Peptide vaccines and associated methods for protection against feline leukemia virus

The present invention discloses peptide vaccines and associated methods for protection against feline leukemia virus (FeLV). The synthetic peptides of the present invention retain their secondary structural identity, stimulate antibodies reactive to the whole virus and elicit a cellular response in vaccinations. These synthetic peptides are inexpensive to produce, stable and permit easy differentiation of vaccinated versus FeLV-infected cats.

1. INTRODUCTION 
The present invention relates to synthetic peptide molecules and their use 
as vaccines to impart immunoprophylaxis against feline leukemia virus 
(FeLV). A limiting factor in utilizing peptides as vaccines, whether 
derived through peptide synthesis or isolation of purified protein 
fragments, is a preference that the peptide retain its structural identity 
in solution in comparison to the corresponding domain of the native 
protein. Retaining structural identity may induce a faithful humoral and 
cytotoxic T-lymphocyte (CTL) response as well as stimulating an 
immunological memory. An especially preferred embodiment of the invention 
discloses the synthetic peptide, PRN60, and methods of use as a vaccine 
against FeLV. The PRN60 peptide may be used either as initial prophylactic 
protection against an FeLV challenge or as a booster to an initial FeLV 
vaccine. The PRN60 peptide substantially maintains an ordered, native-like 
secondary structure similar to that predicted for the corresponding region 
within the immunodominant region of the FeLV protein, gp70. Additionally, 
PRN60 elicits both humoral and cellular responses in experimental 
vaccinations. The peptide vaccines of the present invention are 
inexpensive to produce, stable and less likely than existing FeLV vaccines 
to produce unwanted side effects. In addition, peptides utilized as 
vaccines permit easy differentiation of vaccinated versus FeLV-infected 
cats through simple serological methods. 
2. BACKGROUND OF THE INVENTION 
Feline leukemia virus, a horizontally transmitted retrovirus, was first 
discovered among cats living in an urban environment and having frequent 
social contact (Jarrett, et al., 1964, Nature 202: 566-567). FeLV is 
believed to have been the first naturally occurring retrovirus in which 
contagious spreading was documented (Jarrett, et al., 1964, Nature 202: 
566-567; Kawakami, et al., 1967, Science 158: 1049-1050). The FeLV 
retrovirus can cause either proliferative (lymphosarcoma) or 
antiproliferative diseases (aplastic anemia and immunodeficiency syndrome) 
in cats (Anderson, et al., 1971, J. Natl. Cancer Inst. 47: 807-817; Hardy, 
et al., 1976, Cancer Res. 36: 582-588). Regardless of the clinical course 
of infection, however, the FeLV establishes a permanent infection in the 
cat that is not eliminated. Therefore, prophylactic vaccines are critical 
to prevent FeLV infection in cats. 
A viral vaccine should be designed such that both a humoral and CTL 
response is achieved, as well as stimulating a useful immunological 
memory. Examples of potential vaccines against viral infection include 
attenuated or inactivated whole virus, purified viral macromolecules (such 
as envelope proteins or capsular polysaccharides), recombinant antigen 
vaccines, recombinant vector vaccines and synthetic subunit vaccines. 
The most common type of viral vaccine developed to date have been whole 
organism vaccines that are either attenuated or inactivated. An attenuated 
viral vaccine is a virus that has lost its pathogenicity, but not its 
ability for transient growth within the host. Major advantages of the 
attenuated viral vaccine include prolonged exposure to the immune system 
due its ability for transient growth as well as its ability to induce both 
a humoral and CTL response. Since an attenuated viral vaccine is a mutant, 
avirulent organism selected from culturing of a wild type, virulent 
organism, the possibility exists that the attenuated viral vaccine could 
revert to a virulent form subsequent to host vaccination. An inactivated 
viral vaccine is usually produced by exposing a virulent strain to either 
chemical or radiation treatment. Such inactivated strains cannot, as a 
rule, revert to a virulent strain. However, such vaccines tend to induce 
only a humoral immune response and usually require multiple boosters due 
to an inability to grow transiently within the host. 
Purified macromolecules utilized as a vaccine reduce the risk of reversion 
to a virulent form. However, utilizing a macromolecule such as an envelope 
protein or capsular polysaccharide most likely results only in an 
induction of a humoral response. A CTL response may be induced by proper 
selection and presentation of an immunizing antigen. 
Viral antigen vaccines can be expressed and purified utilizing recombinant 
DNA techniques. A DNA sequence encoding an antigen determinant is 
isolated, characterized and subcloned into an appropriate DNA expression 
vector. The expression vector, (usually plasmid DNA) is transformed into 
an appropriate host (e.g., E. coli, yeast or a mammalian cell line), grown 
under conditions amenable for expression of the cloned antigen 
determinant, and purified for use as a vaccine. The recombinant proteins 
or peptides are usually processed as an exogenous antigen, more often 
resulting in a humoral but not a CTL response. 
A genetically engineered virus can be utilized as a vector. The viral 
vector contains a recombinant DNA sequence encoding an antigen determinant 
and is subcloned downstream of a viral vector promoter. The recombinant 
DNA sequence (again, most likely plasmid DNA) is transferred into the 
viral vector genome such that the DNA sequence encoding the antigenic 
determinant is expressed. The recombinant viral vector is then 
administered, for example, by dermal scratching. A localized infection 
ensues, allowing the antigen determinant to be expressed, inducing both a 
humoral and cellular response within the vaccinated host. For a review of 
these hereinbefore described methods available to the skilled artisan, see 
Kuby, 1992, In: Immunology; Chapter 18, "Vaccines"; W. H. Freeman, New 
York, N.Y. 
Olsen, et al. (1977, Cancer Res., 37: 2082-2085) immunized cats with a 
combined vaccine composed of a killed FeLV virus and killed feline 
oncornavirus-associated cell membrane antigen (FOCMA) tumor cells. The 
combined vaccine did not inhibit the induction of FeLV viremia. (Also, see 
Pederson, et al., 1979, Am. J. Vet. Res. 40: 1120-1126, which exemplifies 
the difficulty in generating immunity via vaccination with killed FeLV 
virus.) 
Hoover, et al., (1991, J. Am. Vet. Med. Assoc. 199: 1392-1401) tested 
inactivated FeLV virus, live FeLV virus and FeLV envelope peptide 
prototypes as potential vaccines. An inactivated vaccine developed from 
the FeLV-FAIDS-61E-A isolate protected cats from both homologous and 
heterologous viral exposure. In contrast, a panel of FeLV-GA-B envelope 
peptides, including peptides representing portions of the immunodominant 
domain, major neutralizing domain and variable neutralizing domain of 
gp70, were unsuccessful in providing resistance against FeLV challenge. 
Nicolaisen-Strouss (1987, J. Virol. 61: 3410-3415) identified an FeLV 
variant that was not neutralized by the 5-amino acid epitope described 
below by Elder, et al. A single amino acid change (proline to leucine) 
three amino acids from the NH.sub.2 -terminus of the 5-amino acid binding 
epitope was implicated in lowering the affinity for binding the 
neutralizing antibody. 
The FeLV envelope protein, gp70, was substantially purified and used to 
vaccinate cats (Pederson, et al., 1986, Vet. Immunol. Immunopathol. 11: 
123-148). Although serum antibodies were produced, no viral neutralizing 
antibodies were detected in vaccinated cats. Additionally, gp70 vaccinated 
cats became more persistently retroviremic than non-immunized cats 
subsequent to a virulent FeLV challenge. 
Gilbert, et al., 1987, Virus Res. 7: 49-67) disclosed a lack of FeLV 
neutralizing activity or immunoprophylaxis following immunization with a 
vaccinia vector expressing the env gp85 protein of FeLV-GA-B. 
Subunit vaccines to combat FeLV infection were developed utilizing FeLV 
antigens released from lymphoid cells persistently infected with the 
Kawakami isolate (Lewis, et al., 1981, Infect. Immun. 34: 888-894; Mastro, 
et al., 1986, Vet. Immunol. Immonopath. 11: 205-213; reviewed in Lewis, et 
al., 1988, Vet. Microbiology 17: 297-308). This cell line produces viral 
coded polypeptides which are subsequently harvested and used to vaccinate 
cats. These viral antigens compose a non-infectious subunit vaccine which 
is approximately 80% effective in preventing FeLV viremia. 
Subunit vaccines may be designed in attempts to structurally mimic domains 
of a viral envelope protein, in part due to the fundamental role these 
proteins play in virus entry into the host cell and subsequent cytopathic 
effects (Kowalski, et al., 1987, Science 237: 1351-1355). For example, the 
retrovirus external surface unit (SU) protein is involved in binding to a 
host cell receptor, followed by a fusion event mediated by the 
transmembrane (TM) protein (Dalgleish, et al., 1984, Nature 312: 763-766; 
Bosch, et al., 1989, Science 244: 694-697; Battini, et al., 1992, J. 
Virol. 66: 1468-1475). Therefore, these proteins may be targeted in 
immunological strategies for prophylactic or therapeutic treatment of a 
viral infection. 
Absent detailed structural characterization by techniques such as NMR 
spectroscopy or crystallography, the structure and function of these 
envelope proteins has been investigated through indirect means. For 
example, inferences concerning structural and functional domains of 
retroviral envelope proteins have been investigated by utilizing (1) 
chimeric viruses (Donahue, et al., 1991, J. Virol. 65: 4461-4469; Battini, 
et al., 1992, J. Virol. 66: 1468-1475), (2) site-directed mutagenesis 
(Bosch, et al., 1989, Science 244: 694-697; Willey, 1989, J. Virol. 63: 
3595-3600), and (3) small synthetic peptides and specific antibodies 
(Palker, et al., 1988, Proc. Natl. Acad. Sci. USA 85: 1932-1936; Dyson, et 
al., 1992, Biochemistry 31: 1458-1463; Nick, et al., 1990, J. Gen. Virol. 
71: 77-83). 
