Methods and compositions for impairing multiplication of HIV-1

A composition is provided which contains a non-naturally occurring peptide or polypeptide comprising at least two or more, and preferably all four amino acid sequences -Asp-Pro-Arg-Leu-Glu-Pro- SEQ ID NO: 6; -Asp-Pro-Lys-Leu-Glu-Pro- SEQ ID NO: 7; -Asp-Pro-Ser-Leu-Glu-Pro- SEQ ID NO: 8; and -Asp-Pro-Asn-Leu-Glu-Pro- SEQ ID NO: 9. This composition, including a number of additional optional peptides or polypeptides, and in a variety of forms, demonstrates a biological activity of inducing antibodies that react with most HIV-1 Tat proteins and impairing the multiplication of HIV-1. Also provided are synthetic genes encoding these peptides, recombinant viruses and commensal bacterium carrying these genes, transfected host cells, and polyclonal or other types of antibodies produced by immunizing other mammals with these aforementioned compositions. Methods for making and using such compositions to lower the viral level of HIV-1 are described.

FIELD OF THE INVENTION 
The present invention relates generally to compositions and methods useful 
for inhibiting the multiplication of human immunodeficiency virus-1 
(HIV-1) in infected patients, symptomatic or asymptomatic, and for 
attenuating HIV-1 multiplication during primary infection in previously 
uninfected subjects, thus minimizing progression to AIDS. 
BACKGROUND OF THE INVENTION 
High plasma levels of human immunodeficiency virus type 1 (HIV-1) RNA are 
found during primary infection with HIV-1, the seroconversion illness, C. 
Baumberger et al, AIDS, 7:(suppl 2):S59 (1993); M. S. Saag et al, Nature 
Med., 2:625 (1996)!, after which they subside as the immune response 
controls the infection to a variable extent. Post seroconversion, lower 
but detectable levels of plasma HIV-1 RNA are present, and these levels 
rise with disease progression to again attain high levels at the AIDS 
stage M. S. Saag et al, Nature Med., 2:265 (1996)!. Approximately 50% of 
subjects have a symptomatic illness at seroconversion B. Tindall and D. 
A. Cooper, AIDS, 5:1 (1991)! and symptomatic seroconversion is associated 
with an increased risk for the development of AIDS, probably because a 
severe primary illness is likely related to an early and extensive spread 
of HIV. 
Inhibition of viral multiplication during the initial infection will likely 
reduce the subsequent development of chronic viremia leading to AIDS. 
Current medical practice, with administration of antiviral drugs for 
defined "at risk" situations, such as needle sticks with contaminated 
blood or pregnancy in HIV infected mothers, supports this concept. 
Post sero-conversion levels of HIV-1 RNA in plasma have proven to be the 
most powerful prognosticator of the likelihood of progression to AIDS J. 
W. Mellors et al, Science, 272:1167 (1996); M. S. Saag et al, Nature Med., 
2:265 (1996); R. W. Coombs et al, J. Inf. Dis., 174:704 (1996); S. L. 
Welles et al, J. Inf. Dis., 174:696 (1990)!. Other measures of viral load, 
such as cellular RNA K. Saksela et al, Proc. Natl. Acad. Sci. USA, 
91:1104 (1994)! and cellular HIV proviral DNA T-H. Lee et al, J. Acq. 
Imm. Def. Syndromes, 7:381 (1994)! similarly establish the importance of 
the initial infection in establishing viral loads that determine future 
disease progression. 
Thus, any intervention that inhibits HIV-1 infectivity during initial 
infection and/or lowers viral load post sero-conversion is likely to have 
a favorable influence on the eventual outcome, delaying or preventing 
progression to AIDS. 
A variety of methods are now employed to treat patients infected with human 
immunodeficiency virus (HIV-1), including treatment with certain 
combinations of protease inhibitor drugs. Unfortunately, however, this 
type of treatment is associated with serious side effects in some 
patients. 
Alternatively, vaccines are under development for control of the spread of 
HIV-1 to uninfected humans. However, this effort has largely been directed 
to proteins of the virus, expressed on the surface of infected cells, 
which are recognized by cytotoxic T cells with elimination of the infected 
cells, while free virus is blocked and cleared by antibody to surface 
antigens of the virion. Limitations of this mode of vaccination are 
readily apparent for HIV-1, which has demonstrated a great diversity in 
immunogenic viral epitopes and rapid mutational variations that occur 
within and between individuals B. D. Preston et al., Science, 
242:1168(1988); J. D. Roberts et al., Science, 242:1171 (1988); A. R. 
Meyerhans et al., Cell, 58:901 (1989); K. Kusumi et al., J. Virol., 66:875 
(1992); B. A. Larder et al., Science, 243:1731 (1989); M. S. Sang et al., 
N. Engl. J. Med., 329:1065 (1993); M. A. Sande, et al., JAMA, 270:2583 
(1993); M. Seligmann et al., Lancet, 343:871 (1994); G. Meyers et al., 
Human retroviruses and AIDS 1993, I-V. A compilation and analysis of 
nucleic acid and amino acid sequences. Los Alamos National Laboratory, Los 
Alamos, N.Mex.! 
Variation in strains of HIV-1 and frequent mutations of virion proteins 
have prevented successful application of conventional vaccine approaches 
W. E. Paul, Cell, 82:177 (1995); J. E. Osborn, J. Acq. Imm. Def. Syndr. 
Hum. Retrovirol., 9:26 (1995)!. Mutation and selection of resistant 
variants is the central problem in developing a successful HIV-1 vaccine 
M. D. Daniel et al., Science, 258:1938 (1992); N. L. Letvin, N. Engl. J. 
Med., 329:1400 (1993); M. Clerici et al., AIDS, 8:1391 (1994); S. M. 
Wolinsky et al, Science, 272:537 (1996)!. 
Other approaches to HIV-1 treatment have focused on the transactivating 
(tat) gene of HIV-1, which produces a protein (Tat) essential for 
transcription of the virus. The tat gene and its protein have been 
sequenced and examined for involvement in proposed treatments of HIV see, 
e.g., U.S. Pat. No. 5,158,877; U.S. Pat. No. 5,238,882; U.S. Pat. No. 
5,110,802; International Patent Application No. WO92/07871, published May 
14, 1992; International Patent Application No. WO91/10453, published Jul. 
25, 1991; International Patent Application No. WO91/09958, published Jul. 
11, 1991; International Patent Application No. WO87/02989, published May 
21, 1987!. Tat protein is released extracellularly, making it available to 
be taken up by other infected cells to enhance transcription of HIV-1 in 
the cells and to be taken up by noninfected cells, altering host cell gene 
activations and rendering the cells susceptible to infection by the virus. 
Uptake of Tat by cells is very strong, and has been reported as mediated 
by a short basic sequence of the protein S. Fawell et al., Proc. Natl. 
Acad. Sci., USA, 91:664-668 (1994)!. 
International Patent Application No. WO92/14755, published Sep. 3, 1992, 
relates to the Tat protein and to the integrin cell surface receptor 
capable of binding to the Tat protein. Two Tat sequences that bind 
integrin are identified, which are the basic region or domain which is the 
dominant binding site for the integrin, having a peptide sequence of 
-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg- SEQ ID NO: 4!, as well as 
-Gly-Arg-Gly-Asp-Ser-Pro- SEQ ID NO: 5!. This specification demonstrates 
that a number of peptides corresponding to these Tat sequences and the 
corresponding integrins block in vitro cell binding to Tat coated plates, 
as do antibodies to the appropriate integrins. However, the specification 
also shows that these reagents do not block uptake of functional Tat by 
cells (see Example 9 in WO92/14755), thus nullifying the proposed 
mechanism of action for therapeutic benefit in HIV infection. The Tat 
sequences described in this international application are distinct from 
the peptide immunogens of the present invention. 
Both monoclonal and polyclonal antibodies to Tat protein have been readily 
produced in animals and shown to block uptake of Tat protein in vitro 
see, e.g., D. Brake et al, J. Virol., 64:962 (1990); D. Mann et al, EMBO 
J., 10:1733 (1991); J. Abraham et al, cited above; P. Auron et al, cited 
above; M. Jaye et al, cited above; G. Zauli et al, cited above!. More 
recent reports showed that monoclonal or polyclonal antibodies to Tat 
protein added to tissue culture medium attenuated HIV-1 infection in vitro 
L. Steinaa et al, Arch. Virol., 139:263 (1994); M. Re et al, J. Acq. Imm. 
Def. Syndr. Hum. Retrovirol., 10:408 (1995); and G. Zauli et al, J. Acq. 
Imm. Def. Syndr. Hum. Retrovirol., 10:306 (1995)!. 
The inventor's own publication G. Goldstein, Nature Med., 2:960 (1996); 
see also, International Patent Application No. WO95/31999, published Nov. 
30, 1995! reviewed the evidence indicating that secretion of HIV-1 Tat 
protein from infected cells and uptake by both infected and uninfected 
cells was important for the infectivity of HIV-1. Previous studies also 
showed that antibodies to Tat protein in vitro blocked uptake of Tat and 
inhibited in vitro infectivity. Goldstein proposed active immunization of 
mammals to induce antibodies to HIV-1 Tat protein as a potential AIDS 
vaccine. 
Despite the growing knowledge about HIV-1 disease progression, there 
remains a need in the art for the development of compositions and methods 
for treatment of HIV-1, both prophylactically and therapeutically, which 
are useful to lower the viral levels of HIV-1 for the treatment and 
possible prevention of the subsequent, generally fatal, AIDS disease. 
SUMMARY OF THE INVENTION 
In one aspect, the invention provides novel compositions designed to induce 
antibodies reactive with almost all known variants of HIV-1 Tat protein. 
In one embodiment the composition contains a non-naturally occurring 
peptide or polypeptide, which contains or has embedded in the 
peptide/polypeptide sequence, at least two, and preferably all four, amino 
acid sequences selected from -Asp-Pro-Arg-Leu-Glu-Pro-; 
-Asp-Pro-Lys-Leu-Glu-Pro; -Asp-Pro-Ser-Leu-Glu-Pro-; and 
-Asp-Pro-Asn-Leu-Glu-Pro- SEQ ID NOS: 6 through 9, respectively!. This 
composition demonstrates a biological activity of inducing in immunized 
mammals antibodies that react with most variants of Tat proteins and 
impair the multiplication of HIV-1 in an acute infection, thus preventing 
high post-seroconversion plasma levels of HIV-1 that are associated with 
progression to AIDS. The peptides or polypeptides of these compositions 
are produced synthetically or recombinantly. 
In another aspect, the above-described composition further contains one or 
more additional peptide or polypeptide(s) which represent other amino acid 
sequences which correspond to amino acid residues 5 to 10 of an HIV-1 Tat 
protein. These optional amino acid sequences are described in detail 
below. These sequences are preferably from an HIV-1 strain with a Tat 
protein variant at that location. 
In still another aspect, the above-described composition further contains 
one or more additional peptide or polypeptide(s) which represent amino 
acid sequences which correspond to amino acid residues 56 to 62 of an 
HIV-1 Tat protein. Such other optional immunogenic peptides and 
polypeptides may contain at least one copy of an amino acid sequence of 
the formula -Arg-Arg-X-Pro-Gln-Y-Ser- SEQ ID NO: 10!, as described in 
detail below. Other peptides or polypeptides representative of aa 56-62, 
but having different sequences from that of the above formula may also be 
included in the composition. 
In one embodiment, the composition described above comprises a peptide or 
polypeptide which contains multiple repeats of one of the above-described 
amino acid sequences. In another embodiment, the composition comprises a 
peptide or polypeptide containing sequentially multiple different amino 
acid sequences. Still another embodiment of the composition comprises 
peptides or polypeptides which contain different amino acid sequences 
repeated multiple times. 
In another aspect, the invention provides a composition as above-described 
wherein each peptide or polypeptide is coupled to the same or different 
carrier protein. 
In still a further aspect, the composition provides the peptides or 
polypeptides in the form of a multiple antigenic peptide. 
In yet a further aspect, the invention provides a synthetic gene which 
encodes sequentially a peptide or polypeptide that contains at least two 
and preferably all four, amino acid sequences selected from 
-Asp-Pro-Arg-Leu-Glu-Pro-; -Asp-Pro-Lys-Leu-Glu-Pro; 
-Asp-Pro-Ser-Leu-Glu-Pro-; and -Asp-Pro-Asn-Leu-Glu-Pro- SEQ ID NOS: 6 
through 9, respectively!, or multiple copies of any of these sequences. 
Synthetic genes which encode any of the peptides or polypeptides 
above-described are also provided. The synthetic gene may contain each 
amino acid sequence separated by a spacer sequence, or may express each 
peptide/polypeptide in an open reading frame with a carrier protein. 
Alternatively the synthetic gene may be separated from the carrier protein 
by a spacer. 
In yet a further aspect, the invention provides a synthetic molecule 
comprising the above-described synthetic gene, operatively linked to 
regulatory nucleic acid sequences, which direct and control expression of 
the product of the synthetic gene in a host cell. 
In another aspect, the invention provides a recombinant virus which 
contains the above described synthetic gene or synthetic molecule, which 
virus is capable of expressing multiple copies of the product of the gene 
or molecule in a host cell. The virus is non-pathogenic to humans. 
In yet another aspect, the invention provides a commensal bacterium which 
contains the above described synthetic gene or synthetic molecule, which 
bacterium is capable of expressing multiple copies of the product of the 
gene or molecule and inducing antibodies in a mammalian host. 
