFORMATION FOR SEQ ID NO:12: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 11 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:12: 
#Proal Glu Val Pro Gly Ser Thr Pro Pro Leu 
# 10 
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