Nunberg, et al. (1990, Proc. Natl. Acad. Sci. USA 81: 3675-3679) disclosed 
a method to map antigenic determinants of a specified protein. DNA 
fragments are generated by DNaseI digestion and subcloned into the 
.beta.-galactosidase gene of the lambda phage Charon 16. Random peptides 
representing the coding region of the entire protein are expressed and 
screened with a specific monoclonal antibody to determine its epitope. 
Binding of a gp70 neutralizing antibody was mapped to a 14 amino acid 
region (213-226) of the gp70 protein. 
Elder, et al. (1987, J. Virol. 61: 8-15) synthesized short peptides 
corresponding to regions of FeLV envelope proteins, gp70 and p15E. These 
peptides were conjugated to keyhole limpit hemocyanin and injected into 
rabbits to induce anti-peptide antibody production. The sera was then 
tested for the ability to neutralize FeLV isolates. A five amino acid 
sequence within the gp70 coding region was determined to be required for 
neutralizing activity. The five amino acid epitope was the core of a 
number of short peptides to which antisera had been raised. The longest of 
these core peptides was 18 amino acids. 
Nick, et al. (1990, J. Gen. Virol. 71: 77-83) synthesized 19 peptides, from 
7-19 amino acid residues in length. These peptides were conjugated to 
keyhole limpit hemocyanin and the anti-peptide sera was tested for 
neutralizing ability against FeLV. One gp70 anti-peptide antibody involved 
in FeLV neutralization was raised against the peptide representing amino 
acids 221-227. This epitope is contained within a portion of the 
neutralization domain described by Nunberg, et al. (1990, Proc. Natl. 
Acad. Sci. USA 81: 3675-3679). However, anti-peptide antibodies raised 
against a peptide representing amino acids 228-234 of gp70, also contained 
within the Nunberg neutralization domain, did not neutralize FeLV in this 
study. The clustering of neutralization sites is located within a 
proline-rich sequence located between amino acids 230-289 of gp70 
(Stewart, et. al., 1986, J. Virol. 58: 825-834; Donahue, et al., 1988 J. 
Virol. 62: 722-731). 
A peptide vaccine providing initial protection against an FeLV challenge or 
as a booster to an initial vaccination against FeLV would be of immense 
value. A humoral response, cellular response and stimulation of an 
immunological memory would most likely result from a peptide retaining a 
secondary structure corresponding to the equivalent amino acid domain of 
an in vivo protein. 
3. SUMMARY OF THE INVENTION 
The present invention relates to peptide vaccines and methods of their use 
in providing initial prophylactic protection against FeLV challenge or as 
a booster to an initial vaccination to provide protection against FeLV 
challenge. 
A peptide vaccine of the present invention is comprised of a plurality of 
proline residues aligned throughout the amino acid sequence such that the 
peptide substantially maintains an ordered, native-like secondary 
structure. 
In a particular embodiment of the invention, the native-like structure in 
which the peptide folds is a polyproline beta turn helix. The peptide is 
induced to form a polyproline beta turn helix in part by comprising a 
percentage of proline residues ranging from about 10% to about 30%. 
In a further embodiment of the invention, the peptide, which forms a 
polyproline beta turn helix, comprising a percentage of proline residues 
ranging from about 10% to about 30%, is at least about 80% homologous to 
the corresponding domain of the native protein at the amino acid level so 
as to induce a proper antibody response. 
In yet a further embodiment of the invention, the peptide, which forms a 
polyproline beta turn helix by comprising a percentage of proline residues 
ranging from about 10% to about 30%, is at least about 80% homologous to 
the corresponding domain of a region from about amino acid 230 to about 
amino acid 290 of gp70 of feline leukemia virus. 
In a preferred embodiment of the invention, the peptide, which forms a 
polyproline beta turn helix by comprising a percentage of proline residues 
ranging from about 10% to about 30%, is at least about 80% homologous to 
the corresponding domain of a region from about amino acid 230 to about 
amino acid 290 of gp70 of feline leukemia virus and is at least about 50 
amino acids in length. 
In an especially preferred embodiment of the invention, the peptide is 
PRN60 (SEQ ID NO:1). 
In another embodiment of the invention, the peptide which forms a 
polyproline beta turn helix, comprising a percentage of proline residues 
ranging from about 10% to about 30%, is at least about 80% homologous to 
the corresponding domain of the native protein at the amino acid level so 
as to induce a proper antibody response and is formed via self assembly of 
two or more peptides. 
In an additional embodiment of the invention, the peptide, which forms a 
polyproline beta turn helix by comprising a percentage of proline residues 
ranging from about 10% to about 30%, is at least about 80% homologous to 
the corresponding domain of the region from about amino acid 230 to about 
amino acid 290 of gp70 of feline leukemia virus and is formed via self 
assembly of two or more peptides. 
In a preferred embodiment of the invention, the peptide, which forms a 
polyproline beta turn helix by comprising a percentage of proline residues 
ranging from about 10% to about 30%, is at least about 80% homologous to 
the corresponding domain of the region from about amino acid 230 to about 
amino acid 290 of gp70 of feline leukemia virus and is formed via self 
assembly of two or more peptides and is at least about 50 amino acids in 
length. 
In an especially preferred embodiment of the invention, the peptide, is 
formed via self assembly of PRN42 (SEQ ID NO:2) and PRN4358 (SEQ ID NO:3). 
The present invention also relates to methods of providing resistance 
against feline leukemia virus. A peptide is generated which comprises an 
amino acid sequence with a plurality of proline residues, the proline 
residues aligned throughout the amino acid sequence such that the peptide 
fragment substantially maintains a native-like structure. The peptide is 
isolated in a substantially pure form and utilized to vaccinate a 
susceptible host so as to provoke a humoral and cellular immune response 
as well as stimulating an immunologic memory. 
In a particular embodiment of the invention, the peptide generated by the 
disclosed method folds into a polyproline beta turn helix. This peptide 
generated by the disclosed method is induced structurally to form a 
polyproline beta turn helix, comprising a percentage of proline residues 
ranging from about 10% to about 30%. 
In a further embodiment of the invention, the peptide generated in the 
disclosed method, which forms a polyproline beta turn helix by comprising 
a percentage of proline residues ranging from about 10% to about 30%, is 
at least about 80% homologous to the corresponding domain of the native 
protein at the amino acid level so as to induce a proper antibody 
response. 
In yet a further embodiment of the invention, the peptide generated in the 
disclosed method, which forms a polyproline beta turn helix by comprising 
a percentage of proline residues ranging from about 10% to about 30%, is 
at least about 80% homologous to a portion of the region from about amino 
acid 230 to about amino acid 290 of gp70 of feline leukemia virus. 
In a preferred embodiment of the invention, the peptide generated in the 
disclosed method, which forms a polyproline beta turn helix by comprising 
a percentage of proline residues ranging from about 10% to about 30%, is 
at least about 80% homologous to the corresponding domain of the region 
from about amino acid 230 to about amino acid 290 of gp70 of feline 
leukemia virus and is at least about 50 amino acids in length. 
In an especially preferred embodiment of the invention, the peptide 
generated in the disclosed method is PRN60 (SEQ ID NO:1). 
In another embodiment of the invention, the peptide generated by the 
disclosed method, which forms a polyproline beta turn helix by comprising 
a percentage of proline residues ranging from about 10% to about 30%, is 
formed via self assembly of two or more peptides. 
In an additional embodiment of the invention, the peptide generated by the 
disclosed method, which forms a polyproline beta turn helix by comprising 
a percentage of proline residues ranging from about 10% to about 30%, is 
at least about 80% homologous to the corresponding domain of the region 
from about amino acid 233 to about amino acid 289 of gp70 of feline 
leukemia virus and is formed via self assembly of two or more peptides. 
In a preferred embodiment of the invention, the peptide generated by the 
disclosed method, which forms a polyproline beta turn helix by comprising 
a percentage of proline residues ranging from about 10% to about 30%, is 
at least about 80% homologous to the corresponding domain of the region 
from about amino acid 233 to about 289 of gp70 of feline leukemia virus 
and is formed via self assembly of two or more peptides and is at least 
about 50 amino acids in length. 
In an especially preferred embodiment of the invention, the peptide of the 
disclosed method is generated via self assembly of PRN42 (SEQ ID NO:2) and 
PRN4358 (SEQ ID NO:3). 
It is an object of the present invention to provide a vaccine, as a 
peptide, which provides initial protection against FeLV challenge or is 
given as a booster to an initial vaccination to provide protection against 
FeLV challenge. 
It is also an object of the invention to provide this vaccine, as a 
peptide, which mimics the native-like structure of the corresponding 
domain of the native protein so as to stimulate a prolonged immune 
response. 
It is a further object of the invention to provide this vaccine, as a 
peptide, forming a polyproline beta turn helix in solution, thus mimicking 
the native-like structure of the corresponding domain of the native 
protein so as to stimulate a prolonged immune response. 
It is an object of the present invention to provide this vaccine, as a 
peptide, which forms a polyproline beta turn helix in solution and is at 
least about 80% homologous at the amino acid level to a portion of the 
region from about amino acid 233 to about amino acid 289 of gp70 of feline 
leukemia virus. 
It is an object of the present invention to provide this vaccine in the 
form of PRN60 (SEQ ID NO:1). 
It is also an object of the present invention to provide this vaccine as a 
peptide formed via self assembly of PRN42 (SEQ ID NO:2) and PRN4358 (SEQ 
ID NO:3). 
It is an object of the present invention to provide a method of initial 
vaccination against FeLV challenge or a booster to an initial vaccination 
to provide protection against FeLV challenge utilizing one or more of the 
peptides disclosed within this specification. 
It is also an object of the present invention to provide a method of 
promoting susceptible host protection against FeLV infection comprising 
use of the PRN60 peptide vaccine, introduced into the susceptible host in 
either a modified or unmodified form such that the peptide vaccine induces 
a humoral and cellular response upon vaccination of the susceptible host. 