In still a further aspect, the invention provides an isolated antibody 
composition which is directed against a non-naturally occurring peptide or 
polypeptide comprising at least two or more copies of an amino acid 
sequence, selected from the group consisting of SEQ ID NOS: 6 through 9, 
respectively!: 
-Asp-Pro-Arg-Leu-Glu-Pro-; 
-Asp-Pro-Lys-Leu-Glu-Pro-; 
-Asp-Pro-Ser-Leu-Glu-Pro-; and 
-Asp-Pro-Asn-Leu-Glu-Pro-. 
The antibody may be directed against the other optional immunogens 
contained in a peptide or polypeptide of this invention, including those 
of formula -Arg-Arg-X-Pro-Gln-Y-Ser- SEQ ID NO: 10!. This antibody is 
produced by immunizing a mammal with a peptide/polypeptide composition of 
the invention, a synthetic gene or synthetic molecule of the invention; a 
recombinant virus or commensal bacterium of the invention; and isolating 
and purifying antibody from said immunized mammal. Alternatively, the 
antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric 
antibody, a humanized antibody, a human antibody, or mixtures thereof. 
Thus, another aspect of the invention is a pharmaceutical composition 
useful for inducing antibodies that react with most HIV-1 Tat proteins and 
impair the multiplication of HIV-1. The pharmaceutical composition 
comprises at least one of the recombinant or synthetic peptide/polypeptide 
compositions described above; the synthetic gene/molecule described above; 
the recombinant virus described herein; or the commensal bacterium 
described herein, in a pharmaceutically acceptable carrier. 
Still a further aspect of the invention is a pharmaceutical composition 
useful for impairing the multiplication of HIV-1, this composition 
containing an above described antibody composition. 
In yet a further aspect of the invention, a method for reducing the viral 
levels of HIV-1 involves exposing a human to antibody-inducing 
pharmaceutical compositions described above, actively inducing antibodies 
that react with most HIV-1 Tat proteins, and impairing the multiplication 
of the virus in vivo. This method is appropriate for an HIV-1 infected 
subject with a competent immune system, or an uninfected or recently 
infected subject. The method induces antibodies which react with HIV-1 Tat 
proteins, which antibodies reduce viral multiplication during any initial 
acute infection with HIV-1 and minimize chronic viremia which leads to 
AIDS. 
In still another aspect, the invention provides a method for reducing the 
viral levels of HIV-1 by administering to a human, who is incapable of 
mounting an effective or rapid immune response to infection with HIV-1, a 
pharmaceutical composition containing the antibody compositions described 
above. The method can involve chronically administering the composition. 
Yet other aspects of the invention include methods for producing the 
compositions described above, as well as host cells transfected with such 
compositions. 
Other aspects and advantages of the present invention are described further 
in the following detailed description of the preferred embodiments thereof 
.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides a solution to the above-stated problem by 
providing compositions which induce antibodies in uninfected or early 
stage infected subjects still capable of mounting an immune response to an 
immunogen, said antibodies reacting with most Tat proteins and inhibiting 
multiplication of HIV-1. This prevents further disease progression to 
AIDS. Antibody compositions are also provided for use in infected or 
non-infected humans, who are incapable of mounting an effective or rapid 
immune response to HIV-1 infection. These compositions are capable of 
reacting with most known Tat proteins, thus reducing viral levels of 
HIV-1, and thus are useful in both a therapeutic and prophylactic context 
to control the development of AIDS in a large population exposed to, or 
infected by, HIV-1 which produce upon infection different Tat proteins. 
The compositions of the present invention may be proteinaceous in nature, 
or may be nucleic acid compositions which encode the peptides and 
polypeptides that induce antibodies to Tat, which in turn impair 
multiplication of HIV-1. 
A. Peptide/Polypeptide Compositions 
1. Primary Compositions 
The present invention provides as its "primary composition", a composition 
containing a non-naturally occurring peptide or polypeptide, which 
comprises at least two "primary immunogens" or "primary amino acid 
sequences". By "immunogen" is meant any sequence or molecule which elicits 
a specific humoral immune response (for the purpose of this invention) in 
a mammal exposed to that molecule in vivo. 
The primary immunogens of this invention are peptides or polypeptides which 
contain embedded in the peptide sequence, at least two of the following 
amino acid sequences SEQ ID NOS: 6 through 9, respectively!: 
-Asp-Pro-Arg-Leu-Glu-Pro-; 
-Asp-Pro-Lys-Leu-Glu-Pro; 
-Asp-Pro-Ser-Leu-Glu-Pro-; and 
-Asp-Pro-Asn-Leu-Glu-Pro-. 
Preferably, this primary composition contains a polypeptide which contains 
all four of these amino acid sequences. Such compositions may also contain 
multiple peptides or polypeptides which contain multiple copies of a 
single peptide, or multiple copies of different of these four peptides in 
any order, or multiple copies of one or two of these peptides. In any 
variation, it is preferred for the efficacy of the primary composition, 
that at least one copy of all four amino acid sequences are present. It is 
not important for the function of the composition, whether these amino 
acid sequences are present in one or more peptides or polypeptides. 
The amino acid sequences identified above may be flanked by other amino 
acids in the primary immunogens; but the identity of the flanking amino 
acids is not essential to the biological function of the primary 
immunogen. These four amino acid sequences which are present in the 
primary immunogens correspond to amino acid residues 5-10 of the Tat 
consensus sequence SEQ ID NO: 1! of FIG. 1 which is derived from a number 
of "Tat sequence variants". The term "Tat sequence variant" means a 
polypeptide or peptide containing Tat protein amino acid residues 5-10 (or 
a sequence from another HIV-1 strain Tat protein corresponding to that 
sequence of SEQ ID NO: 1) or Tat protein residues 56-62 (or a sequence 
from another HIV-1 strain Tat protein corresponding to that sequence of 
SEQ ID NO: 1). Each variant may differ from the consensus sequence of FIG. 
1 SEQ ID NO: 1! and/or from another variant by at least one amino acid 
change within those residues and provides an enhanced immune response to 
that particular Tat protein variant when added to the primary composition 
of the invention. 
This primary composition demonstrates a biological activity of inducing 
antibodies to most Tat proteins, thereby impairing the multiplication of 
HIV-1 in a host cell or a host mammal. This primary composition elicits in 
an immune competent human, i.e., a non-infected human, or an asymptomatic 
infected human, an active immune response which is directed against most 
Tat protein variants of HIV-1. Active induction of antibodies in the early 
asymptomatic phase of HIV infection may reduce viral multiplication, lower 
the plasma viral load and reduce the likelihood of progression to AIDS. 
The composition which contains at least one primary immunogen with all 
four of the above amino acid sequences can elicit an immune response to 
about 97% of the 400 known Tat sequences of the common B subtypes of HIV-1 
and with Tat proteins of all 18 non-B subtype HIV-1 that have been 
sequenced courtesy of Dr. Esther Guzman, Los Alamos NIAID HIV database; 
GenBank database!. 
2. Additional Peptides and Polypeptides 
The above-described primary composition of the invention may also be 
designed to contain a number of additional peptides or polypeptides, which 
contain other sequences which correspond to amino acid residues 5-10 of 
SEQ ID NO: 1 but are derived from other Tat variants which do not 
cross-react well with antibodies to the primary immunogens containing two 
to four of the above-described amino acid sequences. These additional 
peptides and polypeptides are referred to as "optional immunogens". 
Optional immunogens which can be present in compositions of this invention, 
can contain at least one copy of at least one of the following amino acid 
sequences SEQ ID NOS: 11 through 18, respectively!: 
-Gly-Pro-Arg-Leu-Glu-Pro-; 
-Ala-Pro-Arg-Leu-Glu-Pro-; 
-His-Pro-Arg-Leu-Glu-Pro-; 
-Asp-Pro-Gly-Leu-Glu-Pro-; 
-Asp-Pro-Arg-Ile-Glu-Pro-; 
-Asp-Pro-Arg-Leu-Gly-Pro-; 
-Asp-Pro-Arg-Leu-Glu-Ala-; and 
-Asn-Pro-Ser-Leu-Glu-Pro-. 
Still other peptides/polypeptides which may be optionally present in a 
composition of this invention containing two, and preferably four of the 
sequences of the primary immunogens are still other optional immunogenic 
peptides and polypeptides, which may be derived from Tat variant protein 
sequences corresponding to amino acids 56-62 of SEQ ID NO: 1. 
Among such optional immunogens are peptides and polypeptides that contain 
at least one copy of an amino acid sequence of the formula 
-Arg-Arg-X-Pro-Gln-Y-Ser- SEQ ID NO: 10!. According to this formula, 
which is derived from Tat variant protein sequences corresponding to amino 
acids 56-62 of SEQ ID NO: 1, X may be Ala, Pro, Ser or Gln; and Y may be 
Asp, Asn, Gly or Ser. A preferred peptide/polypeptide in a composition of 
this invention contains the optional immunogen 
-Arg-Arg-Ala-Pro-Gln-Asp-Ser- SEQ ID NO: 19!. Still other amino acid 
sequences which may be included in optional immunogens are 
-Arg-Arg-Ala-Pro-Pro-Asp-Ser-, -Arg-Arg-Ala-His-Gln-Asp-Ser- or 
-Arg-Arg-Ala-His-Gln-Asn-Ser- SEQ ID NOS: 20 through 22, respectively!. 
Yet other amino acid sequences useful in optional immunogens for inclusion 
in the composition with the primary immunogens are 
-Arg-Arg-Pro-Pro-Gln-Asp-Asn, -Arg-Arg-Ala-Pro-Gln-Asp-Arg-; 
-Arg-Gly-Ala-Pro-Gln-Asp-Ser-; -Arg-Arg-Ala-Pro-Glu-Asp-Ser-; or 
-Arg-Arg-Ala-Ser-Gln-Asp-Ser- SEQ ID NOS: 23 through 27, respectively!. 
As can be determined from review of the examples below, the inclusion of 
these optional immunogens into the primary composition can induce 
antibodies that react with rare Tat proteins of HIV-1 which are not 
cross-reactive with, or do not have a sufficiently strong cross-reactivity 
to, antibodies induced by the primary immunogens. 
With regard to compositions of the invention which include optional 
immunogens, the composition may contain multiple peptides or polypeptides 
which contain multiple copies of a single optional immunogen sequence or 
multiple copies of different optional immunogen amino acid sequences in 
any order, or multiple copies of one or two of these optional sequences 
with some or all of the primary immunogens. In any variation, it is 
preferred for the efficacy of the primary composition, that at least one 
copy of all four primary amino acid sequences are present in the 
composition. It is not important for the function of the composition, 
whether the optional amino acid sequences are present in one or more 
peptides or polypeptides. 
The amino acid sequences identified above may be flanked by other amino 
acids in the optional immunogen peptides or polypeptides; but the identity 
of the flanking amino acids is not essential to the biological function of 
the composition of this invention. 
While the amino acid sequences useful in the primary immunogens and 
optional immunogens identified herein were obtained by rigorous analysis 
of over 400 known Tat sequences of HIV-1, it should be understood by one 
of skill in the art that similar compositions may be obtained using Tat 
proteins, the nucleic acid sequences encoding them, and fragments thereof 
from newly isolated Tat proteins of HIV-1 subtype B, or from Tat proteins 
of the other subtypes, or from other HIV strains. 
Thus, the compositions of this invention, i.e., the peptide/polypeptides 
containing the above-identified amino acid sequences, when provided to a 
human subject, are useful in the immunologic interdiction of extracellular 
Tat proteins of most HIV-1 strains. These compositions function to 
critically reduce explosive multiplication of the virus and permit 
effective immune control of the virus. 
The primary immunogens, with or without any optional immunogens may be 
presented in a variety of forms, for example, chemically synthesized or as 
recombinant peptides, polypeptides, proteins, fusion proteins or fused 
peptides. As pharmaceutical compositions, these primary compositions are 
admixed with a pharmaceutically acceptable vehicle, such as saline or 
phosphate buffered saline, suitable for administration as a protein 
composition for prophylaxis or treatment of virus infections. These 
proteins may be combined in a single pharmaceutical preparation for 
administration. Suitable adjuvants may also be employed in the 
protein-containing primary compositions of this invention. 
As one embodiment, a composition of the present invention may be a 
synthetic peptide, containing single or multiple copies of the same or 
different primary immunogen amino acid sequences, and amino acid sequences 
of the optional immunogens, coupled to a selected carrier protein. In this 
embodiment of a composition of this invention, multiple different 
above-described primary and optional amino acid sequences with or without 
flanking sequences, may be combined sequentially in a polypeptide and 
coupled to the same carrier. Alternatively, the primary immunogens, and 
any optional immunogens may be coupled individually as peptides to the 
same or a different carrier, and the resulting immunogen-carrier 
constructs blended together to form a single composition. 
For this embodiment, the carrier protein is desirably a protein or other 
molecule which can enhance the immunogenicity of the primary or optional 
immunogen. Such a carrier may be a larger molecule which has an 
adjuvanting effect. Exemplary conventional protein carriers include, 
without limitation, E. coli DnaK protein, galactokinase (galK, which 
catalyzes the first step of galactose metabolism in bacteria), ubiquitin, 
.alpha.-mating factor, .beta.-galactosidase, and influenza NS-1 protein. 
Toxoids (i.e., the sequence which encodes the naturally occurring toxin, 
with sufficient modifications to eliminate its toxic activity) such as 
diphtheria toxoid and tetanus toxoid may also be employed as carriers. 
Similarly a variety of bacterial heat shock proteins, e.g., mycobacterial 
hsp-70 may be used. Glutathione reductase (GST) is another useful carrier. 
One of skill in the art can readily select an appropriate carrier. 
In particularly desirable immunogen-carrier protein construct, one or more 
primary immunogen and optional immunogen peptides/polypeptides may be 
covalently linked to a mycobacterial E. coli heat shock protein 70 (hsp70) 
K. Suzue et al, J. Immunol., 156:873 (1996)!. In another desirable 
embodiment, the composition is formed by covalently linking the 
immunogen-containing peptide or polypeptide sequences to diphtheria 
toxoid. 
In yet another embodiment, the peptides or polypeptide primary immunogens 
and any selected optional immunogens may be in the form of a multiple 
antigenic peptide ("MAP", also referred to as an octameric lysine core 
peptide) construct. Such a construct may be designed employing the MAP 
system described by Tam, Proc. Natl. Acad. Sci. USA, 85:5409-5413 (1988). 
This system makes use of a core matrix of lysine residues onto which 
multiple copies of the same primary or optional immunogens of the 
invention are synthesized as described D. Posnett et al., J. Biol. Chem., 
263(4):1719-1725 (1988); J. Tam, "Chemically Defined Synthetic Immunogens 
and Vaccines by the Multiple Antigen Peptide Approach", Vaccine Research 
and Developments, Vol. 1, ed. W. Koff and H. Six, pp. 51-87 (Marcel 
Deblau, Inc., New York 1992)!. Each MAP contains multiple copies of only 
one peptide. Therefore a primary composition of this invention can include 
a MAP in which the peptide or polypeptide primary immunogen attached to 
the lysine core contains one or sequential repeats of the four "primary" 
amino acid sequences identified above. Multiple different MAPs may be 
employed to obtain all four primary sequences and a selected number of 
optional immunogen sequences. Preferably these MAP constructs are 
associated with other T cell stimulatory sequences, or as pharmaceutical 
compositions, administered in conjunction with T cell stimulatory agents, 
such as known adjuvants. 
In either of the above compositions, e.g., as peptide/polypeptide-carrier 
constructs or MAPs, each peptide/polypeptide immunogen, or each amino acid 
sequence in the immunogen, may be optionally separated by an optional 
amino acid sequences called "spacers". Spacers are sequences of between 1 
to about 4 amino acids which are interposed between two sequences to 
permit linkage therebetween without adversely effecting the three 
dimensional structure of the immunogen. Spacers may also contain 
restriction endonuclease cleavage sites to enable separation of the 
sequences, where desired. Suitable spacers or linkers are known and may be 
readily designed and selected by one of skill in the art. Preferred 
spacers are sequences containing Gly or Ser amino acids. 
B. Nucleic Acid Compositions of the Invention 
Other embodiments of this invention include nucleic acid sequences, which 
encode the compositions, including the peptide and polypeptide immunogens 
of the compositions described above, including those peptides and 
polypeptides fused to carrier proteins. The nucleic acid sequences may 
also include sequences encoding the carrier proteins. 
Thus, one preferred embodiment of the invention is a "synthetic gene" which 
encodes sequentially at least one or more copies of primary immunogen 
peptides/polypeptides comprising at least two of the amino acid sequences 
SEQ ID NOS: 6 through 9, respectively!: 
-Asp-Pro-Arg-Leu-Glu-Pro-; 
-Asp-Pro-Lys-Leu-Glu-Pro-; 
-Asp-Pro-Ser-Leu-Glu-Pro-; and 
-Asp-Pro-Asn-Leu-Glu-Pro-. 
As for the primary composition in protein/peptide form, the synthetic gene 
preferably encodes all four amino acid sequences of the primary 
immunogens. The synthetic gene can also encode any selection of the 
optional immunogens identified above. The synthetic gene may, as for the 
peptide/polypeptides described above, encode multiple copies of the same 
amino acid sequence, copies of multiple different immunogens or amino acid 
sequences, or multiple copies of multiple different immunogens or amino 
acid sequences. The synthetic gene may encode a desired peptide or 
polypeptide immunogen, or multiple peptide or polypeptide immunogens 
containing the selected amino acid sequences, which peptide/polypeptide is 
expressed in an open reading frame with, or fused to, a carrier protein. A 
further characteristic of the synthetic gene may be that it encodes a 
spacer between each immunogen or between each of the amino acid sequences 
in the immunogen, or between the immunogen and the carrier protein. 
The synthetic gene of the present invention may also be part of a synthetic 
or recombinant molecule. The synthetic molecule may be a nucleic acid 
construct, such as a vector or plasmid which contains the synthetic gene 
encoding the protein, peptide, polypeptide, fusion protein or fusion 
peptide under the operative control of nucleic acid sequences encoding 
regulatory elements such as promoters, termination signals, and the like. 
Such synthetic molecules may be used to produce the polypeptide/peptide 
immunogen compositions recombinantly. 
The synthetic gene or synthetic molecules can be prepared by the use of 
chemical synthesis methods or preferably, by recombinant techniques. For 
example, the synthetic gene or molecules may contain certain preference 
codons for the species of the indicated host cell. 
The synthetic gene or molecules, preferably in the form of DNA, may be used 
in a variety of ways. For example, these synthetic nucleic acid sequences 
may be employed to express the peptide/polypeptides of the invention in 
vitro in a host cell culture. The expressed immunogens, after suitable 
purification, may then be incorporated into a pharmaceutical reagent or 
vaccine. 
Alternatively, the synthetic gene or synthetic molecule of this invention 
may be administered directly into a mammalian, preferably human subject, 
as so-called `naked DNA` to express the protein/peptide immunogen in vivo 
in a patient. See, e.g., J. Cohen, Science, 259:1691-1692 (Mar. 19, 1993); 
E. Fynan et al., Proc. Natl. Acad. Sci., USA, 90:11478-11482 (December 
1993); and J. A. Wolff et al., Biotechniques, 11:474-485 (1991), all 
incorporated by reference herein. The synthetic molecule, e.g., a vector 
or plasmid, may be used for direct injection into the mammalian host. This 
results in expression of the protein by host cells and subsequent 
presentation to the immune system to induce antibody formation in vivo. 
In still another aspect of the present invention, the synthetic genes or 
molecules of this invention may be incorporated into a non-pathogenic 
microorganism. The resulting microorganism, when administered to a 
mammalian host expresses and multiplies the expressed compositions of this 
invention in vivo to induced specific antibody formation. For example, 
non-pathogenic recombinant viruses or commensal bacterium which carry the 
compositions or synthetic genes of this invention and are useful for 
administration to a mammalian patient may be prepared by use of 
conventional methodology and selected from among known non-pathogenic 
microorganisms. 
Among commensal bacterium which may be useful for exogenous delivery of the 
synthetic molecule to the patient, and/or for carrying the synthetic gene 
into the patient in vivo, include, for example, various strains of 
Streptococcus, e.g., S. gordonii, or E. coli, Bacillus, Streptomyces, and 
Saccharomyces. 
Suitable non-pathogenic viruses which may be engineered to carry the 
synthetic gene into the cells of the host include poxviruses, such as 
vaccinia, adenovirus, canarypox, retroviruses and the like. A number of 
such non-pathogenic viruses are commonly used for human gene therapy, and 
as carrier for other vaccine agents, and are known and selectable by one 
of skill in the art. 
C. Preparation or Manufacture of Compositions of the Invention 
The compositions of the invention, and the individual polypeptides/peptides 
containing the primary and optional immunogens of this invention, the 
synthetic genes, and synthetic molecules of the invention, may be prepared 
conventionally by resort to known chemical synthesis techniques, such as 
described by Merrifield, J. Amer. Chem. Soc., 85:2149-2154 (1963). 
Alternatively, the compositions of this invention may be prepared by known 
recombinant DNA techniques by cloning and expressing within a host 
microorganism or cell a DNA fragment carrying a sequence encoding a 
peptide/polypeptide containing a primary and/or optional immunogen and 
optional carrier protein. Coding sequences for the primary and optional 
immunogens can be prepared synthetically W. P. C. Stemmer et al, Gene, 
164:49 (1995) or can be derived from viral RNA by known techniques, or 
from available cDNA-containing plasmids. 
Combinations of these techniques may be used, such as for production of the 
synthetic gene, which may require assembly of sequential immunogens by 
conventional molecular biology techniques, and site-directed mutagenesis 
to provide desired sequences of immunogens. The product of the synthetic 
gene is then produced recombinantly. All of these manipulations may be 
performed by conventional methodology. 
Systems for cloning and expressing the peptide/polypeptide compositions of 
this invention using the synthetic genes or molecules, include various 
microorganisms and cells which are well known in recombinant technology. 
These include, for example, various strains of E. coli, Bacillus, 
Streptomyces, and Saccharomyces, as well as mammalian, yeast and insect 
cells. Suitable vectors therefor are known and available from private and 
public laboratories and depositories and from commercial vendors. 
Currently, the most preferred host is a mammalian cell such as Chinese 
Hamster ovary cells (CHO) or COS-1 cells. These hosts may be used in 
connection with poxvirus vectors, such as vaccinia or swinepox. The 
selection of other suitable host cells and methods for transformation, 
culture, amplification, screening and product production and purification 
can be performed by one of skill in the art by reference to known 
techniques. See, e.g., Gething and Sambrook, Nature, 293:620-625 (1981). 
Another preferred system includes the baculovirus expression system and 
vectors. 
When produced by conventional recombinant means, the compositions of this 
invention, i.e., the polypeptide/peptides containing the indicated copies 
of the primary immunogens and optional immunogens may be isolated either 
from the cellular contents by conventional lysis techniques or from cell 
medium by conventional methods, such as chromatography. See, e.g., 
Sambrook et al., Molecular Cloning. A Laboratory Manual., 2d Edit., Cold 
Spring Harbor Laboratory, New York (1989). 
Suitable plasmid and viral vectors used either for production of the 
peptide/polypeptide components as DNA vaccines are well known to those of 
skill in the art and are not a limitation of the present invention. See, 
Sambrook et al., cited above and the references above to production of the 
protein. See, also International Patent Application PCT WO94/01139, 
published Jan. 20, 1994. 
Briefly, the DNA encoding the selected peptide/polypeptide is inserted into 
a vector or plasmid which contains other optional flanking sequences, a 
promoter, an mRNA leader sequence, an initiation site and other regulatory 
sequences capable of directing the multiplication and expression of that 
sequence in vivo or in vitro. These vectors permit infection of patient's 
cells and expression of the synthetic gene sequence in vivo or expression 
of it as a protein/peptide or fusion protein/peptide in vitro. 
The resulting composition may be formulated into a primary composition with 
any number of optional immunogens and screened for efficacy by in vivo 
assays. Such assays employ immunization of an animal, e.g., a rabbit or a 
simian, with the composition, and evaluation of titers of antibody to the 
Tat proteins of HIV-1 or to synthetic detector peptides corresponding to 
variant Tat sequences (as shown in the examples below). 
D. Antibody Compositions of the Invention 
An isolated mammalian antibody composition which is directed against a 
peptide or polypeptide of the invention, as described above, is also an 
aspect of this invention. Such polyclonal antibody compositions are 
produced by immunizing a mammal with a peptide/polypeptide composition 
containing an assortment of primary immunogens and optional immunogens, as 
described above. Suitable mammals include primates, such as monkeys; 
smaller laboratory animals, such as rabbits and mice, as well as larger 
animals, such as horse, sheep, and cows. Such antibodies may also be 
produced in transgenic animals. However, a desirable host for raising 
polyclonal antibodies to a composition of this invention includes humans. 
The polyclonal antibodies raised in the mammal exposed to the composition 
are isolated and purified from the plasma or serum of the immunized mammal 
by conventional techniques. Conventional harvesting techniques can include 
plasmapheresis, among others. 
Such polyclonal antibody compositions may themselves be employed as 
pharmaceutical compositions of this invention. Alternatively, other forms 
of antibodies may be developed using conventional techniques, including 
monoclonal antibodies, chimeric antibodies, humanized antibodies and fully 
human antibodies. See, e.g., Harlow et al., Antibodies A Laboratory 
Manual, Cold Spring Harbor Laboratory, (1988); Queen et al., Proc. Nat'l. 
Acad. Sci. USA, 86:10029-10032 (1989); Hodgson et al., Bio/Technology, 
9:421 (1991); International PCT Application PCT/GB91/01554, Publication 
No. WO92/04381 and International PCT Application PCT/GB93/00725, 
Publication No. WO93/20210!. Other anti-Tat antibodies may be developed by 
screening hybridomas or combinatorial libraries, or antibody phage 
displays W. D. Huse et al., Science, 246:1275-1281 (1988)! using the 
polyclonal or monoclonal antibodies produced according to this invention 
and the amino acid sequences of the primary or optional immunogens. 
These antibody compositions bind to most Tat protein variants of HIV-1, and 
prevent the Tat proteins from supporting further HIV-1 multiplication. 
Thus, these antibodies are useful in pharmaceutical methods and 
formulations described below. 
E. Pharmaceutical Compositions of the Invention 
As another aspect of this invention, a pharmaceutical composition useful 
for inducing antibodies that react with most HIV-1 Tat proteins and impair 
the multiplication of HIV-1 is provided. This composition can comprise as 
its active agents, one of the following above-described components: 
(a) a peptide/polypeptide primary immunogen which contains at least two, 
and preferably all four of the primary amino acid sequences and optionally 
contains other optional immunogens. These primary/optional immunogens may 
be in the form of recombinant proteins. Alternatively, they may be in the 
form of a mixture of carrier protein conjugates or MAPs. 