Is it also an object of the present invention to provide a method of 
promoting susceptible host protection against FeLV infection comprising 
use a peptide vaccine formed via self-assembly of two peptides such that 
the peptide vaccine, introduced into the susceptible host in either a 
linked or unlinked form, induces a humoral and cellular response upon 
vaccination of the susceptible host. 
These and other objects of the invention will be more fully understood from 
the following description of the invention and disclosed examples, the 
referenced figures and tables attached hereto and the claims appended 
hereto. 
3.1 Definitions 
The terms listed throughout this specification will have the meanings 
indicated. 
FeLV--Feline Leukemia Virus 
CTL--Cytotoxic T--Lymphocyte 
DPI--Days Post Infection 
UVD--Unintegrated Viral Deoxyribonucleic Acid 
HIV--Human Immunodeficiency Virus 
EIAV--Equine Infectious Anemia Virus 
LTR--Long Terminal Repeat 
PLL--Poly-L-Lysine 
KHL--Keyhole Limpit Hemocyanin

5. DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to peptide vaccines and methods of their use 
in providing initial protection against FeLV challenge or as a booster to 
an initial vaccination to provide protection against FeLV challenge. 
Peptide vaccines are disclosed which elicit a humoral and cellular immune 
response in experimental vaccinations against host exposure to feline 
leukemia virus. These peptide vaccines comprise an amino acid sequence 
with a plurality of proline residues, the proline residues aligned 
throughout the amino acid sequence such that the peptide fragment 
substantially maintains a native-like structure. As discussed in the 
Background of the Invention, synthetic peptides for the most part have 
historically been unable to substantially maintain the native-like 
secondary structure in solution, thus limiting their usefulness as 
vaccines. However, the present invention discloses a synthetic peptide 
containing a plurality of proline residues synthesized to a surprising 
length and purity, these peptides shown to substantially maintain the 
ordered secondary structure of the corresponding protein domain in 
solution. 
In a particular embodiment of the invention, the native-like structure in 
which the peptide folds is a polyproline beta turn helix. The peptide is 
induced to form a polyproline beta turn helix by comprising a percentage 
of proline residues ranging from about 10% to about 30%. 
In a further embodiment of the invention, the peptide, which forms a 
polyproline beta turn helix, comprising a percentage of proline residues 
ranging from about 10% to about 29%, is at least about 80% homologous to 
the corresponding domain of the native protein at the amino acid level so 
as to induce a proper antibody response. 
In another embodiment of the invention, the peptide vaccine, comprising 
from about 10% to about 30% proline residues; and at least about 80% 
homologous to the corresponding protein domain; is at least about 50 amino 
acids in length. 
Several embodiments of the invention are disclosed through the synthesis, 
isolation and utilization of peptides corresponding to at least a portion 
of the proline-rich neutralization domain of FeLV gp70. The envelope 
glycoproteins of FeLV were modeled according to the rules of Chou and 
Fasman (1978, Ann. Rev. Biochem. 47: 251-276), as well as considering 
amphipathic character (Margalit, et al., J. Immunol. 138: 2213-2229) and 
surface potential (Parker, et al., 1986, Biochemistry 25: 5425-5431) of 
the proteins sequence. The experimentally determined structural model of 
the FeLV 61-E gp70 protein (hereinafter referred to as gp70) is disclosed 
in FIG. 1 (gp70 is SEQ ID NO:6). This model indicates a domain of the gp70 
amino acid sequence consisting of 10 sequential reverse turns which is 
rich in proline residues and determined experimentally to form a 
polyproline beta turn helix. 
In one embodiment of the invention, a peptide synthesized from at least a 
portion of this gp70 coding region, from about amino acid 233 to about 
amino acid 289 (FIG. 1) maintains an ordered, native-like secondary 
structure. 
This embodiment of the invention is exemplified, but not limited to, the 
peptide PRN60 as a vaccine against FeLV challenge. The PRN60 peptide spans 
amino acid 230-290 of the gp70 amino acid sequence (FIG. 1). The PRN60 
peptide vaccine may be generated by a number of known procedures, 
including but not limited to automated peptide synthesis, manual peptide 
synthesis or by controlled digestion of peptide fragments. This peptide 
vaccine is then isolated in a substantially pure form, substantially 
maintaining an order structure in solution. The present invention 
discloses a conformational model of gp70 and the region represented by 
PRN60 has been predicted to consist of sequential reverse turns. This 
peptide contains 18% proline, 11% threonine, 9% serine, and 9% glycine and 
9% glutamine. This sequential reverse turn motif predicted by our model is 
consistent with the polyproline beta turn helix form of secondary 
structure associated with proline-rich repeat sequences. NMR spectroscopy 
measurement of intrinsic viscosity, as well as determining the circular 
dichroism spectrum of PRN60 has been utilized to determine that this 
peptide retains a highly ordered confirmation in solution. Therefore, it 
is disclosed in the present invention that PRN60 forms an ordered 
structure in solution with large confirmation mobility that is long lived, 
and that peptide fragments of this structure will self assemble into the 
native-like confirmation experimentally predicted in the model depicted in 
FIG. 1. The PRN60 peptide was utilized to immunize cats, mice and rabbits 
with both free peptide and carrier bound peptide. As discussed further in 
Example Section 7, PRN60 induces both humoral and cellular immune 
responses that may protect against FeLV infection. Therefore, it is 
disclosed by way of example, and not of limitation, that an extremely long 
peptide corresponding to a domain of gp70, experimentally predicted by 
computer modeling to possess a polyproline beta turn helix region, both 
retains that structure as well as inducing in both humoral and cellular 
immune responses subsequent to vaccination of a susceptible host. 
In an additional embodiment of the invention, the peptide, which forms a 
polyproline beta turn helix by comprising a percentage of proline residues 
ranging from about 10% to about 30%, is at least about 80% homologous to a 
portion of the region from about amino acid 230 to about amino acid 290 of 
gp70 of feline leukemia virus and is formed via self assembly of two or 
more peptides. 
In a preferred embodiment of the invention, the peptide, which forms a 
polyproline beta turn helix by comprising a percentage of proline residues 
ranging from about 10% to about 30%, is at least about 80% homologous to a 
portion of the region from about amino acid 230 to about amino acid 290 of 
gp70 of feline leukemia virus and is formed via self assembly of two or 
more shorter peptides and is at least about 50 amino acids in length. 
In still another embodiment of the invention, two or more peptide fragments 
are mixed to self-assemble into a larger peptide fragment. It is disclosed 
that smaller peptides corresponding to sub-regions of the PRN60 domain do 
not share secondary structural characteristics with PRN60. These smaller 
peptides self-assemble into a larger peptide fragment in vitro. The 
self-assembly of these smaller peptides results in a larger peptide with 
an ordered, native-like secondary structure similar to PRN60. In a 
specific embodiment of the invention, the peptide fragment PRN42 (Table 2; 
SEQ ID NO:2) is mixed with the peptide fragment PRN4358 (Table 2; SEQ ID 
NO:5). These two peptide fragments self-assemble into a peptide fragment 
exhibiting secondary structural characteristics substantially similar to 
PRN60. Therefore, smaller peptides may be utilized to construct the 
extremely long, structurally ordered peptides (such as PRN60) to be 
utilized as vaccines. 
The present invention also relates to methods of providing resistance 
against feline leukemia virus. A peptide is generated which comprises an 
amino acid sequence with a plurality of proline residues, the proline 
residues aligned throughout the amino acid sequence such that the peptide 
fragment substantially maintains a native-like structure. The peptide is 
isolated in a substantially pure form and utilized to vaccinate a 
susceptible host so as to provoke a humoral and cellular immune response 
as well as stimulating an immunologic memory. 
In a particular embodiment of the invention, the peptide generated by the 
disclosed method folds into a polyproline beta turn helix. This peptide 
generated by the disclosed method is induced to form a polyproline beta 
turn helix, comprising a percentage of proline residues ranging from about 
10% to about 30%. 
In a further embodiment of the invention, the peptide generated in the 
disclosed method, which forms a polyproline beta turn helix by comprising 
a percentage of proline residues ranging from about 10% to about 30%, is 
at least about 80% homologous to the corresponding domain of the native 
protein at the amino acid level so as to induce a proper antibody 
response. 
In yet a further embodiment of the invention, the peptide generated in the 
disclosed method, which forms a polyproline beta turn helix by comprising 
a percentage of proline residues ranging from about 10% to about 30%, is 
at least about 80% homologous to the corresponding domain of the region 
from about amino acid 230 to about amino acid 290 of gp70 of feline 
leukemia virus. 
In a preferred embodiment of the invention, the peptide generated peptide 
in the disclosed method, which forms a polyproline beta turn helix by 
comprising a percentage of proline residues ranging from about 10% to 
about 30%, is at least about 80% homologous to the corresponding domain of 
the region from about amino acid 230 to about amino acid 290 of gp70 of 
feline leukemia virus and is at least about 50 amino acids in length. 
In an especially preferred embodiment of the invention, the peptide 
generated in the disclosed method is PRN60 (SEQ ID NO:1). 
In an additional embodiment of the invention, the peptide generated by the 
disclosed method, which forms a polyproline beta turn helix by comprising 
a percentage of proline residues ranging from about 10% to about 30%, is 
at least about 80% homologous to a portion of the region from about amino 
acid 230 to about amino acid 290 of gp70 of feline leukemia virus and is 
formed via self assembly of two or more peptides. 
In a preferred embodiment of the invention, the peptide generated by the 
disclosed method, which forms a polyproline beta turn helix by comprising 
a percentage of proline residues ranging from about 10% to about 30%, is 
at least about 80% homologous to a portion of the region from about amino 
acid 230 to about 290 of gp70 of feline leukemia virus and is formed via 
self assembly of two or more peptides and is at least about 50 amino acids 
in length. 
In an especially preferred embodiment of the invention, the peptide of the 
disclosed method is generated via self assembly of PRN42 (SEQ ID NO:2) and 
PRN4358 (SEQ ID NO:3). 