(b) a synthetic gene described above; 
(c) a synthetic molecule described above; 
(d) a recombinant virus carrying the synthetic gene or molecule; and 
(e) a commensal bacterial carrying the synthetic gene or molecule. The 
selected active component(s) is present in a pharmaceutically acceptable 
carrier, and the composition may contain additional ingredients. All of 
these pharmaceutical compositions can operate to lower the viral levels of 
a mammal. 
The peptide/polypeptide compositions and synthetic genes or molecules in 
vivo are capable of eliciting in an immunized host mammal, e.g., a human, 
an immune response capable of interdicting most extracellular Tat protein 
variants from HIV-1 and thereby lowering the viral levels. 
Pharmaceutical formulations containing the compositions of this invention 
may contain other active agents, such as T cell stimulatory agents for the 
MAPs, adjuvants and immunostimulatory cytokines, such as IL-12 and other 
well-known cytokines, for the protein/peptide compositions. 
Suitable pharmaceutically acceptable carriers for use in an immunogenic 
proteinaceous composition of the invention are well known to those of 
skill in the art. such carriers include, for example, saline, a selected 
adjuvant, such as aqueous suspensions of aluminum and magnesium 
hydroxides, liposomes, oil in water emulsions and others. The present 
invention is not limited by the selection of the carrier or adjuvant. 
Yet another pharmaceutical composition useful for impairing the 
multiplication of HIV-1 comprises an antibody composition as described in 
detail above. In a pharmaceutical composition, the antibodies may be 
carried in a saline solution of other suitable carrier. The antibody 
compositions are capable of providing an immediate, exogenously provided 
interdiction of Tat. 
The preparation of these pharmaceutically acceptable compositions, from the 
above-described components, having appropriate pH isotonicity, stability 
and other conventional characteristics is within the skill of the art. 
F. Method of the Invention--Impairing Multiplication of HIV-1 
According to the present invention, a method for reducing the viral levels 
of HIV-1 involves exposing a human to the Tat antibody-inducing 
pharmaceutical compositions described above, actively inducing antibodies 
that react with most HIV-1 Tat proteins, and impairing the multiplication 
of the virus in vivo. This method is appropriate for an HIV-1 infected 
subject with a competent immune system, or an uninfected or recently 
infected subject. The method induces antibodies which react with HIV-1 Tat 
proteins, which antibodies reduce viral multiplication during any initial 
acute infection with HIV-1 and minimize chronic viremia leading to AIDS. 
This method also lowers chronic viral multiplication in infected subjects, 
again minimizing progression to AIDS. 
In one embodiment, the pharmaceutical compositions may be therapeutically 
administered to an HIV-1 infected human with a competent immune system for 
treatment or control of viral infection. Such an infected human may be 
asymptomatic. In a similar embodiment, the pharmaceutical compositions may 
be administered to an uninfected human for prophylaxis. 
In these two instances, the pharmaceutical compositions preferably contain 
the peptide/polypeptide compositions, the synthetic genes or molecules, 
the recombinant virus or the commensal recombinant bacterium. Each of 
these active components of the pharmaceutical composition actively induces 
in the exposed human the formation of anti-Tat antibodies which block the 
transfer of Tat from infected cells to other infected or uninfected cells. 
This action reduces the multiplicity of infection and blocks the burst of 
HIV-1 viral expansion, and thus lowers viral levels. In already infected 
patients, this method of reduction of viral levels can reduce chronic 
viremia and progression to AIDS. In uninfected humans, this administration 
of the compositions of the invention can reduce acute infection and thus 
minimize chronic viremia leading to progression to AIDS. 
Yet another aspect of the invention is a method for reducing the viral 
levels of HIV-1 by administering to a human, who is incapable of mounting 
an effective or rapid immune response to infection with HIV-1, a 
pharmaceutical composition containing the antibody compositions described 
above. The method can involve chronically administering the composition. 
Among such patients suitable for treatment with this method are HIV-1 
infected patients who are immunocompromised by disease and unable to mount 
a strong immune response. In later stages of HIV infection, the likelihood 
of generating effective titers of antibodies is less, due to the immune 
impairment associated with the disease. Also among such patients are HIV-1 
infected pregnant women, neonates of infected mothers, and unimmunized 
patients with putative exposure (e.g., a human who has been inadvertently 
"stuck" with a needle used by an HIV-1 infected human). 
For such patients, the method of the invention preferably employs as the 
pharmaceutical composition the antibody composition of the invention, 
which is a polyclonal antibody composition prepared in other mammals, 
preferably normal humans. Alternatively, the other forms of antibody 
described above may be employed. These antibody compositions are 
administered as passive immunotherapy to inhibit viral multiplication and 
lower the viral load. The exogenous antibodies which react with most Tat 
proteins from HIV-1 provide in the patient an immediate interdiction of 
the transfer of Tat from virally infected cells to other infected or 
uninfected cells. According to this method, the patient may be chronically 
treated with the antibody composition for a long treatment regimen. 
In each of the above-described methods, these compositions of the present 
invention are administered by an appropriate route, e.g., by the 
subcutaneous, oral, intravenous, intraperitoneal, intramuscular, rectal or 
vaginal routes. The presently preferred route of administration is 
intramuscular for the immunizing (active induction) compositions and 
intravenous or intramuscular for the antibody (passive therapy) 
compositions. The recombinant viral vectors and/or live commensal bacteria 
may be delivered orally. 
The amount of the protein, peptide or nucleic acid sequences of the 
invention present in each vaccine dose is selected with regard to 
consideration of the patient's age, weight, sex, general physical 
condition and the like. The amount of active component required to induce 
an immune response, preferably a protective response, or produce an 
exogenous effect in the patient without significant adverse side effects 
varies depending upon the pharmaceutical composition employed and the 
optional presence of an adjuvant (for the protein-containing 
compositions). 
Generally, for the compositions containing protein/peptide, fusion protein, 
MAP or coupled protein, or antibody composition, each dose will comprise 
between about 50 .mu.g to about 1 mg of the peptide/polypeptide immunogens 
per mL of a sterile solution. A more preferred dosage may be about 200 
.mu.g of immunogen. Other dosage ranges may also be contemplated by one of 
skill in the art. Initial doses may be optionally followed by repeated 
boosts, where desirable. 
The antibody compositions of the present invention can be employed in 
chronic treatments for subjects at risk of acute infection due to needle 
sticks or maternal infection. A dosage frequency for such "acute" 
infections may range from daily dosages to once or twice a week i.v. or 
i.m., for a duration of about 6 weeks. The antibody compositions of the 
present invention can also be employed in chronic treatments for infected 
patients, or patients with advanced HIV. In infected patients, the 
frequency of chronic administration may range from daily dosages to once 
or twice a week i.v. or i.m., and may depend upon the half-life of the 
immunogen (e.g., about 7-21 days). However, the duration of chronic 
treatment for such infected patients is anticipated to be an indefinite, 
but prolonged period. 
Alternatively, compositions of this invention may be designed for direct 
administration of synthetic genes or molecules of this invention as "naked 
DNA". Suitable vehicles for direct DNA, plasmid nucleic acid, or 
recombinant vector administration include, without limitation, saline, or 
sucrose, protamine, polybrene, polylysine, polycations, proteins, 
CaPO.sub.4 or spermidine. See e.g, PCT application WO94/01139 and the 
references cited above. As with the protein immunogenic compositions, the 
amounts of components in the DNA and vector compositions and the mode of 
administration, e.g., injection or intranasal, may be selected and 
adjusted by one of skill in the art. Generally, each dose will comprise 
between about 50 .mu.g to about 1 mg of immunogen-encoding DNA per mL of a 
sterile solution. 
For recombinant viruses containing the synthetic genes or molecules, the 
doses may range from about 20 to about 50 ml of saline solution containing 
concentrations of from about 1.times.10.sup.7 to 1.times.10.sup.10 pfu/ml 
recombinant virus of the present invention. A preferred human dosage is 
about 20 ml saline solution at the above concentrations. However, it is 
understood that one of skill in the art may alter such dosages depending 
upon the identity of the recombinant virus and the make-up of the 
immunogen that it is delivering to the host. 
The amounts of the commensal bacteria carrying the synthetic gene or 
molecules to be delivered to the patient will generally range between 
about 10.sup.3 to about 10.sup.12 cells/kg. These dosages, will of course, 
be altered by one of skill in the art depending upon the bacterium being 
used and the particular composition containing primary and optional 
immunogens being delivered by the live bacterium. 
Thus, the compositions of this invention are designed to retard or minimize 
infection by the selected virus of an uninfected mammal, e.g., human. Such 
compositions thus have utility as vaccines. Anti-Tat protein antibodies 
are not reactive with the HIV-1 proteins used in diagnostic assays to 
detect seroconversion after infection. Thus, subjects treated with the 
compositions of this invention would not be stigmatized with 
false-positive tests for HIV-1 infection, and it would remain possible to 
detect seroconversion if treated subjects did become infected with HIV-1. 
Providing a mammal with the compositions of this invention, whether as a 
protein/peptide-containing composition or by administration of a novel 
nucleic acid sequence encoding the immunogen, affords a radically 
different strategy for AIDS vaccination because it permits the lowering of 
viral levels by biological interdiction of most known Tat protein variants 
of HIV-1, lowering multiplication of HIV-1. 
The use of the Tat immunogen-containing compositions has a particularly 
desirable advantage in contrast to other treatments and prophylactic 
methods employed against such viruses. Because interdiction of the Tat 
protein extracellularly inhibits the multiplication of all HIV 
quasi-species or strains indiscriminately, it does not create a selective 
pressure on the parent virus itself for selection of mutant virus 
variants. Thus, blocking the uptake of Tat protein by the patient's cells 
not only reduces the level of viremia, but does so in a manner that 
precludes the selection of "escape variants". 
Additionally, the invention comprises a method of actively treating 
asymptomatic HIV-1 infected subjects with viremia, since during the course 
of the disease, extracellular Tat protein likely contributes to the 
persistent infection and immune abnormalities that are present at this 
stage of HIV-1 infection. Interdiction of extracellular Tat protein by 
antibodies induced by immunization according to this invention can reduce 
viremia with more effective immune control, and result in delay or 
prevention of progression to AIDS. 
The mechanism of the present invention as described above is useful in 
impeding the course of viral infection and producing desirable clinical 
results. More specifically, the compositions of this invention are capable 
of reducing viremia in patients already infected with the virus by 
blocking further uptake of the Tat protein by uninfected cells. The 
compositions of the present invention, used either alone or in conjunction 
with other therapeutic regimens for HIV infected patients, are anticipated 
to assist in the reduction of viremia and prevention of clinical 
deterioration. 
For such therapeutic uses, the formulations and modes of administration are 
substantially identical to those described specifically above and may be 
administered concurrently or simultaneously with other conventional 
therapeutics for the specific viral infection. For therapeutic use or 
prophylactic use, repeated dosages of the immunizing compositions may be 
desirable, such as a yearly booster or a booster at other intervals. 
G. Advantages of the Invention 
One of the advantages of the compositions of this invention is the small 
number of immunogens required for inclusion into a composition of this 
invention to cross-react with greater than 97% of known Tat protein 
variants of HIV-1 of the common B subtype. As illustrated in the examples 
below, the primary immunogenic composition containing all four primary 
amino acid sequences cross-reacts with 387 of 399 Tat proteins of HIV-1 of 
the common B subtype, as well as with all 18 Tat protein sequences from 
less frequent non-B subtypes of HIV-1. Thus, a single composition may be 
usefully employed in protecting against or treating infection, caused by 
the vast majority of HIV-1 strains that can be encountered. 
Further, having identified the precise epitopes on Tat against which 
binding is desired (i.e., AA5-10 or AA56-62 of SEQ ID NO: 1) new desirable 
Tat peptide immunogens from newly occurring HIV-1 strains or newly 
discovered strains may be easily identified using the methods described 
herein, and included in the compositions. This flexibility enables the 
compositions of this invention to be useful prophylactically against any 
new strain or strains of HIV-1 identified in the future. In view of the 
teachings herein, one of skill in the art is expected to be readily able 
to incorporate new combinations of Tat immunogens (and the nucleic acid 
constructs encoding them) into the compositions. 
For example, the use of conventional techniques such as PCR and high 
density oligonucleotide arrays M. J. Kozal et al, Nature Med., 2:753 
(1996)! enables one of skill in the art to obtain the amino acid sequences 
of a large array of HIV-1 Tat proteins representing variants of clinical 
isolates of HIV-1 strains and subtypes. Using such techniques permits 
determination of other variants of the HIV-1 B subtype as well as other 
subtypes in undeveloped countries, which have not been so intensively 
studied to date. The determination of new Tat sequences will enable ready 
inclusion of the corresponding peptides as immunogens into compositions of 
this invention, allowing the induction of an antibody response against 
other rare Tat proteins of HIV-1. 
Cross-reactivity studies with antibodies raised to synthetic peptides 
corresponding to each Tat variant can be utilized to eliminate the need 
for immunizing with Tat variants in which the sequence changes are 
immunologically silent, in that these peptides are strongly bound by 
antibodies to the consensus sequence or other variants. 
The following examples illustrate preferred methods for preparing the 
compositions of the invention and utilizing these compositions to induce 
antibodies to Tat proteins of the virus in an immunized host. These 
examples are illustrative only and do not limit the scope of the 
invention. 
EXAMPLE 1 
IMMUNOLOGICAL STUDIES ON MINIMAL TAT PROTEIN AMINO ACID SEQUENCES NECESSARY 
FOR BINDING TO ANTIBODY FOR TWO B CELL EPITOPES IN HIV-1 TAT PROTEIN 
Two peptides were synthesized as described below, and these corresponded to 
amino acids 4-16 and 53-62 of SEQ ID NO: 1 illustrated in FIG. 1. The 
sequences are the most frequent sequence representations at these 
positions in 31 Tat protein sequences of the common B subtype reported in 
the NIAID HIV database. The sequences were chosen as putative immunogens. 