The peptide vaccines utilized in accordance with this invention may be 
prepared by methods other than manual or automated peptide synthesis. By 
way of example and not of limitation, the peptides may be prepared by 
peptide isolation, such as the controlled enzymatic digestion of the 
proline-rich region of the FeLV-gp70 transmembrane protein, followed by 
purification of the peptide fragment in a substantially pure form. 
Alternatively, the amino acid sequence comprising this envelope peptide 
may be encoded as a portion of a larger peptide sequence, the larger 
peptide sequence having been expressed by recombinant DNA techniques known 
to one of ordinary skill in the art. 
It also will be well known to one of ordinary skill in the art that a 
susceptible host may be immunized using the appropriate peptide vaccine 
formulated in adjuvant to increase the immune response. Such adjuvants 
include but are not limited to Freund's (complete and incomplete), mineral 
gels, such as aluminum hydroxide, surface active substances such as 
keyhole limpet hemocyanin, lysolecithin, pluronic polyols, polyanions, 
peptides, BCG (Bacille Calmette-Guerin), oil emulsions and dinotrophenols. 
Immunization can be carried out with additional various presentation and 
cross-linking permutations. By way of example and not of limitation, such 
permutations include PRN60 cross-linked to KLH as a carrier, PRN60 
cross-linked to FeLV core protein as carrier, PRN60 cross-linked to 
itself, and these combinations presented by the various adjuvants listed 
above. It will become evident that such permutations are available in 
regard to other peptides and self-assembled peptides disclosed throughout 
this specification. 
It will also be known to one of ordinary skill in the art that use of the 
term "susceptible host" includes any such mammalian host susceptible to 
infection by feline leukemia virus. It will be further evident that any 
such susceptible host is a candidate for treatment to promote protection 
from FeLV utilizing the peptide vaccines and associated methods described 
in this specification. A primary target for treatment utilizing these 
peptide vaccines and associated methods are cats, with primary emphasis on 
treating domestic cats. 
The following examples are offered by way of illustration of the present 
invention, and not by way of limitation. 
6. EXAMPLE: A PROLINE RICH NEUTRALIZATION DOMAIN OF FeLV SURFACE UNIT 
PROTEIN (gp70) FORMS A POLYPROLINE .beta.-TURN HELIX SECONDARY MOTIF 
6.1. Materials and Methods 
6.1.2. Peptide Synthesis 
Synthetic peptides were prepared by a manual solid-phase strategy using 
9-fluorenylmethyloxycarbonyl protected amino acids. Dimethylformamide 
(DMF) was sequencing grade from FisherBiotech (Fairlawn, N.J.). TFA was 
sequencing grade from MilliGen/Biosearch, Div. of Millipore (Burlington, 
Mass.). Thioanisole (TA), ethanedithiol (EDT) and Hobt were peptide 
synthesis grade and purchased form DuPont (Boston, Mass.). Methanol, 
dichloromethane (DCM), acetonitrile and toluene were HPLC grade and 
purchased from Mallinkrodt (Paris, Ky.). 
The techniques for producing, purifying and characterizing peptides are 
described in Fontenot, et al. (1991, Peptide Research 4: 19-25). Briefly, 
peptides were synthesized by a manual solid-phase method using 
9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids. Fmoc amino acid 
side-chain protecting groups include t-butyl ethers for serine, threonine, 
tyrosine, aspartic acid, and glutamic acid; cysteine t-butyl or trityl for 
cysteine; Boc for lysine; 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) 
and N-(2,2,5,7,8-penthamethyl) chroman-6-sulfonyl (PMC) for arginine; and 
trityl for histine. The resulting synthetic peptides were deprotected and 
cleaved from the resin support in trifluoroacetic acid with the 
appropriate scavengers, followed by sequential extractions with organic 
solvents and purification by conventional gel filtration and reverse phase 
HPLC. Molecular weight analysis of the purified peptide was performed 
using electrospray mass spectroscopy. 
Briefly, peptides were synthesized manually on a Rapid Multiple Peptide 
Synthesizer (RaMPS) from DuPont. The syntheses were performed in groups of 
5-10 peptides utilizing 0.1 mM Rapid Amine (2,3-dimethoxybenzhydrylamine 
resin) cartridges from DuPont which yield C-terminal amides. The standard 
coupling procedure was performed using 0.25 mmol preformed 
pentafluorophenyl esters of fluorenylmethoxycarbonyl amino acids 
(Fmoc-AA-opfp) and 0.1 mmol of Hobt in 3 ml DMF. The coupling times were a 
standard 2 h, followed by extensive DMF and methanol washes. The 
completeness of the coupling reaction was monitored by the method of 
Kaiser et al. (1970, Anal. Biochem. 34: 595-598). The Kaiser reagents were 
purchased from DuPont. Exceptions to the standard opfp ester activation 
coupling include alanine, histidine, arginine and trityl-protected 
cysteine, which were each coupled as the symmetric anhydride. Cysteine was 
also coupled as a Hobt ester for t-butyl-protected cysteine. Serine and 
threonine were coupled as 3-hydroxy-4-oxo-3,4 dihydrobenzotriazine (odhbt) 
esters purchased from DuPont. 
Upon completion of the synthetic regimen, the resins were deblocked in 3 ml 
of 50:50 DMF:piperidine for 9 minutes, followed by extensive washing in 
DMF and methanol. Following the final methanol wash, the resin was 
air-dried by suction for 10 minutes. Final TFA cleavage and deprotection 
were performed by the manufacturer's suggested procedures. Briefly, 
Arg-containing peptides were shaken vigorously for 16 hours in a solution 
of 90% TFA, 5% EDT, 4% water and 1% TA. Peptides without Arg were cleaved 
in a solution of 90% TFA, 5% EDT and 5% water. Finally the TFA was 
evaporated, and the peptides were extracted sequentially with ethyl ether, 
ethyl acetate and water, followed by lyophilization. 
Automated peptide synthesis may be performed with a MilliGen/Biosearch SAM 
II peptide synthesizer according to manufacturer-specified protocols. 
Briefly, peptides are synthesized on Fmoc-L-amino acid-p-benzyloxybenzyl 
alcohol resins generating C-terminal acids. Amino acids are activated by 
the addition of 0.4M diisopropylcarbodimide (DIC) in the presence of 
equimolar Hobt and amino acid. Coupling times are a standard 1 hours. 
Exceptions include opfp ester activation of asparagine and glutamine, and 
symmetric anhydride for leucine. Deblocking is performed in a solution of 
30% piperidine in 50:50 toluene:DMF for 3.15 minutes. Final cleavage is 
performed in a solution of TFA:TA:EDT:anisole at a ratio of 90:5:3:2 for 8 
hours with Arg-containing peptides. Final cleavage for peptides without 
Arg and Trp is in a solution of TFA:DCM:DMS at a ratio of 70:25:5 for 2 
hours. Peptides with Trp, but without Arg, are cleaved in a solution of 
TFA:DCM:indole at a ratio of 70:28:2. 
6.1.3. NMR Spectroscopy 
All peptides samples for NMR analysis were prepared from HPLC purified and 
lyophilized peptide. The sample concentrations were all 5 mM in 0.1M 
phosphate buffer pH 7.2 with either 90%/10%, H.sub.2 O/D.sub.2 O or 99.5% 
D.sub.2 O. High ionic strength buffer was used to reduce the electrostatic 
interactions. A pH of 7.2 was chosen to approximate native conditions and 
to maximize the rate of proton exchange into D.sub.2 O, thereby insuring 
that only the most long-lived protons would be present when the .sup.1 
H-NMR spectra were recorded. 
The NMR spectra were recorded on a Bruker AM-500 spectrometer equipped with 
an Aspect 3000 computer and a 5-mm .sup.1 H probe. The temperature of the 
probe was regulated with a BVT-1000 unit and calibrated with a sample of 
methanol. The spectra in D.sub.2 O were obtained 5-10 minutes after 
dissolution of the peptides. The water signal was suppressed during the 
repetition delay of 1.5 seconds for samples in H.sub.2 O and D.sub.2 O. A 
control spectrum of the D.sub.2 O sample was taken without water 
pre-saturation to make sure that none of the amide protons are affected by 
pre-saturation of water at any given power level. A total of 1024 
transients were collected for each spectrum. The proton chemical shift is 
referred to the proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate 
(DSS) at 0.0 ppm. 
6.1.4. Circular Dichroism 
Circular dichroism spectra were recorded on a Jasco model J-710 
spectropolarimeter equipped with a PTC-343 peltier-type thermostatic cell 
holder and temperature control program. The spectra were recorded from 
195-260 nm, with readings every 0.1 nm. The peptide concentrations were 
0.1 mg/ml of HPLC purified peptide in 0.01M phosphate buffer at pH 7.2. A 
0.1-cm path length strain free quartz cuvette was used for all 
measurements. 
6.1.5. Intrinsic Viscosity 
Viscometry measurements were performed in a Cannon-Fenske-Ostwald type 
capillary viscometer using HPLC purified peptide in 0.1M phosphate buffer 
at pH 7.0 and 30.degree. C. The procedure used was as described by Tanford 
& Buzzell (1956, J. Phys. Chem. 60: 225-231) and Buzzell & Tanford (1956, 
J. Phys. Chem. 60:1204-1207). The capillary constant was calculated as 
described by Tanford & Buzzell (1956, J. Phys. Chem. 60: 225-231). All 
kinematic viscosity measurements were repeated at least ten times and the 
averages used to calculate the intrinsic viscosity. Intrinsic viscosity 
was calculated from kinematic viscosity and the appropriate density 
correction (0.0029 ml/g) was applied as described by Tanford (1955, J. 
Phys. Chem. 59: 798-799). The Simha shape factor and the peptide axial 
ratios were calculated as described Tanford (1961, In: Physical chemistry 
and macromolecules. John Wiley and Sons, New York, N.Y. pp 390-405) and 
Cantor & Schimmel (1980, Biophysical Chemistry. Part 2: Techniques for the 
Study of Biological Structure and Function: W. H. Freeman and Co, New 
York, N.Y.). 