A. Peptide synthesis--immunizing peptides 
The two immunogens, amino acid sequences 
-Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser- SEQ ID NO: 28! and 
-Arg-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- SEQ ID NO: 29!, respectively, 
were synthesized by solid phase methodology on polypropylene pegs 
according to the methods of H. M. Geysen et al., J. Immunol. Meth., 
102:259 (1987), with an N-terminal cysteinyl being incorporated to 
facilitate coupling to a carrier protein. The N-terminus was left as a 
free amine and the C-terminus was amidated. 
Immunizing peptides were generally purified to greater than 95% purity by 
reverse phase HPLC, and purity was further confirmed by mass spectometry 
(MS). 
Immunizing peptides were covalently coupled to diphtheria toxoid (DT) 
carrier protein via the cysteinyl side chain by the method of A. C. J. Lee 
et al., Molec. Immunol., 17:749 (1980), using a ratio of 6-8 moles peptide 
per mole of diphtheria toxoid. 
B. Peptide synthesis--Detector peptides 
Peptides corresponding to the amino acid sequences of the two immunogen 
peptides were synthesized by the method of Geysen, cited above, for use in 
ELISA assays for detection of reactivity and cross-reactivity. Additional 
peptides with N- and C-terminal truncations were also synthesized. 
Detector peptides had an N-terminal -Ser-Gly-Ser-Gly- SEQ ID NO: 30! 
added, with biotinylation of the new N-terminus, and the C-terminal 
remained a free acid. These detector peptides had a purity exceeding 70% 
by mass spectometry and were not purified further. 
C. Immunization of rabbits 
The peptide conjugates were taken up in purified water and emulsified 1:1 
with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant 
(IFA) ANTIBODIES--A LABORATORY MANUAL, Eds. E. Harlow and P. Lane, Cold 
Spring Harbor Laboratory (1998)!. Total volume per rabbit was 1 ml, and 
this contained 100 .mu.g of peptide coupled to DT. 
Two rabbits were used for each immunizing peptide, with the initial 
intramuscular (IM) injection with conjugate in CFA and a subsequent IM 
boost at 2 weeks with conjugate in IFA. A pre-bleed was drawn before the 
first injection and larger bleeds were taken 3 and 5 weeks after the 
booster injection. 
D. ELISA determination of binding of antiserums to biotinylated peptides 
These assays were performed as described by H. M. Geysen et al., Proc. 
Natl. Acad. Sci. USA, 81:3998 (1983). Briefly, using Nunc Immuno 
Maxisorb.TM. 96 well plates, biotinylated peptides were bound to 
streptaviden coated plates and, with washing with phosphate buffered 
saline (PBS) between steps, successive incubations were performed with 
antiserum dilutions and horseradish peroxidase conjugated anti-rabbit 
immunoglobulin to detect bound antibody. Plated were developed with ABTS, 
with an O.D. reading at 405 nm. Absorbance greater than O.D. 1.0 was taken 
as positive and titers were determined from doubling dilutions of each 
antiserum. The geometric mean titer (GMT) was calculated for each 
antiserum pair for a given immunogen. 
E. Determination of antibody binding sequences 
ELISA results demonstrated that the antibodies to the first immunogen were 
reacting with the sequence -Asp-Pro-Arg-Leu-Glu-Pro AA 5-10 of SEQ ID NO: 
1! and that antibodies to the second immunogen were reacting with the 
sequence -Arg-Arg-Ala-Pro-Gln-Asp-Ser-AA 56-62 of SEQ ID NO: 1!. N- or 
C-terminal truncation of these sequences reduced the ELISA titer, so these 
hexapeptide and heptapeptide sequences constituted the antibody binding 
region and were termed HIV-1 Tat protein B cell epitope 1 and epitope 2, 
respectively. These results are summarized in Tables 1 and 2 below. From 
the results of Table 1, the minimal epitope 1 was 
-Asp-Pro-Arg-Leu-Glu-Pro- SEQ ID NO: 6!. From the results of Table 2, the 
minimal epitope 2 was -Arg-Arg-Ala-Pro-Gln-Asp-Ser- SEQ ID NO: 19!. 
TABLE 1 
__________________________________________________________________________ 
Antiserum to: 
Val-Asp-Pro-Arg-Leu-Glu- 
Pro-Trp-Lys-His-Pro- 
Gly-Ser-; GMT (% binding 
SEQ ID 
Detector Sequence* versus immunogen) 
NO 
__________________________________________________________________________ 
Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser- 
65,885 (100) 
28/31 
Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser- 
83,753 (127) 
32 
Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp 
96,627 (147) 
33 
Val-Asp-Pro-Arg-Leu-Glu-Glu-Pro- 
80,960 (123) 
34 
Val-Asp-Pro-Arg-Leu-Glu 32,016 (49) 35 
__________________________________________________________________________ 
*Substitution of Asp to Gly, Ala or His reduced titer to &lt;1% (See Example 
2) 
TABLE 2 
__________________________________________________________________________ 
Antiserum to: 
Arg-Gln-Arg-Arg- 
Arg-Ala-Pro-Gln- 
Asp-Ser; GMT (% binding) 
SEQ ID 
Detector Sequence versus immunogen) 
NO 
__________________________________________________________________________ 
Arg-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- 
34,305 (100) 29 
Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- 
32,119 (91) 36 
Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- 
32,029 (91) 37 
Arg-Arg-Ala-Pro-Gln-Asp-Ser- 
36,165 (102) 19 
Arg-Ala-Pro-Gln-Asp-Ser- 
10,357 (29) 38 
Arg-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp- 
2,440 (7) 39 
Arg-Gln-Arg-Arg-Arg-Ala-Pro-Gln- 
704 (2) 40 
__________________________________________________________________________ 
EXAMPLE 2 
SEQUENCE VARIATIONS IN EPITOPE 1 OF HIV-1 TAT PROTEIN AND IMMUNOLOGICAL 
CROSS-REACTIVITIES OF ANTISERUMS TO THESE SEQUENCES 
Variations in the sequence of Tat protein AA 5-10 of SEQ ID NO: 1 were 
analyzed in sequences available in HUMAN RETROVIRUSES and AIDS 1996, 
published by the Theoretical Biology and Biophysics Group of the Los 
Alamos National Laboratory, Los Alamos, N.Mex., and additional sequences 
kindly obtained from GenBank by Esther Guzman of the Los Alamos 
Laboratory. 
A. Variations in sequences 
399 aa 5-10 Tat hexapeptide sequences of the common B subtype of HIV-1 were 
obtained, as were 18 from the non-B subtypes (6 from subtype A, 2 from 
subtype C, 7 from subtype D, 2 from subtype F and 1 from subtype U). 
For the B subtype, 386 of the total 399 (97%) hexapeptides had either Arg 
(289, 74%), or Lys (45, 11%), or Ser (36, 9%) or Asn (16, 4%) in position 
3 as the only variation in the hexapeptides. The remaining variations (3%) 
comprised: 
-Gly-Pro-Arg-Leu-Glu-Pro-(4) SEQ ID NO: 11!, 
-Asp-Pro-Gly-Leu-Glu-Pro-(2) SEQ ID NO: 14!, and single examples of: 
-Asp-His-Arg-Leu-Glu-Pro- SEQ ID NO: 41!, 
-Ala-Pro-Arg-Leu-Glu-Pro- SEQ ID NO: 12!, 
-His-Pro-Arg-Leu-Glu-Pro- SEQ ID NO: 13!, 
-Asp-Pro-Arg-Ile-Glu-Pro- SEQ ID NO: 15!, 
-Asp-Pro-Arg-Leu-Gly-Pro- SEQ ID NO: 16!, 
-Asp-Pro-Arg-Leu-Glu-Ala- SEQ ID NO: 17! and 
-Asn-Pro-Ser-Leu-Glu-Pro- SEQ ID NO: 18!. 
For the 18 non-B subtype sequences, 2 had Arg, 1 had Lys, 2 had Ser and 9 
had Asn at position 3 of the hexapeptides aa5-10, and other variants were 
-Asp-Pro-Asn-Leu-Asp-Pro-(2) SEQ ID NO: 42! and single examples of 
-Asp-Pro-Asn-Ile-Glu-Pro- SEQ ID NO: 43! and 
-Asp-Pro-Asn-Leu-Glu-Ser- SEQ ID NO: 44!. 
B. Assessment of immunological reactivity and cross-reactivity of the four 
primary immunogens 
Immunizing and detector sequences were synthesized, as described in Example 
1, for the following sequences SEQ ID NOS: 28 and 45 through 47, 
respectively!: 
-Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser-, 
-Val-Asp-Pro-LYs-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser-, 
-Val-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser-, 
-Val-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser-. 
Rabbits were immunized and the antiserums were tested by ELISA, as 
described in Example 1, for reactivity and cross-reactivity. 
Self-reactivities are summarized in Table 3. 
TABLE 3 
______________________________________ 
SEQ ID 
Immunogen and detector sequence 
GMT NO 
______________________________________ 
--Val--Asp--Pro--Arg--Leu--Glu--Pro--Trp-- 
88,000 28 
Lys--His--Pro--Gly--Ser-- 
--Val--Asp--Pro--Lys--Leu--Glu--Pro--Trp-- 
132,000 45 
Lys--His--Pro--Gly--Ser-- 
--Val--Asp--Pro--Ser--Leu--Glu--Pro--Trp-- 
166,355 46 
Lys--His--Pro--Gly--Ser-- 
--Val--Asp--Pro--Asn--Leu--Glu--Pro--Trp-- 
173,097 47 
Lys--His--Pro--Gly--Ser-- 
______________________________________ 
Cross-reactivities between these primary immunogens are displayed in Table 
4. 
TABLE 4 
______________________________________ 
Antiserums to primary immunogens (figures 
denote % reactivity v. self-reactivity) 
Detectors Arg3 Lys3 Ser3 Asn3 
______________________________________ 
Arg3 100 49 3 4 
Lys3 24 100 6 5 
Ser3 11 16 100 15 
Asn3 11 22 10 100 
______________________________________ 
Tables 3 and 4 demonstrate that each variant is an effective immunogen, but 
in general there is only modest cross-reactivity between variants. This 
implies that optimal coverage would require inclusion of all four variants 
as immunogens in a primary composition as described above. 
C. Assessment of cross-reactivities of other variants. 
Detector peptides were made for all remaining epitope variants and tested 
for cross-reactivity with the antiserums to the appropriate position 3 
primary hexapeptide immunogen. The cross-reactivities versus 
self-reactivity with the appropriate position 3 primary immunogen are 
displayed in Table 5. 
TABLE 5 
______________________________________ 
Detector sequence % cross- SEQ ID 
(variations in epitope 1) 
reactivity 
NO 
______________________________________ 
Cross-reactive 
--Asp--His--Arg--Leu--Glu--Pro-- 
55 41 
--Asp--Pro--Asn--Ile--Glu--Pro-- 
70 43 
--Asp--Pro--Asn--Leu--Asp--Pro-- 
100 42 
--Asp--Pro--Asn--Leu--Glu--Ser-- 
78 44 
Non cross-reactive 
--Gly--Pro--Arg--Leu--Glu--Pro-- 
1 11 
--Ala--Pro--Arg--Leu--Glu--Pro-- 
1 12 
--His--Pro--Arg--Leu--Glu--Pro-- 
1 13 
--Asp--Pro--Gly--Leu--Glu--Pro-- 
1 14 
--Asp--Pro--Arg--Ile--Glu--Pro-- 
9 15 
--Asp--Pro--Arg--Leu--Gly--Pro-- 
10 16 
--Asp--Pro--Arg--Leu--Glu--Ala-- 
1 17 
--Asn--Pro--Ser--Leu--Glu--Pro-- 
10 18 
______________________________________ 
The results of Tables 3-5 indicates that immunization with the four primary 
immunogens would generate antibodies reactive with greater than 97% of 
HIV-1 Tat proteins of the common B subtype. Interestingly all 18 non-B 
subtypes in the databases had epitope 1 sequences reactive with antibodies 
to the primary immunogens. 
D. Immunogenicity of certain epitope 1 variants 
Immunizing and detecting peptides were synthesized for the following 2 
variant epitope 1 peptides, with immunizations and ELISA testing as in 
Example 1. The self titers (GMT) are displayed in Table 6. 
TABLE 6 
______________________________________ 
Detecting and Self-titer 
SEQ ID 
Immunizing Peptides (GMT) NO 
______________________________________ 
--Val--Asn--Pro--Ser--Leu-- 
94,919 48 
Glu--Pro--Trp--Lys--His-- 
Pro--Gly--Ser-- 
--Val--Asp--His--Arg--Leu-- 
72,686 49 
Glu--Pro--Trp--Lys--His-- 
Pro--Gly--Ser-- 
______________________________________ 
These data show that inclusion of rare variants along with the primary 
immunogens expands antibody coverage to such rare epitope variants. 
Immunization with the four primary epitope 1 sequences can induce high 
titer antibodies reactive with Tat proteins of &gt;97% of all HIV-1 strains. 
This coverage can be optionally extended with the inclusion of additional 
rare epitope 1 variant sequences in the immunizing composition. 