6.1.6. Molecular Modeling 
Potential structural domains of the surface unit protein of gp-70 (Stewart, 
et al., 1986, J. Virol. 58 825-834; Overbaugh, et al., 1988, Science 239: 
906-910) was determined experimentally through the use of various models 
utilized to calculate secondary structure (Chou and Fasman, 1978, Ann. 
Rev. Biochem 47:251-276); surface potential (Parker, 1986, Biochemistry 
25: 5425-5431) and amphipathic alpha helical regions (Margalit et al., 
1987, J. Immunol. 138: 2213-2229). 
The sequence of PRN60 was modeled into a poly type I turn conformation on a 
Silicon Graphics model INDIGO (Mountain View, Calif.) terminal using the 
Tripos molecular graphic program Sybyl (St. Louis, Mich.). From this 
model, the distance of the longitudinal axis and cross sectional axis were 
estimated. 
6.2. Results and Discussion 
6.2.1. Molecular Model of gp70-PRN60 
While experimentally constructing a conformational model of gp70 an unusual 
sequence was detected spanning amino acids 230-289 that was predicted to 
consist of 10 sequential reverse turns (Table 1). This peptide segment is 
18% proline, 11% threonine, 9% serine, 9% glycine and 9% glutamine and 
appears to be a mucin-like sequence (Jentoft, 1990, Trends Biochem Sci. 
15: 291-294; Strouss and Dekker, 1992, Critical Review in Biochemistry and 
Molecular Biology 27: 1/2:57-92). This segment contains disclosed 
neutralizing sites for FeLV and has been designated the proline-rich 
neutralization domain. The proline-rich sequence is entirely incompatible 
with either .alpha.-helix or .beta.-sheet secondary structure by 
Chou-Fasman analysis. The pattern of (PXXV)A(PXXV)G(PXXI) appears in amino 
acids 271-289 and (PXXV) again at amino acids 239-242 of FeLV gp-70E, with 
XX being two hydrophilic amino acids. Six of the potential turns are 
predicted with a probability greater than twice the value necessary to 
predict a turn, and all ten proposed turn sequences have a turn 
conformational potential (P.sub.t) greater than 1.00 and P.sub.t greater 
than P.sub..alpha. of P.sub..beta.. In addition, there are no aromatic 
amino acids within this domain. The 13 amino acids N-terminal (219-310) to 
PRN60 and 12 amino acids C-terminal (290-301) are predicted to be 
.beta.-sheet. There are no cysteines within either PRN60 or the flanking 
.beta.-sheet regions. The nearest cysteines to the proline-rich sequence 
are cys 185 and cys 312. The lack of cysteine residues is in distinct 
contrast to the approximately 180 residues at the N-terminus and 
C-terminus of gp70, each with 8 cysteine residues. In total there are 8 
cysteines in the N-terminal 185 amino acids and 8 cysteines from amino 
acid 312 through 445. This arrangement may result in the entire 
proline-rich domain with flanking .beta.-sheets forming an exposed loop 
structure in the native gp70 molecule. 
The sequential reverse turn motif formulated by our modeling is consistent 
with the polyproline, .beta.-turn helix form of secondary structure 
associated with the proline-rich repeat sequences of proteins as proposed 
by Matsushima et al. (1990, Proteins: Structure, Function and Genetics 7: 
125-155). In the present disclosure, the predicted structural motif of the 
FeLV proline-rich sequence is evaluated directly using .sup.1 H-NMR and CD 
spectroscopy as well as measurements of intrinsic viscosity. 
6.2.2. Peptides 
The PRN peptides that were synthesized are summarized in Table 2. These 
include the entire 60 amino acid region (PRN60) taken from FeLV gp70 amino 
acids 230-289, the N-terminal 42 amino acids (PRN42), the N-terminal 21 
amino acids (PRN21), the middle 20 amino acids (PRN2242) and the 
C-terminal 16 amino acids (PRN4358). The molecular weights of the peptides 
were confirmed by electrospray mass spectroscopy, and in each case the 
molecular weight obtained was the expected weight. 
6.2.3. NMR Spectroscopy 
.sup.1 H-NMR spectroscopy was chosen to investigate the folded state of the 
PRN peptides. In this study, the test peptides were dissolved in 
deuterated phosphate buffer in D.sub.2 O and the region of the .sup.1 
H-NMR spectrum that is characteristic of amide protons was monitored for 
protected protons. The occurrence of amide proton resonances in D.sub.2 O 
indicates that the peptide is folded into a compact structure in which 
amides are buried and inaccessible to solvent D.sub.2 O molecules. 
The .sup.1 H-NMR spectrum of each PRN peptide was determined in 0.1M 
phosphate buffer, pH 7.2 in 90%/10%, H.sub.2 O/D.sub.2 O, and in 0.1M 
deuterated phosphate buffer, pH 7.2 in 99.9% D.sub.2 O. The results of the 
peptides dissolved in D.sub.2 O are shown in FIG. 2. The amide proton 
region of the .sup.1 H-NMR spectrum reveals that there are protons 
protected from exchange with D.sub.2 O in each of the peptides except the 
C-terminal 16 amino acid peptide. This indicates that the peptide forms a 
folded structure in solution and that there are amide protons which are 
stable and protected from solvent. Many of the features of the .sup.1 
H-NMR spectrum of the 60 amino acid peptide (PRN60; FIG. 2a) are distinct 
from the pattern seen in the 2 twenty amino acid peptides PRN21 (FIG. 2c) 
and PRN2242 (FIG. 2d), most noticeably the large resonance at 6.9 ppm is 
absent in the 60-mer and is present in the D.sub.2 O spectrum of the two 
20-mers. The resonances in the spectrum of PRN60 from 7.1 to 7.6 ppm are 
present but shifted and less complex in the two 20-mers. In addition, the 
sixty amino acid peptide shows a complex series of resonances from 7.9 to 
8.4 ppm that are absent in the two 20-mers. The .sup.1 H-NMR spectrum is 
D.sub.2 O of the peptide corresponding to the N-terminal 42 amino acids 
(FIG. 2b) of PRN60 contains resonances similar to both the 60 amino acid 
peptide .sup.1 H-NMR spectrum and the two twenty amino acid peptide .sup.1 
H-NMR spectra. The spectrum of the 42 amino acid peptide contains a broad 
resonance at 6.9 ppm which is present in PRN21 (FIG. 2c) and PRN2242 (FIG. 
2d). However, the spectrum of the 42 amino acid peptide also contains a 
series of resonances from 7.9 to 8.4 ppm, that are not as complex as that 
seen in the spectrum of the 60 amino acid peptide in D.sub.2 O and yet are 
absent in the spectrum of the two 20 amino acid peptides. The .sup.1 H-NMR 
spectrum in D.sub.2 O of the C-terminal segment (PRN 4358; FIG. 2e) does 
not show any protection of protons alone in D.sub.2 O, indicating the lack 
of significant conformation in this particular peptide. The line-widths of 
the proton resonances increase as the frequency increases and as the 
peptide size decreases. This indicates that there is a large 
conformational mobility for PRN60 and the mobility increases for the 
peptide fragments (Chaffotte, et al., 1991, Biochemistry 30: 8067-8074). 
The resonances seen in PRN60, PRN21, and PRN2242 in D.sub.2 O were followed 
for three days and decreased in intensity only slightly when kept at 
4.degree. C. in between measurements. Upon heating the PRN60 in D.sub.2 O 
sample to 4.degree. C., and equilibrating for one hour, there was a 
significant decrease in intensity of the resonances from 7.9 to 8.4 ppm 
and in the two peaks in between 6.7 and 6.8 ppm. The series of resonances 
from 7.1 to 7.6 ppm were remarkably stable to increased temperature, even 
60.degree. C. for 12 hours failed to completely allow exchange of these 
protons. The effect of increased temperature on the .sup.1 H-NMR spectrum 
of PRN60 in the region of 0.0 to 5.0 ppm was to sharpen the lines of the 
alpha-, beta-, and gamma-proton resonances indicating that the side chains 
of the amino acids are exposed to the solvent and not buried in a globular 
fold. Increasing the temperature of 60.degree. C. in the presence of 6M 
urea caused multiple shifts of resonances in the region of the alpha and 
beta protons indicating an unfolding process was occurring. These results 
demonstrate that the PRN peptide sequences assume a high ordered stable 
secondary structure compatible with a polyproline .beta.-turn helix. 
6.2.4. Self-Assembly of PRN Peptides 
The N-terminal 42 amino acid peptide (PRN42) and the C-terminal 16 amino 
acid peptide (PRN4358) self assemble into some state approaching the 
native fold when mixed together. When an equimolar (5 mM) amount of 
PRN4358 (FIG. 3a) is mixed with PRN42 (FIG. 3b) in H.sub.2 O, followed by 
exchange into D.sub.2 O, the result is the native-like .sup.1 H-NMR 
spectrum seen in FIG. 3c. The NMR spectrum of the mixture (FIG. 3c) 
indicates that the peptides form a complex that is very similar to that of 
PRN60. The most striking feature of the NMR spectrum of the self assembled 
mixture is the complete loss of the largest peak associated with the 42 
amino acid peptide at 6.9 ppm. This peak at 6.9 ppm which is present in 
PRN21, PRN2242, and PRN42 is the most distinguishing difference between 
the spectrum of these peptides and the spectrum of the 60 amino acid 
peptide. In addition, the large band of resonances from 7.2 to 7.6 ppm 
present in the .sup.1 H-NMR spectrum of the 42 amino acid peptide is 
replaced by a complex series of sharper peaks which are similar to the 
pattern seen in the spectrum of PRN60. A similar result is obtained if the 
two peptides are mixed in H.sub.2 O, is allowed to equilibrated for 2 
hours, followed by freezing and lyophilization before dissolution in 
D.sub.2 O (FIG. 3d). Again, the large peak at 6.9 ppm which is 
characteristic of the 42 amino acid peptide is completely absent in the 
.sup.1 H-NMR spectrum of the mixture (FIG. 3d). The NMR spectrum of the 
region from 7.2 to 7.6 ppm is intermediate in spectral detail when 
compared to PRN60 (FIG. 3e) and PRN42 (FIG. 3b) in the lyophilized sample. 
However, the details of the NMR spectrum from 7.9 to 8.4 ppm more closely 
resemble PRN60 when the sample is lyophilized. Thus, these NMR data 
indicate that synthetic peptide segments of the proline-rich domain can 
self-assemble into complex ordered multimers with the structure of the 
full length PRN60 peptide. The propensity for self-assembly in 
proline-rich repetitive proteins is compatible with a .beta.-turn helix 
structure, as described for the dynamic .beta.-spirals of bovine elastin 
(Urry, 1987, J. Prot. Chem. 7: 1-34). In the case of elastin, the sequence 
(VPAVG)n forms repeating type II .beta.-turns (Bhandary, et al., 1990, 
Int. J. Peptide Protein Res. 36: 122-127). The repetitive turn motif of 
elastin displays some very unusual physical properties such as: (i) 
self-assembly to form fibrils, (ii) increasing order with increasing 
temperature up to 60.degree. C., and (iii) development of viscoelastic 
force coincident with molecular ordering (Urry, 1987, J. Prot. Chem. 7: 
1-34; Urry, et al., 1988, Proc. Natl. Acad. Sci: USA 85: 3407-3411). 
6.2.5. Intrinsic Viscosity 
The intrinsic viscosity of a protein is exquisitely sensitive to the folded 
state and shape of a protein (Tanford, 1961, In: Physical Chemistry and 
Macromolecules; John Wiley and Sons, New York, N.Y.; 390-405; Tanford, et 
al., 1967, J. Am. Chem. Soc. 89: 729-736). The intrinsic viscosity is 
extrapolated from the reduced viscosity at zero concentration and that 
value is 7.49 ml/g for PRN60 (Table 3). Tanford has shown that the 
intrinsic viscosity of a protein in the random coil state is the highest 
viscosity state achievable and is given by the equation [.eta.]=0.716 (n) 
0.66, where n is the number of residues in the protein. The theoretical 
value of [.eta.] calculated for PRN60 in a random coil state is 10.7 ml/g. 
The measured value of [.eta.] for PRN60 was determined to be 7.49 ml/g 
(Table 3). This measured value is significantly less than the theoretical 
intrinsic viscosity predicted for random coil 60 amino acid peptide. In 
fact, the [.eta.] value of 7.49 ml/g predicts a random coil peptide of 36 
amino acid residues. The relatively low intrinsic viscosity for the PRM 
peptide indicates a highly ordered conformation for this peptide in 
solution. This result agrees with the .sup.1 H-NMR experiments indicating 
that PRN60 forms a folded structure PRN in solution. 
Intrinsic viscosity measurements also provide information on the shape of 
macromolecules. The values of intrinsic viscosity obtained for spherical, 
globular proteins are in the range of 3.3 to 3.9 ml/g and are independent 
of molecular weight (Tanford, 1961, In: Physical Chemistry and 
Macromolecules; John Wiley and Sons, New York, N.Y.; 390-405). The value 
of 7.49 ml/g for the intrinsic viscosity of PRN60 is inconsistent with a 
globular folded state for this molecule. However, this value is consistent 
with a prolate ellipsoid shape of axial ratio equal to 9.1 (Table 3) 
(Cantor & Schimmel, 1980, In: Biophysical Chemistry; Part 2: Techniques 
for the Study of Biological Structure and Function. W. H. Freeman and Co, 
New York, N.Y.). This value for the axial ratio is in good agreement with 
the value of 9.7 (longitudinal axis/cross sectional axis) obtained from a 
computer model of this sequence in a poly-type I turn secondary structural 
motif. 
6.2.6. Circular Dichroism 
The circular dichroism spectrum of PRN60 in 0.01M phosphate buffer at 
25.degree. C. is shown in FIG. 4. The spectrum reveals a characteristic 
large negative peak with a minimum at 198 nm (FIG. 4). The shape of the 
spectrum was unaffected by increasing the temperature sequentially to 
55.degree. C., 75.degree. C. and 90.degree. C. (FIG. 4). However, the 
intensity at 198 nm decreased quite dramatically with increasing 
temperature. The molar ellipticity of PRN60 at 90.degree. C. was 28.8% 
less than the value at 25.degree. C. The ability to reduce the intensity 
at 198 nm indicates that PRN60 contains significant structure in solution. 
The lack of two separate negative bands at 220 and 208 nm and the absence 
of the large positive band at 192 nm rules out any .alpha.-helical 
character for this peptide in solution. The CD spectrum of PRN60 (FIG. 4) 
also lacks the negative band near 216 nm and the large positive band 
between 195 and 200 nm which are characteristic of the spectra for a 
.beta.-sheet structure (Woody, 1985, Circular Dichroism of Peptides. In: 
The Peptides: Analysis, Synthesis, Biology, 7:16-104 Academic Press, Inc. 
Orlando, Fla.; Johnson, 1988, Ann. Rev. Biophys. Chem. 17:145-166) 
A previous model peptide study concluded that a CD spectrum similar to that 
observed here for PRN60 was characteristic of a random-coil peptide 
(Brahms and Brahms, 1980, J. Mol. Biol. 138: 149-178). This interpretation 
can now be reconsidered if one considers the model peptide used for this 
study was poly (PKLKL)n. This peptide motif, originally intended to be 
non-helical and non-beta sheet, is most compatible with the motif for a 
proline-rich poly-reverse turn protein. In fact, a turn is predicted to 
occur every (LPKL) with pt=0.9.times.10.sup.-4 when the Chou-Fasman 
criteria are applied to this sequence. Thus, this peptide sequence may 
actually be a standard for a poly-reverse turn protein and not a 
random-coil conformation. 
Other proteins have been shown to possess a large negative peak at 198 nm 
(e.g., Ausio, et al., 1987, Biochemistry 26: 975-982; Johnson, 1988, Ann. 
Rev. Biophys. Chem 17: 145-166; Tatham, et al., 1985, Biochem. J. 226: 
557-562; Madison and Schellman, 1990, Biopolymers 9: 65-94; Urry, 1987, J. 
Prot. Chem. 7: 1-34). An alternative interpretation for the CD spectrum of 
PRN60 is that this is the characteristic CD spectra of proline-rich poly 
.beta.-turn structures. This CD spectrum is identical to that observed 
with the model compound N-acetyl-L-proline-N,N-diethylamide (AcProDMA) in 
which the CD spectrum is dominated by a large negative Cotton effect at 
198 nm and is attributed to three .pi.-.pi.* transitions and a large 
n-.pi.* transition in the tertiary amide (Madison & Schellman, 1970, 
Biopolymers 9: 511-567, 569-588). The large negative Cotton effect at 198 
nm in H.sub.2 O was shown to be characteristic of AcProDMA in the trans 
conformation. In contrast, the CD spectrum of AcProDMA in the cis 
conformation yields a large positive Cotton effect at 198 nm. The recorded 
spectrum could be represented as linear combination of the spectrum of the 
trans and cis isomers (Madison & Schellman, 1970, Biopolymers 9: 511-567, 
569-588). The decrease in intensity at 198 nm observed with PRN60 at 
increasing temperatures indicates either that more cis proline is being 
formed as the temperature increases or that the .beta.-turn helix in which 
the proline residues would be preferentially in the trans conformation 
(Matsushima, et al., 1990, Proteins: Structure, Function and Genetics 7: 
125-155). 
6.2.7. The PRN Domain of FeLV gp70 
The present invention discloses a peptide corresponding to the proline-rich 
neutralization domain from the surface unit of gp70 of the feline leukemia 
virus forms a polyproline .beta.-turn helix. A proline-rich domain is 
documented for retroviruses such as the murine leukemia virus (Ott, et 
al., 1990, J. Virol. 64: 757-766; Battini, et al., 1992, J. Virol. 66: 
1468-1475) and the Gibbon ape leukemia virus (GaLV) (Delassus, et al., 
1989, Virology 53: 205-213). The amphotrophic murine leukemia virus (MuLv) 
4070A contains a proline-rich region (PR) that is composed of 33% proline, 
18% serine, 11% threonine, 8% valine and 7% glycine. A similar domain is 
located in the external glycoprotein of Gibbon ape leukemia virus (GaLV) 
(Delassus, et al., 1989, Virology 53: 205-213) and contains 27% proline, 
15% threonine, 10% leucine, 9% alanine and 9% serine. The retroviral 
proline-rich domains resemble in sequence composition and predicted 
repetitive structure, without the same sequence repeats, the human breast 
and pancreatic mucin (Gendler, et al., 1988, J. Biol. Chem. 26: 
12820-12823; Lan, et al., 1990, Cancer Res. 50: 2997-3001). These 
sequences appear to represent retroviral versions of mucin type 
glycoproteins (Jentoft, 1990, Trends Biochem. Sci. 15: 291-294; Strouss 
and Dekker, 1992, Critical Reviews in Biochemistry and Molecular Biology 
27(1/2): 57-92). 
Examples of other proline-rich peptide fragments that have been studied 
include human breast and pancreatic epithelial duct mucin (Tendler, 1990, 
Biochem. J. 267: 733-737), Type IV and Type I collagen (Mayo, et al., 
1991, Biochemistry 30: 8251-8267; Otter, et al., 1989, Biochemistry 28: 
8003-8010), LC1 alkali light chain of skeletal myosin (Bhandari, et al., 
1986, Eur. J. Biochem. 160: 349-356), the Spisula solidissima nuclear 
sperm-specific protein (Ausio, et el., 1987, Biochemistry 26: 975-982), 
and bovine elastin (Chang, et al., 1989, J. Biomol. Struct. and Dynam. 36: 
122-127; Bhandary, et al., 1990, Int. J. Peptide Protein Res. 36: 
122-127). In particular, the solution structure of peptide fragments of 
larger proteins which are immunogenic as fragments are of interest in 
examining the relationship between peptide structure in solution and 
induction of functional antibodies (Dyson, et al., 1988, J. Mol. Biol. 