EXAMPLE 3 
SEQUENCE VARIATIONS IN EPITOPE 2 OF HIV-1 Tat PROTEIN AND IMMUNOLOGICAL 
CROSS-REACTIVITIES OF ANTISERUMS TO THESE SEQUENCES 
Variations in the sequence of Tat protein AA 56-72 were analyzed in 
sequences available in HUMAN RETROVIRUSES and AIDS 1996, published by the 
Theoretical Biology and Biophysics Group of the Los Alamos National 
Laboratory, Los Alamos, N.Mex., and additional sequences kindly obtained 
from GenBank by Esther Guzman of the Los Alamos Laboratory. 
A. Variations in epitope 2 sequences 
482 sequences of epitope 2 of the common B subtype of HIV-1 were available 
for analysis. The most frequent sequence found conformed to the formula 
-Arg-Arg-X-Pro-Gln-Y-Ser- SEQ ID NO: 10!, where X is Ala, Pro, Ser, or 
Gln, and Y is Asp, Asn, Gly or Ser. This sequence type, which appears 
immunologically cross-reactive (see below), was found in 292 of 482 (61%) 
of available Tat sequences. 
Other sequence variants occurred in lower incidence and these included: 
-Arg-Arg-Ala-Pro-Pro-Asp-Ser- (20, 4%) SEQ ID NO: 20!, 
-Arg-Arg-Ala-Pro-Pro-Asp-Asn- (21, 4%) SEQ ID NO: 50!, 
-Arg-Arg-Ala-His-Gln-Asp-Ser- (20, 4%) SEQ ID NO: 21!, 
-Arg-Arg-Ala-His-Gln-Asn-Ser- (17, 3.5%) SEQ ID NO: 22!, 
-Arg-Arg-Ala-Pro-Gln-Gly-Asn- (10, 2%) SEQ ID NO: 51!, 
-Arg-Gly-Ala-Pro-Gln-Asp-Ser- (9, 2%) SEQ ID NO: 25!, 
-Arg-Arg-Ala-Pro-Glu-Asp-Ser- (8, 2%) SEQ ID NO: 26!, 
-Arg-Arg-Ala-Ser-Gln-Asp-Ser- (8, 2%) SEQ ID NO: 27!, 
-Arg-Arg-Pro-Pro-Gln-Asp-Asn- (9, 2%) SEQ ID NO: 23!, and 
-Arg-Arg-Ala-Pro-Gln-Asp-Arg- (8, 2%) SEQ ID NO: 24!. 
Together these sequences account for 85% of epitope 2 variants, with the 
balance comprised of a large number of low incidence variations. 
Epitope 2 sequences were only available from 18 examples of HIV-1 non-B 
subtypes. Unlike epitope 1, they showed divergence from the B subtype 
sequences and optionally a larger number of sequences can be selected for 
inclusion into the composition of this invention, if additional non-B 
subtype epitope 2 sequences are determined and are desirable in an 
immunogenic composition of this invention. 
B. Assessment of immunological reactivity and cross reactivity of selected 
epitope 2 sequences 
Immunizing and detector sequences were synthesized, as described in Example 
1, for the following sequences: 
-Arg-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- SEQ ID NO: 29!, 
-Arg-Gln-Arg-Arg-Arg-Ala-His-Gln-Asn-Ser- SEQ ID NO: 52!, 
-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Asp-Ser- SEQ ID NO: 53!, 
-Gly-Arg-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- SEQ ID NO: 54!, 
-Gly-Arg-Arg-Ala-His-Gln-Asp-Ser-Gly- SEQ ID NO: 55!, 
-Gly-Arg-Arg-Ala-Pro-Pro-Asn-Ser-Gly- SEQ ID NO: 56!, 
-Gly-Arg-Arg-Ala-Pro-Pro-Asp-Ser-Gly- SEQ ID NO: 57!, and 
-Arg-Arg-Ala-Pro-Gln-Asp-Ser-Gln-Thr-His-Gln- SEQ ID NO: 58!. 
Rabbits were immunized and antiserums were tested by ELISA, as described in 
Example 1, for reactivity and cross-reactivity. 
Additional detector peptides were synthesized as follows: 
-Arg-Gln-Arg-Arg-Arg-Ala-Pro-Glu-Asp-Ser- SEQ ID NO: 83!, 
-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Gly-Ser- SEQ ID NO: 59!, 
-Arg-Gln-Arg-Arg-Gly-Pro-Pro-Gln-Gly-Ser- SEQ ID NO: 60!, 
-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Asn-Ser- SEQ ID NO: 61!, 
-Arg-Gln-Arg-Arg-Arg-Ser-Pro-Gln-Asp-Ser- SEQ ID NO: 62!, 
-Arg-Gln-Arg-Arg-Arg-Ser-Pro-Gln-Asn-Ser- SEQ ID NO: 63!, 
-Arg-Gln-Arg-Arg-Arg-Thr-Pro-Gln-Ser-Ser- SEQ ID NO: 64!, 
-Arg-Gln-Arg-Arg-Arg-Ala-His-Gln-Asp-Ser- SEQ ID NO: 65!, 
-Arg-Gln-Arg-Arg-Arg-Ala-Pro-Pro-Asp-Ser- SEQ ID NO: 66!, 
-Arg-Arg-Ala-Pro-Pro-Asp-Asn- SEQ ID NO: 50!, 
-Arg-Arg-Ala-Pro-Gln-Asp-Arg- SEQ ID NO: 24!, 
-Arg-Arg-Ala-Pro-Gln-Asp-Asn- SEQ ID NO: 67!, 
-Arg-Arg-Ala-Pro-Gln-Gly-Asn- SEQ ID NO: 51!, 
-Arg-Arg-Thr-Pro-Gln-Gly-Ser- SEQ ID NO: 68!, 
-Arg-Arg-Ala-Pro-Gln-Gly-Ser- SEQ ID NO: 69!, 
-Arg-Arg-Thr-Pro-Gln-Asp-Ser- SEQ ID NO: 70!, 
-Arg-Arg-Pro-Pro-Gln-Ser-Ser- SEQ ID NO: 71!, 
-Arg-Arg-Ala-Pro-Gln-Asn-Ser- SEQ ID NO: 72!, and 
-Arg-Arg-Ser-Pro-Gln-Asp-Ser- SEQ ID NO: 73!. 
The various antiserums and detector peptides were utilized to determine 
immunogenicity of the various sequences and the extent of immunological 
cross reactivity. 
The incidence and immunological reactivity of epitope 2 sequences of the 
formula -Arg-Arg-X-Pro-Gln-Y-Ser- SEQ ID NO: 10! (see above) are shown in 
Table 7. In the Table 7 below, percent cross-reactivity was measured with 
antiserum to Cys-Arg-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- SEQ ID NO: 74!, 
self titer=46,115. The results of Table 7 below demonstrate that 
immunization with -Arg-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- SEQ ID NO: 
29! should provide effective cross-reactivity with most of these variants, 
represented in 61% of HIV-1 strains. 
TABLE 7 
______________________________________ 
Incidence in 
% Cross- SEQ ID 
Epitope 2 sequence 
482 sequences 
reactivity 
NO 
______________________________________ 
Arg-Arg-Ala-Pro-Gln-Asp-Ser- 
93 100 19 
Arg-Arg-Pro-Pro-Gln-Asp-Ser- 
50 111 75 
Arg-Arg-Pro-Pro-Gln-Asn-Ser- 
41 96 76 
Arg-Arg-Pro-Pro-Gln-Gly-Ser- 
37 97 77 
Arg-Arg-Ser-Pro-Gln-Asp-Ser- 
19 93 73 
Arg-Arg-Thr-Pro-Gln-Gly-Ser- 
14 56 68 
Arg-Arg-Ala-Pro-Gln-Gly-Ser- 
9 87 69 
Arg-Arg-Thr-Pro-Gln-Asp-Ser- 
7 116 70 
Arg-Arg-Ala-Pro-Gln-Asn-Ser- 
5 128 72 
Arg-Arg-Ser-Pro-Gln-Asp-Ser- 
4 110 73 
Arg-Arg-Ser-Pro-Gln-Asn-Ser- 
2 142 78 
Arg-Arg-Ala-Pro-Gln-Ser-Ser- 
1 97 79 
Arg-Arg-Ser-Pro-Gln-Gly-Ser- 
1 78 80 
Arg-Arg-Thr-Pro-Gln-Asn-Ser- 
1 43 81 
292/482 (61%) 
______________________________________ 
Immunization with -Arg-Arg-Ala-Pro-Pro-Asp-Asn- SEQ ID NO: 50! and 
-Arg-Arg-Ala-Pro-Pro-Asp-Ser- SEQ ID NO: 20! yielded antibodies that 
cross-reacted with both detector peptides, as shown in Table 8. Thus, 
inclusion of either sequence in an immunizing composition of this 
invention would provide antibodies against Tat protein epitope 2 variants 
in a further 41/482 (8.5%) of HIV-1 strains. 
Immunization with -Arg-Gln-Arg-Arg-Arg-Ala-His-Gln-Asn-Ser- SEQ ID NO: 52! 
induced antibodies that gave a self titer of 209,286 and a 
cross-reactivity of 5,356 (2.5%) with -Arg-Arg-Ala-His-Gln-Asp-Ser- SEQ 
ID NO: 21!. Thus inclusion of this sequence in an immunogen would cover an 
additional 17/482 (3.5%) of HIV-1 strains. 
Thus, immunization with three epitope 2 variants, 
-Arg-Arg-Ala-Pro-Gln-Asp-Ser- SEQ ID NO: 19!, 
-Arg-Arg-Ala-Pro-Pro-Asp-Asn- SEQ ID NO: 50!, 
and 
-Arg-Arg-Ala-His-Gln-Asn-Ser- SEQ ID NO: 22!, could provide antibodies 
reactive with the Tat proteins of 73% of HIV-1 strains. 
TABLE 8 
__________________________________________________________________________ 
Detector peptides ELISA GMT 
Arg-Arg-Ala-Pro-Pro-Asp-Asn- 
Arg-Arg-Ala-Pro-Pro-Asp-Ser- 
Antiserum to: 
SEQ ID NO: 50! 
SEQ ID NO: 20! 
__________________________________________________________________________ 
Arg-Arg-Ala-Pro-Pro- 
11,056 12,230 
Asp-Asn- SEQ ID NO: 50! 
Arg-Arg-Ala-Pro-Pro- 
9,340 7,865 
Asp-Ser- SEQ ID NO: 20! 
__________________________________________________________________________ 
EXAMPLE 4 
CONSTRUCTION OF A SYNTHETIC GENE OF THE INVENTION 
A synthetic gene was constructed that incorporated in frame eight epitope 1 
variants (including the four primary immunogens of the invention) and 
thirteen epitope 2 variants, these constituting all the variant epitope 1 
and epitope 2 sequences found in the Tat protein sequences of 31 HIV-1 B 
subtype strains reported in the 1996 HUMAN RETROVIRUSES and AIDS 
compilation, published by the Theoretical Biology and Biophysics Group of 
the Los Alamos National Laboratory, Los Alamos, N.Mex. These included 
amino acids 4-16 for epitope 1 and 53-62 for epitope 2, using the 
numbering of SEQ ID NO: 1 illustrated in FIG. 1. The epitope sequences 
were separated by dipeptide spacers containing Gly and/or Ser residues. 
The sequence of this one exemplary gene of this invention is shown in FIGS. 
2A-2C SEQ ID NOS: 2 and 3!. The gene was assembled as described in W. P. 
C. Stemmer et al., Gene, 164:49 (1995). Briefly, eleven top strand 60-mer 
oligonucleotides (oligos) and eleven bottom strand oligos with 20 
nucleotide (nt) overlaps were synthesized along with two end 50-mers. The 
twenty-two 60-mers were incubated together under hybridizing conditions 
and polymerase chain reaction (PCR) was used to fill in the sequence and 
amplify it. The end 50-mers were then added and the assembly completed by 
PCR, with isolation of the full length gene on agarose gel. 
The gene was sequenced and found to have the correct sequence within the 
actual epitopes, with the exception of an Ala to Thr substitution at 
position 136 (see FIGS. 2A-2C). This was accepted since this change does 
not affect antibody binding of epitope 2 (see Example 3). 
This gene was then excised with restriction enzymes and inserted into the 
expression vector pBAD L-M. Guzman et al., J. Bacteriol, 177:4121 (1950)! 
containing, in frame, the sequence for green fluorescent protein (GFP) A. 
Crameri et al., Nature Biotech, 14:315 (1996)!. TG1 E coli were 
transfected and green-fluorescent colonies were isolated. 
The isolated colonies were grown and expression was induced. Protein from 
each of three colonies had fluorescent bands on Western blotting with the 
expected molecular size (i.e., twice that of GFP alone). The resulting 
protein was soluble and was purified by nickel column affinity 
purification utilizing a hexa-histidyl that had been incorporated in the 
sequence. 
Yield was approximately 1 mg protein per liter of supernatant after double 
affinity purification to yield &gt;90% purity. 
EXAMPLE 5 
IMMUNOLOGICAL CHARACTERIZATION OF THE RECOMBINANT FUSION PROTEIN EXPRESSING 
HIV-1 Tat PROTEIN EPITOPE VARIANTS 
A. Reactivity of fusion protein with rabbit antiserums to Epitope 1 and 2 
variants. 
Rabbit antiserums generated to synthetic peptides corresponding to the four 
primary epitope 1 sequences (see Example 2 and below) and four epitope 2 
sequences (see below) were tested by ELISA, using the methodology 
described in Example 1 except that the plates were initially directly 
coated with a 100 .mu.g/ml solution of the fusion protein described in 
Example 4 above. Table 9 summarizes the ELISA titers of these antiserums 
with the fusion protein. 
TABLE 9 
______________________________________ 
Titers on SEQ ID 
Antiserum to: fusion protein 
NO 
______________________________________ 
--Val--Asp--Pro--Arg--Leu-- 
&gt;&gt;8,000 28 
Glu--Pro--Trp--Lys--His-- 
Pro--Gly--Ser-- 
--Val--Asp--Pro--Asn--Leu-- 
&gt;&gt;8,000 47 
Glu--Pro--Trp--Lys--His-- 
Pro--Gly--Ser-- 
--Val--Asp--Pro--Lys--Leu-- 
&gt;&gt;8,000 45 
Glu--Pro--Trp--Lys--His-- 
Pro--Gly--Ser-- 
--Val--Asp--Pro--Ser--Leu-- 
&gt;8,000 46 
Glu--Pro--Trp--Lys--His-- 
Pro--Gly--Ser-- 
--Arg--Gln--Arg--Arg--Arg-- 
7,000 29 
Ala--Pro--Gln--Asp--Ser-- 
--Arg--Gln--Arg--Arg--Arg-- 
&gt;&gt;8,000 52 
Ala--His--Gln--Asn--Ser-- 
--Arg--Gln--Arg--Arg--Arg-- 
&gt;8,000 53 
Pro--Pro--Gln--Asp--Ser-- 
--Arg--Gln--Arg--Gln--Arg-- 
8,000 82 
Ala--Pro--Asp--Ser--Ser-- 
______________________________________ 
These data show that the variant epitope sequences, expressed as a linear 
recombinant fusion protein, are expressed in a conformation recognizable 
by antibodies to the corresponding synthetic peptides. 
B. Immunization of mice with the fusion protein 
Three mice were immunized with 10 .mu.g each of an aqueous solution of the 
fusion protein of Example 4 emulsified with an equal volume of Freund's 
complete adjuvant, given intraperitoneally. Two weeks later they were 
similarly boosted, except that Freund's incomplete adjuvant was used. 
Serums were obtained three weeks later. 
C. ELISA testing of antiserums to the fusion protein with synthetic 
peptides corresponding to the epitope variants incorporated in the fusion 
protein 
ELISA testing was performed as described in Example 1 except that 
horseradish peroxidase conjugated anti-mouse immunoglobulin was used to 
detect antibody binding. The results are summarized in Table 10 below. 
These data demonstrate that both epitope 1 and epitope 2 sequences are 
expressed in the linear fusion protein, and react with antibodies to the 
synthetic sequences (see above). Antibodies to epitope 1 were detectably 
induced by the recombinant fusion protein under the conditions of this 
experiment in mice. Thus recombinant linear expression is effective for 
induction of specific antibodies to epitope 1. The apparent failure of 
this experiment to induce antibodies to epitope 2 sequences is believed to 
be probably due to low immunogenicity in mice or some other experimental 
factor. It is anticipated that additional experiments with a more 
immunogenic fusion partner than GFP will demonstrate that the epitope 2 
sequences also induce antibody responses, and thus are useful components 
of the compositions of this invention. 
TABLE 10A 
______________________________________ 
Titer with antiserum 
SEQ ID 
Detector peptides to fusion protein 
NO 
______________________________________ 
--Val--Asp--Pro--Arg--Leu-- 
2218 28 
Glu--Pro--Trp--Lys--His-- 
Pro--Gly--Ser-- 
--Val--Asp--Pro--Asn--Leu-- 
3158 47 
Glu--Pro--Trp--Lys--His-- 
Pro--Gly--Ser-- 
--Val--Asp--Pro--Lys--Leu-- 
2440 45 
Glu--Pro--Trp--Lys--His-- 
Pro--Gly--Ser-- 
--Val--Asp--Pro--Ser--Leu-- 
3031 46 
Glu--Pro--Trp--Lys--His-- 
Pro--Gly--Ser-- 
--Val--Asn--Pro--Ser--Leu-- 
3718 48 
Glu--Pro--Trp--Lys--His-- 
Pro--Gly--Ser-- 
--Val--Asp--His--Arg--Leu-- 
3223 49 
Glu--Pro--Trp--Lys--His-- 
Pro--Gly--Ser-- 
--Arg--Gln--Arg--Arg--Arg-- 
Background 29 
Ala--Pro--Gln--Asp--Ser-- 
--Arg--Gln--Arg--Arg--Arg-- 
Background 52 
Ala--His--Gln--Asn--Ser-- 
--Arg--Gln--Arg--Arg--Arg-- 
Background 53 
Pro--Pro--Gln--Asp--Ser-- 
--Arg--Gln--Arg--Gln--Arg-- 
Background 82 
Ala--Pro--Asp--Ser--Ser-- 
--Arg--Gln--Arg--Arg--Arg-- 
Background 83 
Ala--Pro--Glu--Asp--Ser-- 
--Arg--Gln--Arg--Arg--Arg-- 
Background 59 
Pro--Pro--Gln--Gly--Ser-- 
______________________________________ 
TABLE 10B 
______________________________________ 
Titer with antiserum 
SEQ ID 
Detector peptides to fusion protein 
NO 
______________________________________ 
--Arg--Gln--Arg--Arg--Gly-- 
Background 60 
Pro--Pro--Gln--Gly--Ser-- 
--Arg--Gln--Arg--Arg--Arg-- 
Background 61 
Pro--Pro--Gln--Asn--Ser-- 
--Arg--Gln--Arg--Arg--Arg-- 
Background 62 
Ser--Pro--Gln--Asp--Ser-- 
--Arg--Gln--Arg--Arg--Arg-- 
Background 63 
Ser--Pro--Gln--Asn--Ser-- 
--Arg--Gln--Arg--Arg--Arg-- 
Background 64 
Thr--Pro--Gln--Ser--Ser-- 
--Arg--Gln--Arg--Arg--Arg-- 
Background 65 
Ala--His--Gln--Asp--Ser-- 
--Arg--Gln--Arg--Arg--Arg-- 
Background 66 
Ala--Pro--Pro--Asp--Ser-- 
______________________________________ 
EXAMPLE 6 
PRIMATE ANIMAL STUDY 
A study was conducted in ten juvenile male rhesus macaques to determine if 
the presence of antibodies to Tat protein, induced by a synthetic peptide 
of this invention prior to infection with immunodeficiency virus would 
attenuate infection and reduce levels of virus in plasma. HIV-1 does not 
infect monkeys, but a corresponding simian immunodeficiency virus (SIV) 
does. P. A. Luciw et al., Proc. Natl. Acad. Sci. USA, 92:7490 (1995) 
constructed an infectious recombinant virus (chimera) of SIV.sub.mac239 
and HIV-1.sub.SF33 that does infect monkeys, typically causing an acute 
viremia that peaks around 2 weeks and subsequently subsides by week 8. In 
this chimeric construct, termed SHIV.sub.SF33, the SIV nucleotides 
encoding tat, rev and env (gp160) of SIV.sub.mac239 have been replaced 
with the corresponding region of HIV-1.sub.SF33. 
A. Immunization of monkeys 
The monkeys were randomized into two groups. 
Each monkey of group 1 (control group) was immunized with 0.4 mg diphtheria 
toxoid (Commonwealth Scrum Laboratories, Victoria, Australia) with 0.25 mg 
threonyl muramyl dipeptide (T-MDP) in 0.5 ml water, this being emulsified 
with 0.5 ml MF75 adjuvant (Chiron Corp, Emeryville Calif.). 
Each monkey of group 2 (test group) was immunized with 0.1 mg of the 
synthetic peptide 
Cys-Val-Asp-Pro-Asn-Leu-Glu-Pro-Trp-His-Pro-Gly-Ser-amide SEQ ID NO: 84! 
coupled to 0.4 mg diphtheria toxoid A. C. Lee et al., Mol. Immunol., 
17:749 (1980)!. The conjugate was dissolved in 0.5 ml water containing 
0.25 mg T-MDP and emulsified with 0.5 ml MF75 adjuvant. 
Each monkey was immunized at day 0 and day 28 (week 4) with two 0.5 ml 
intramuscular injections at two distinct sites. The synthetic peptide 
immunogen contained the B cell epitope, -Asp-Pro-Asn-Leu-Glu-Pro- SEQ ID 
NO: 9! of the Tat protein of SF33 HIV-1 that is incorporated in the 
SHIV.sub.SF33 molecular clone that was used to challenge the monkeys (see 
above). 
B. Testing for antibodies to Tat protein 
At day 42 (week 6), 2 weeks after the booster injection, serums were drawn 
and tested by ELISA for binding to 
Ser-Gly-Ser-Gly-Val-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser-OH 
SEQ ID NO: 85!, as described above in Example 1. 
The control monkeys had background titers ranging from 25 to 44, while the 
test group had titers of 1788 to 9588, as shown in Table 11 below. 
TABLE 11 
______________________________________ 
CONTROL GROUP TEST GROUP 
Monkey # Titer Monkey # Titer 
______________________________________ 
18782 30 18759 1788 
18785 25 18789 5780 
18786 30 18790 2718 
18859 54 18863 4139 
18908 46 18945 9588 
GEOMETRIC MEAN 
35 4068 
TITER (GMT): 
______________________________________ 
C. Viral Challenge 
At day 49 (week 7) after initial immunization, all monkeys were given 1 ml 
of a 1/1000 dilution of animal titered SHIV.sub.SF33 stock intravenously 
(challenge day 0). This corresponded to 50 animal infectious doses.sub.50% 
(50 AID.sub.50) or 200 tissue culture infectious doses.sub.50% (200 
TCID.sub.50). 
D. Assessment of infection 
Plasma was drawn in EDTA at weeks 2, 4 and 8, and copies of viral RNA per 
ml of plasma were measured by QR-RT-PCR, using SIV probes for the SIV 
component of SHIV.sub.SF33 A. J. Conrad et al., J. Acq. Imm. Def. 
Syndrome and Hum. Retrovirol., 10:425 (1995)!. 
The results are summarized as follows in Table 12. 
TABLE 12 
______________________________________ 
SHIV RNA copies/ml plasma 
Monkey # 2 weeks 4 weeks 8 weeks 
______________________________________ 
CONTROL GROUP 
18782 880,000 30,000 &lt;500 
18785 610,000 80,000 &lt;500 
18786 500,000 50,000 &lt;500 
18859 22,000,000 120,000 &lt;500 
18908 20,000,000 100,000 1,000 
GEOMETRIC MEAN: 2,596,851 67,869 
TEST GROUP 
18759 920,000 60,000 &lt;500 
18789 950,000 50,000 &lt;500 
18790 390,000 17,000 &lt;500 
18863 2,000,000 27,000 &lt;500 
18945 330,000 65,000 500 
GEOMETRIC MEAN: 742,034 38,938 
INHIBITION, 71% 43% 
TEST VERSUS CONTROLS: 
______________________________________ 
As expected, SHIV.sub.SF33 caused an acute infection, with peak levels of 
viral RNA at 2 weeks and barely or non-detectable levels by week 8. 
Monkeys immunized with a synthetic peptide conjugate that induced 
antibodies to the Tat protein of the challenge SHIV.sub.SF33 virus had, by 
comparison with control immunized monkeys, a 71% reduction in peak virus 
levels in plasma 2 weeks after viral challenge, with a 43% inhibition 
being still detectable in the subsiding plasma viral levels at 4 weeks. 
This shows that SHIV multiplication in vivo was inhibited in the presence 
of antibodies to the Tat protein being utilized by the virus, and suggests 
that a similar effect would prevail in HIV infected humans. 
E. Assessment of seroconversion 
Subjects infected with HIV develop antibodies to virion surface proteins 
and this is detected by ELISA and used to diagnose infection. Monkey 
serums were tested prior to virus challenge and 8 weeks after challenge, 
using the HIVAB.RTM.HIV-1/HIV-2(rDNA)EIA (Abbott Labs, Illinois). All 
pre-challenge serums were negative and all 8 week post challenge serums 
were positive. These findings provide additional support for the fact that 
antibodies to Tat protein do not register in diagnostic assays for HIV 
seroconversion. 