210: 201-217; Richman and Reese, 1988, Proc. Natl. Acad. Sci. USA 85: 
1662-1666; Kotake, et al., 1990, Cell. Immunol. 126: 331-342; Dyson, et 
al., 1992, Biochemistry 31: 1458-1463). The sequence of PRN60 is 
incompatible with either .alpha.-helical or .beta.-sheet secondary 
structure and is determined by our modeling to form ten reverse turns. 
Matsushima, et al. (1990, Proteins: Structure, Function and Genetics 
7:125-155) has proposed that certain proline-rich repeat sequences form a 
secondary structure similar to poly-type I or III reverse turn motif 
called a polyproline .beta.-turn helix Previous .sup.1 H-NMR studies with 
peptides of collagen (Mayo, et al., 1991, Biochemistry 30: 8251-8267; 
Otter, et al., 1989, Biochemistry 28: 8003-8010 and mucin (Tendler, 1990, 
Biochem. J. 267: 733-737) indicate that a multiple-turn motif might be 
stable in a peptide corresponding to a larger protein. Our .sup.1 H-NMR 
results show that protons in PRN60 are protected from D.sub.2 O exchange. 
In fact, exchange was remarkably resistant to time and temperature. 
The PRN peptides self-assemble in solution and form a complex whose .sup.1 
H-NMR spectrum is very much like native PRN60 in D.sub.2 O. This type of 
behavior was recently shown to occur with E. coli Trp repressor protein 
(Tasayco and Carey, 1992, Science 225: 594-597). The self-assembly of Trp 
repressor peptides generated proteolytically could be followed by 
observing the proton chemical shift dispersion of the native protein, 
individual peptide fragments and reconstituted mixture in the amide proton 
region of the .sup.1 H-NMR spectrum. Peptide fragments of a bovine 
pancreatic trypsin inhibitor (BPTI) folding intermediate [5-55] also 
display the ability to fold into a native-like structure as determined by 
the chemical shift dispersion in the amide proton region and circular 
dichroism (Staley and Kim, 1990, Nature 344: 685-688). 
Therefore, the structure formed by PRN60 in solution represents the native 
conformation found in the virion for three reasons: (i) sequence 
composition and modeling studies of intact surface unit (FeLV gp70) 
indicates that this sequence is only compatible with a .beta.-turn helix 
motif, (ii) antibodies generated to the peptide free in solution and 
conjugated to KLH recognize the virion very well in ELISA, and (iii) 
monoclonal antibodies generated to native gp70 also bind to the peptide. 
Therefore, it is demonstrated by .sup.1 H-NMR that PRN60 forms an ordered 
structure in solution with large conformational mobility that is long 
lived, and that peptide fragments of this structure will self-assemble 
into a native like conformation. The circular dichroism results verify 
that the solution conformation is not .alpha.-helical or .beta.-sheet, and 
that the structure assumed contains primarily proline in the trans 
conformation. Proline in the trans conformation is consistent with proline 
in reverse turns. The intrinsic viscosity results suggest a non-random 
coil structure that is rod shaped. Therefore, PRN60 forms a polyproline 
.beta.-turn helix and that region of gp70 is a separate folding domain of 
the surface unit protein. 
TABLE 1 
______________________________________ 
Synthetic peptides corresponding to FeLV-gp7O proline-rich 
neutralization domain (SEQ ID NO:6). Start and stop sites for 
the fragment peptides were determined by the intervals of the 
predicted turns. 
PREDICTED TURNS IN PROLINE RICH 
NEUTRALIZATION DOMAIN 
.sup.a Bend Probability 
Amino Acid # Sequence .times. 10.sup.-4 
.sup.b P.sub.t 
______________________________________ 
233-236 PPQA 0.66 1.17 
238-241 GPNL 4.10 1.31 
243-246 LPDQ 1.98 1.15 
248-251 PPSR 3.26 1.38 
255-258 TGSK 0.87 1.23 
263-266 RPQT 0.62 1.11 
270-273 APRS 1.90 1.15 
275-278 APTT 0.93 1.04 
280-283 GPKR 1.88 1.26 
285-288 GTGD 1.69 1.39 
______________________________________ 
.sup.a probability of a bend at residue i through i + 4 
.sup.b average conformational potential of this sequence; P.sub.t &gt; 1.00 
and P.sub.t &gt; P.sub..alpha. & P.sub..beta. for a turn to be predicted. 
TABLE 2 
__________________________________________________________________________ 
PROLINE RICH NEUTRALIZATION DOMAIN PEPTIDES 
__________________________________________________________________________ 
PRN60 
(SEQ ID NO: 1) 
TITPPQAMGP 
NLVLPDQKPP 
SRQSQTGSKV 
ATQRPQTNES 
APRSVAPTTV 
GPKRIGTGDR 
PRN42 
(SEQ ID NO: 2) 
TPPQAMGP NLVLPDQKPP 
SRQSQTGSKV 
ATQRPQTNES 
APRS 
PRN21 
(SEQ ID NO: 3) 
TPPQAMGP NLVLPDQKPP 
SRQ 
PRN2242 
(SEQ ID NO: 4) 
SQTGSKV ATQRPQTNES APRS 
PRN4358 
(SEQ ID NO: 5) 
VAPTTV GPKRIGTGDR 
__________________________________________________________________________ 
The sequences are underneath the name of each respective peptide from the 
amino to carboxy terminal. Peptide fragment start and stop sites 
correspond to intervals between predicted turns. 
TABLE 3 
______________________________________ 
MOLECULAR DIMENSION OF PRN60 FROM 
INTRINSIC VISCOSITY 
Intrinsic Viscosity 
Simha shape 
[.eta.] ml/g factor v A/B.sup.b 
A/B.sup.c 
______________________________________ 
7.49 10.7 9.1 9.7 
______________________________________ 
.sup.a Summary of the molecular dimensions determined from instrinsic 
viscosity measurements of PRN60. 
.sup.b Axial ratio determined by intrinsic viscosity. 
.sup.c Axial ratio determined by molecular graphics program sybyl. 
7. EXAMPLE: PRODUCTION OF ANTI-PRN60 PEPTIDE ANTIBODIES AND EXPERIMENTAL 
VACCINATIONS WITH PRN60 
7.1. Materials and Materials 
Various procedures known in the art may be used for the production of 
anti-PRN60 peptide antibodies that bind to and neutralize FeLV. Any of the 
antibodies generated for use in the invention include but are not limited 
to polyclonal, monoclonal, chimeric, single chain and Fab fragments. 
For the production of polyclonal antibodies, a vertebrate host may be 
immunized with the peptide alone or a peptide joined to a carrier molecule 
to promote an immune response. The immunogen may then be mixed with an 
adjuvant and injected into the vertebrate host according to a 
predetermined schedule. The animal is then periodically bled and the titer 
tested by PLL-ELISA. In the present example, the PRN60 peptide was either 
linked to KLH or left unlinked and utilized to immunize rabbits, mice and 
cats. In the case of rabbits and cats the adjuvant was initially Freund's 
complete adjuvant and followed by Freund's incomplete adjuvant. 
Peptides were reacted in a solid-phase PLL-ELISA, which has been optimized 
for use with peptide antigens (Ball, J. M., 1990, Ph.D thesis, Louisiana 
State University, Baton Rouge). Briefly, poly-L-lysine (Mr 45-50K) is 
adsorbed onto the plastic surface of the microtiter plate. A low 
background with adequate signal (high signal to noise ratio) is achieved 
when PLL is bound to the wells of Immulon I (Dynatech Laboratories; 
Chantilly, Va.) 96-well microtiter plates and is coupled to the antigen 
with 1% glutaraldehyde in PBS. The PLL treated microtiter plates were then 
used directly in the ELISA based assay described below. Whole virus 
(FeLV-Theilen) was adsorbed directly onto the plastic surface of the 
microtiter plate with 1% gluteraldehyde in PBS. The whole virus containing 
microtiter plate was then used directly in the ELISA based assay described 
below. 
The primary antibodies were diluted serially at 1/100, 1/1,000 and 1/10,000 
in 10% BLOTTO (Carnation dry milk) and 10% horse serum in 0.01M phosphate 
buffer (pH 7.4) with 0.5M NaCl and 0.2% Tween 20. Primary antibody 
incubations were at room temperature for 30 minutes. The secondary 
antibody was goat anti-cat immunoglobulin G-horseradish peroxidase 
(Jackson Immuno Research Laboratories, Inc., West Grove, Pa.) with 
o-phenylenediamine dihydrochloride as the substrate. The secondary 
antibody was diluted at 1/5,000 in 10% BLOTTO (Carnation dry milk) and 10% 
horse serum in 0.01M phosphate buffer (pH 7.4), with 0.5M NaCl and 0.2% 
Tween 20 and was incubated for 30 minutes at room temperature. The ELISA 
wash solution was 1M NaCl and 0.2% Tween 20. 
Mice were immunized subcutaneously with four doses of unlinked PRN60 
peptide (100 micrograms per dose) in incomplete Freund's adjuvant at 2 
week intervals. Two weeks following the last immunization, the mice were 
sacrificed and blood and spleens were collected for assays of humoral and 
cellular immune responses, respectively, elicited by the PRN60 immunogen. 
Antibody responses were measured in standard PLL-ELISA assays using PRN60 
as substrate. Cellular immune responses were assayed by measuring in vitro 
proliferative responses (.sup.3 H-thymidine incorporation) of spleen cells 
from immunized mice in response to different amount of PRN60 peptide. 