Numerous modifications and variations of the present invention are included 
in the above-identified specification and are expected to be obvious to 
one of skill in the art. Such modifications and alterations to the 
compositions and processes of the present invention are believed to be 
encompassed in the scope of the claims appended hereto. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 85 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 72 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
MetGluProValAspProArgLeuGluProTrpLysHisProGlySer 
151015 
GlnProLysThrAlaCysThrAsnCysTyrCysLysLysCysCysPhe 
202530 
HisCysGlnValCysPheThrThrLysGlyLeuGlyIleSerTyrGly 
354045 
ArgLysLysArgArgGlnArgArgArgAlaProGlnAspSerGlnThr 
505560 
HisGlnValSerLeuSerLysGln 
6570 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 912 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: join(1..876, 883..912) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GAGCTCTACAAATCCGGGGATCCGGGTGAAGATCCGCGTTTAGAGCCG48 
GluLeuTyrLysSerGlyAspProGlyGluAspProArgLeuGluPro 
151015 
TGGAAACACCCGGGTTCTGGTTCTGTTGACCCTAACCTTGAACCTTGG96 
TrpLysHisProGlySerGlySerValAspProAsnLeuGluProTrp 
202530 
AAGCATCCTGGCAGCTCCGGAGTCGATCCCAAACTCGAGCCCTGGAAA144 
LysHisProGlySerSerGlyValAspProLysLeuGluProTrpLys 
354045 
CACCCCGGAAGTTCGGGGGTAGACCCATCTCTGGAACCATGGAAGCAT192 
HisProGlySerSerGlyValAspProSerLeuGluProTrpLysHis 
505560 
CCAGGGAGTGGTAGCGTGAATCCGTCATTAGAGCCGTGGAAACACCCG240 
ProGlySerGlySerValAsnProSerLeuGluProTrpLysHisPro 
65707580 
GGTTCATCTGGAGTTGATCCTCGCTTGGAACCTTGGGAGCATCCTGGT288 
GlySerSerGlyValAspProArgLeuGluProTrpGluHisProGly 
859095 
TCGTCCGGTGTAGACCCCCGACTTGAGCCCTGGAATCACCTCGGGAGT336 
SerSerGlyValAspProArgLeuGluProTrpAsnHisLeuGlySer 
100105110 
TCAGGCGTAGATCATCGGCTCGAACCATGGAAACATCCAGGTTCTGGA384 
SerGlyValAspHisArgLeuGluProTrpLysHisProGlySerGly 
115120125 
GATCTGCGCCAGCGGCGACGTACTCCTCAGGATTCTGGATCTCGACAA432 
AspLeuArgGlnArgArgArgThrProGlnAspSerGlySerArgGln 
130135140 
CGTCGGCGCCCTCCCCAAGACTCCTCAGGACGGCAGCGCCGACGACCC480 
ArgArgArgProProGlnAspSerSerGlyArgGlnArgArgArgPro 
145150155160 
CCACAGGGTTCAGGTTCACGTCAACGACGCGGTCCACCCCAAGGCTCG528 
ProGlnGlySerGlySerArgGlnArgArgGlyProProGlnGlySer 
165170175 
GGTTCGCGCCAGCGGCGACGTCCGCCTCAGAACTCTAGTGGACGACAA576 
GlySerArgGlnArgArgArgProProGlnAsnSerSerGlyArgGln 
180185190 
CGTCGGCGCTCTCCCCAAGATTCCGGCGGGCGGCAGCGCCGTCGATCA624 
ArgArgArgSerProGlnAspSerGlyGlyArgGlnArgArgArgSer 
195200205 
CCACAGAACTCAGGTGGGCGTCAACGACGCCGGACTCCGCAATCTTCA672 
ProGlnAsnSerGlyGlyArgGlnArgArgArgThrProGlnSerSer 
210215220 
TCCGGCCGCCAGCGGCGACGTGCCCATCAGAATAGCGGCAGCCGACAA720 
SerGlyArgGlnArgArgArgAlaHisGlnAsnSerGlySerArgGln 
225230235240 
CGTCGGCGCGCACACCAAGACAGCAGTGGGCGGCAGCGCCGTCGAGCG768 
ArgArgArgAlaHisGlnAspSerSerGlyArgGlnArgArgArgAla 
245250255 
CCTGAAGATAGTGGTTCTCGTCAACGACGCCGGGCTCCCCCTGACAGC816 
ProGluAspSerGlySerArgGlnArgArgArgAlaProProAspSer 
260265270 
TCCGGACGCCAGCGGCAACGTGCACCAGATAGTTCCTCAGGTCATCAC864 
SerGlyArgGlnArgGlnArgAlaProAspSerSerSerGlyHisHis 
275280285 
CACCATCATCACTAATAAGAATTCGGATCCTCTAGAGTCGACAAGCTT912 
HisHisHisHisGluPheGlySerSerArgValAspLysLeu 
290295300 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 302 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GluLeuTyrLysSerGlyAspProGlyGluAspProArgLeuGluPro 
151015 
TrpLysHisProGlySerGlySerValAspProAsnLeuGluProTrp 
202530 
LysHisProGlySerSerGlyValAspProLysLeuGluProTrpLys 
354045 
HisProGlySerSerGlyValAspProSerLeuGluProTrpLysHis 
505560 
ProGlySerGlySerValAsnProSerLeuGluProTrpLysHisPro 
65707580 
GlySerSerGlyValAspProArgLeuGluProTrpGluHisProGly 
859095 
SerSerGlyValAspProArgLeuGluProTrpAsnHisLeuGlySer 
100105110 
SerGlyValAspHisArgLeuGluProTrpLysHisProGlySerGly 
115120125 
AspLeuArgGlnArgArgArgThrProGlnAspSerGlySerArgGln 
130135140 
ArgArgArgProProGlnAspSerSerGlyArgGlnArgArgArgPro 
145150155160 
ProGlnGlySerGlySerArgGlnArgArgGlyProProGlnGlySer 
165170175 
GlySerArgGlnArgArgArgProProGlnAsnSerSerGlyArgGln 
180185190 
ArgArgArgSerProGlnAspSerGlyGlyArgGlnArgArgArgSer 
195200205 
ProGlnAsnSerGlyGlyArgGlnArgArgArgThrProGlnSerSer 
210215220 
SerGlyArgGlnArgArgArgAlaHisGlnAsnSerGlySerArgGln 
225230235240 
ArgArgArgAlaHisGlnAspSerSerGlyArgGlnArgArgArgAla 
245250255 
ProGluAspSerGlySerArgGlnArgArgArgAlaProProAspSer 
260265270 
SerGlyArgGlnArgGlnArgAlaProAspSerSerSerGlyHisHis 
275280285 
HisHisHisHisGluPheGlySerSerArgValAspLysLeu 
290295300 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
ArgLysLysArgArgGlnArgArgArg 
15 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GlyArgGlyAspSerPro 
15 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
AspProArgLeuGluPro 
15 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
AspProLysLeuGluPro 
15 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
AspProSerLeuGluPro 
15 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
AspProAsnLeuGluPro 
15 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Active-site 
(B) LOCATION: 3 
(D) OTHER INFORMATION: /note= "the amino acid in position 
3 may be Ala, Pro, Ser or Gln" 
(ix) FEATURE: 
(A) NAME/KEY: Active-site 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /note= "the amino acid in position 
6 may be Asp, Asn, Gly or Ser" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
ArgArgXaaProGlnXaaSer 
15 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
GlyProArgLeuGluPro 
15 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
AlaProArgLeuGluPro 
15 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
HisProArgLeuGluPro 
15 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
AspProGlyLeuGluPro 
15 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
AspProArgIleGluPro 
15 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
AspProArgLeuGlyPro 
15 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
AspProArgLeuGluAla 
15 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
AsnProSerLeuGluPro 
15 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
ArgArgAlaProGlnAspSer 
15 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
ArgArgAlaProProAspSer 
15 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
ArgArgAlaHisGlnAspSer 
15 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
ArgArgAlaHisGlnAsnSer 
15 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
ArgArgProProGlnAspAsn 
15 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
ArgArgAlaProGlnAspArg 
15 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
ArgGlyAlaProGlnAspSer 
15 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
ArgArgAlaProGluAspSer 
15 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
ArgArgAlaSerGlnAspSer 
15 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
ValAspProArgLeuGluProTrpLysHisProGlySer 
1510 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
ArgGlnArgArgArgAlaProGlnAspSer 
1510 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
SerGlySerGly 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
ValAspProArgLeuProTrpLysHisProGlySer 
1510 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
AspProArgLeuProTrpLysHisProGlySer 
1510 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
ValAspProArgLeuProTrp 
15 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
ValAspProArgLeuPro 
15 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
ValAspProArgLeu 
15 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
GlnArgArgArgAlaProGlnAspSer 
15 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
ArgArgArgAlaProGlnAspSer 
15 
(2) INFORMATION FOR SEQ ID NO:38: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
ArgAlaProGlnAspSer 
15 
(2) INFORMATION FOR SEQ ID NO:39: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
ArgGlnArgArgArgAlaProGlnAsp 
15 
(2) INFORMATION FOR SEQ ID NO:40: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
ArgGlnArgArgArgAlaProGln 
15 
(2) INFORMATION FOR SEQ ID NO:41: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
AspHisArgLeuGluPro 
15 
(2) INFORMATION FOR SEQ ID NO:42: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
AspProAsnLeuAspPro 
15 
(2) INFORMATION FOR SEQ ID NO:43: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
AspProAsnIleGluPro 
15 
(2) INFORMATION FOR SEQ ID NO:44: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
AspProAsnLeuGluSer 
15 
(2) INFORMATION FOR SEQ ID NO:45: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
ValAspProLysLeuGluProTrpLysHisProGlySer 
1510 
(2) INFORMATION FOR SEQ ID NO:46: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 
ValAspProSerLeuGluProTrpLysHisProGlySer 
1510 
(2) INFORMATION FOR SEQ ID NO:47: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: 
ValAspProAsnLeuGluProTrpLysHisProGlySer 
1510 
(2) INFORMATION FOR SEQ ID NO:48: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 
ValAsnProSerLeuGluProTrpLysHisProGlySer 
1510 
(2) INFORMATION FOR SEQ ID NO:49: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 
ValAspHisArgLeuGluProTrpLysHisProGlySer 
1510 
(2) INFORMATION FOR SEQ ID NO:50: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: 
ArgArgAlaProProAspAsn 
15 
(2) INFORMATION FOR SEQ ID NO:51: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: 
ArgArgAlaProGlnGlyAsn 
15 
(2) INFORMATION FOR SEQ ID NO:52: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: 
ArgGlnArgArgArgAlaHisGlnAsnSer 
1510 
(2) INFORMATION FOR SEQ ID NO:53: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: 
ArgGlnArgArgArgProProGlnAspSer 
1510 
(2) INFORMATION FOR SEQ ID NO:54: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: 
GlyArgGlnArgArgArgAlaProGlnAspSer 
1510 
(2) INFORMATION FOR SEQ ID NO:55: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: 
GlyArgArgAlaHisGlnAspSerGly 
15 
(2) INFORMATION FOR SEQ ID NO:56: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: 
GlyArgArgAlaProProAsnSerGly 
15 
(2) INFORMATION FOR SEQ ID NO:57: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: 
GlyArgArgAlaProProAsnSerGly 
15 
(2) INFORMATION FOR SEQ ID NO:58: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: 
ArgArgAlaProGlnAspSerGlnThrHisGln 
1510 
(2) INFORMATION FOR SEQ ID NO:59: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: 
ArgGlnArgArgArgProProGlnGlySer 
1510 
(2) INFORMATION FOR SEQ ID NO:60: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: 
ArgGlnArgArgGlyProProGlnGlySer 
1510 
(2) INFORMATION FOR SEQ ID NO:61: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: 
ArgGlnArgArgArgProProGlnAsnSer 
1510 
(2) INFORMATION FOR SEQ ID NO:62: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: 
ArgGlnArgArgArgSerProGlnAspSer 
1510 
(2) INFORMATION FOR SEQ ID NO:63: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: 
ArgGlnArgArgArgSerProGlnAsnSer 
1510 
(2) INFORMATION FOR SEQ ID NO:64: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: 
ArgGlnArgArgArgThrProGlnSerSer 
1510 
(2) INFORMATION FOR SEQ ID NO:65: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: 
ArgGlnArgArgArgAlaHisGlnAspSer 
1510 
(2) INFORMATION FOR SEQ ID NO:66: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: 
ArgGlnArgArgArgAlaProProAspSer 
1510 
(2) INFORMATION FOR SEQ ID NO:67: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: 
ArgArgAlaProGlnAspAsn 
15 
(2) INFORMATION FOR SEQ ID NO:68: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: 
ArgArgThrProGlnGlySer 
15 
(2) INFORMATION FOR SEQ ID NO:69: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: 
ArgArgAlaProGlnGlySer 
15 
(2) INFORMATION FOR SEQ ID NO:70: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: 
ArgArgThrProGlnAspSer 
15 
(2) INFORMATION FOR SEQ ID NO:71: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: 
ArgArgProProGlnSerSer 
15 
(2) INFORMATION FOR SEQ ID NO:72: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: 
ArgArgAlaProGlnAsnSer 
15 
(2) INFORMATION FOR SEQ ID NO:73: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: 
ArgArgSerProGlnAspSer 
15 
(2) INFORMATION FOR SEQ ID NO:74: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: 
CysArgGlnArgArgArgAlaProGlnAspSer 
1510 
(2) INFORMATION FOR SEQ ID NO:75: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75: 
ArgArgProProGlnAspSer 
15 
(2) INFORMATION FOR SEQ ID NO:76: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: 
ArgArgProProGlnAsnSer 
15 
(2) INFORMATION FOR SEQ ID NO:77: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: 
ArgArgProProGlnGlySer 
15 
(2) INFORMATION FOR SEQ ID NO:78: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: 
ArgArgSerProGlnAsnSer 
15 
(2) INFORMATION FOR SEQ ID NO:79: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: 
ArgArgAlaProGlnSerSer 
15 
(2) INFORMATION FOR SEQ ID NO:80: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80: 
ArgArgSerProGlnGlySer 
15 
(2) INFORMATION FOR SEQ ID NO:81: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: 
ArgArgThrProGlnAsnSer 
15 
(2) INFORMATION FOR SEQ ID NO:82: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: 
ArgGlnArgGlnArgAlaProAspSerSer 
1510 
(2) INFORMATION FOR SEQ ID NO:83: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: 
ArgGlnArgArgArgAlaProGluAspSer 
1510 
(2) INFORMATION FOR SEQ ID NO:84: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Binding-site 
(B) LOCATION: 13 
(D) OTHER INFORMATION: /note= "an amide is attached to the 
Ser in position 13" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: 
CysValAspProAsnLeuGluProTrpHisProGlySer 
1510 
(2) INFORMATION FOR SEQ ID NO:85: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Binding-site 
(B) LOCATION: 17 
(D) OTHER INFORMATION: /note= "OH is attached to the Ser 
in position 17" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85: 
SerGlySerGlyValAspProAsnLeuGluProTrpLysHisProGly 
151015 
Ser 
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