7.2. Results 
Table 4 represents bleeds taken one month (1 boost) after immunization for 
rabbits and six weeks (2 boosts) after immunization for cats. In each case 
KHL linked peptide induced higher titer antibodies that reacted well with 
PRN60 and whole FeLV-Theilen. Unconjugated peptides induced lower titer 
antibodies to PRN60 and to FeLV-Theilen. 
The ratio of the whole virus titer divided by the PRN60 titer provides a 
measure of the polyclonal antibodies ability to react with native protein 
(Table 5). Cats generated lower titer antibodies to PRN60, yet these 
antibodies had a 16 times greater ability to bind FeLV-Theilen. For 
example, PRN60 conjugated to KLH induced in a cat a titer of 4000 to PRN60 
and a titer of 1500 to the whole virus (Table 4). In contrast, PRN60 
conjugated to KLH induced a rabbit titer of 2200 to PRN60 and 520 to the 
whole virus. This disparity in the ability to induce virus reactive 
antibodies reduces to a factor of seven when unlinked titers are 
considered. However, the pattern of cats inducing lower peptide titers and 
higher virus titers is maintained with unlinked antigen (Tables 4 and 5). 
The covalently conjugated peptide induced antibodies that reacted with 
higher titers to the individual peptide fragments of PRN60 (PRN21, 
PRN2242, PRN4358), and this pattern was observed in rabbits and cats. The 
C-terminus of PRN60 appears to dominate the antibody response, especially 
in cats. The C-terminal segment induced three times higher titer with 
conjugated peptide and ten times higher titer with unconjugated peptide in 
cats. This pattern is broken somewhat in rabbits immunized with conjugated 
peptide. In this case, the middle segment and C-terminal segment induced 
5-7 times higher titers that the N-terminal segment. The pattern of 
antibody reactivity induced by unconjugated peptide as immunogen is 
similar in rabbits and cats, with the C-terminal segment dominating. 
Table 6 summarizes the humoral and cellular immune responses elicited by 
PRN60 immunization of mice. All of the immunized mice produced relatively 
high levels of PRN60 -specific antibody, with calculated titers of 
2000-3000. In addition, all of the mice appeared to develop significant 
cellular immune responses, as evidenced by the proliferative responses 
(2-3 stimulation index) to 1 microgram of PRN60 per 10,000 cells. These 
mouse immunization studies demonstrate that unlinked PRN60 peptide is 
highly immunogenic, even in the absence of a strong adjuvant and with a 
relatively mild immunization regimen. The induction of proliferative 
T-cell responses indicates that the PRN60 is able to bind to MHC class II 
molecules and stimulate T-helper cells that support the production of high 
levels of antibody. 
Therefore, the present invention discloses peptides comprising a secondary 
structure which mimics the native structure of an FeLV envelope protein. 
These peptides may be utilized as immunogens in a variety of fashions as 
indicated throughout this specification. Disclosed in this specification 
is the ability of large synthetic peptides to (1) retain a native 
configuration; (2) stimulate antibodies reactive with the whole virus; and 
(3) elicit a cellular response in experimental vaccinations, leading to 
the conclusion that such peptides are candidates for use as a prophylactic 
vaccine as well as an immune enhancer to an initial vaccine. The PRN60 is 
exemplified in this specification as one such candidate for use in these 
vaccine applications. 
The present invention is not to be limited in scope by the embodiments 
disclosed in the examples which are intended as illustrations of a few 
aspects of the invention, and any embodiments which are functionally 
equivalent are within the scope of the invention. Indeed, various 
modifications of the invention in addition to those shown and described 
herein will become apparent to those skilled in the art and are intended 
to fall within the scope of the appended claims. 
TABLE 4 
______________________________________ 
Titers.sup.a of Anti-PRN60 antibodies 
Whole.sup.b 
Sp./ Conj(? ) 
PRN60 Virus 21.sup.c 
2242.sup.d 
43580.sup.e 
______________________________________ 
cat yes 4000 1500 420 420 1500 
no 1200 90 41 290 470 
rabbit 
yes 22000 520 330 2000 1500 
no 7500 80 28 380 2700 
______________________________________ 
.sup.a titer is defined as the inverse of the dilution to yield an 
absorbance of 1.0 in a peptide ELISA 
.sup.b FeLV theilen 
.sup.c Amino acids 1 through 21 of PRN60 (PRN21) 
.sup.d Amino acids 22 through 42 of PRN60 (PRN2242) 
.sup.e Amino acids 43 through 58 of PRN60 (PRN4358) 
TABLE 5 
______________________________________ 
Antibody Reactivity to Native Protein 
Ratios 
Species Conjugation 
FeLV/PRN60.sup.a 
______________________________________ 
cat yes .375 
no .075 
rabbit yes .024 
no .011 
______________________________________ 
.sup.a Titer of antiPRN60 to FeLVTheilen divided by the titer to the 
immunizing antigen (PRN60). 
TABLE 6 
______________________________________ 
Antibody and Cellular Immune Responses in Mice 
Immunized with PRN60 
Mouse 
Index ELISA Reactivity.sup.a 
Antibody Titer.sup.b 
Stimulation.sup.c 
______________________________________ 
1 1.3 2,500 2.0 
2 1.1 2,500 2.3 
3 1.0 3,000 3.0 
______________________________________ 
.sup.a Absorbance in PLLELISA against PRN60 at a serum dilution of 1:500. 
.sup.b Calculated end point titer against PRN60 in PLLELISA. 
.sup.c Tcell proliferation of spleen cells (10,000) incubated with 1 mg/m 
of PRN60. The stimulation index is calculated as the ratio of .sup.3 
Hthymidine incorporation in cells incubated with peptide antigen compared 
to cells in the absence of antigen. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 60 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Feline leukemia virus 
(B) STRAIN: 61E 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ThrIleThrProProGlnAlaMetGlyProAsnLeuValLeuProAsp 
151015 
GlnLysProProSerArgGlnSerGlnThrGlySerLysValAlaThr 
202530 
GlnArgProGlnThrAsnGluSerAlaProArgSerValAlaProThr 
354045 
ThrValGlyProLysArgIleGlyThrGlyAspArg 
505560 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Feline leukemia virus 
(B) STRAIN: 61E 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ThrProProGlnAlaMetGlyProAsnLeuValLeuProAspGlnLys 
151015 
ProProSerArgGlnSerGlnThrGlySerLysValAlaThrGlnArg 
202530 
ProGlnThrAsnGluSerAlaProArgSer 
3540 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Feline leukemia virus 
(B) STRAIN: 61E 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ThrProProGlnAlaMetGlyProAsnLeuValLeuProAspGlnLys 
151015 
ProProSerArgGln 
20 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Feline leukemia virus 
(B) STRAIN: 61E 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
SerGlnThrGlySerLysValAlaThrGlnArgProGlnThrAsnGlu 
151015 
SerAlaProArgSer 
20 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Feline leukemia virus 
(B) STRAIN: 61E 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
ValAlaProThrThrValGlyProLysArgIleGlyThrGlyAspArg 
151015 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 445 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Feline leukemia virus 
(B) STRAIN: 61E 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
MetGluSerProThrHisProLysProSerLysAspLysThrLeuSer 
151015 
TrpAsnLeuValPheLeuValGlyIleLeuPheThrIleAspIleGly 
202530 
MetAlaAspProSerProHisGlnIleTyrAsnValThrTrpValIle 
354045 
ThrAsnValGlnThrAsnThrGlnAlaAsnAlaThrSerMetLeuGly 
505560 
ThrLeuThrAspValTyrProThrLeuHisValAspLeuCysAspLeu 
65707580 
ValGlyAspThrTrpGluProIleValLeuSerProThrAsnValLys 
859095 
HisGlyAlaArgTyrProSerSerLysTyrGlyCysLysThrThrAsp 
100105110 
ArgLysLysGlnGlnGlnThrTyrProPheTyrValCysProGlyHis 
115120125 
AlaProSerLeuGlyProLysGlyThrHisCysGlyGlyAlaGlnAsp 
130135140 
GlyPheCysAlaAlaTrpGlyCysGluThrThrGlyGluAlaTrpTrp 
145150155160 
LysProSerSerSerTrpAspTyrIleThrValLysArgGlySerSer 
165170175 
GlnAspAsnAsnCysGluGlyLysCysAsnProLeuIleLeuGlnPhe 
180185190 
ThrGlnLysGlyLysGlnAlaSerTrpAspGlyProLysMetTrpGly 
195200205 
LeuArgLeuTyrArgThrGlyTyrAspProIleAlaLeuPheThrVal 
210215220 
SerArgGlnValSerThrIleThrProProGlnAlaMetGlyProAsn 
225230235240 
LeuValLeuProAspGlnLysProProSerArgGlnSerGlnThrGly 
245250255 
SerLysValAlaThrGlnArgProGlnThrAsnGluSerAlaProArg 
260265270 
SerValAlaProThrThrValGlyProLysArgIleGlyThrGlyAsp 
275280285 
ArgLeuIleAsnLeuValGlnGlyThrTyrLeuAlaLeuAsnAlaThr 
290295300 
AspProAsnLysThrLysAspCysTrpLeuCysLeuValSerArgPro 
305310315320 
ProTyrTyrGluGlyIleAlaIleLeuGlyAsnTyrSerAsnGlnThr 
325330335 
AsnProProProSerCysLeuSerIleProGlnHisLysLeuThrIle 
340345350 
SerGluValSerGlyGlnGlyLeuCysIleGlyThrValProLysThr 
355360365 
HisGlnAlaLeuCysAsnLysThrGlnGlnGlyHisThrGlyAlaHis 
370375380 
TyrLeuAlaAlaProAsnGlyThrTyrTrpAlaCysAsnThrGlyLeu 
385390395400 
ThrProCysIleSerMetAlaValLeuAsnTrpThrSerAspPheCys 
405410415 
ValLeuIleGluLeuTrpProArgValThrTyrHisGlnProGluTyr 
420425430 
ValTyrThrHisPheAlaLysAlaValArgPheArgArg 
435440445 
__________________________________________________________________________