CA2190972A1 - Compositions of transactivating proteins of human immunodeficiency virus - Google Patents

Abstract

The inventions provides compositions and a novel method of immunization directed against released transactivating proteins of certain target viruses that are taken up by other cells, including particularly HIV and other integrating and chronically infecting viruses. The method employs TAT immunogens, which are capable of eliciting high titer antibody to the native TAT protein, especially the regions involved in cellular uptake.

Description

Wo 95/31999 P~ r -n77 21~972 .
COMPOSlTrONS OF TRANSACTIVATING PROTEINS OF HUMAN
IMMUNODEFICIENCY VIRUS

Field of the Tnvention The pre6ent invention relates generally to methods for immunizing humans or other mammals against infection by viruses that produce proteins that are released extr;~c~ rly and are taken up by other noninfected cells, preparing them for infection by the virus.
Backaround of the Invention Certain viruses possess genes which are involved in the expression and secretion of viral proteins which function to transfer susceptibility to infection by the virus to other non-infected host cells. One primary example of such a viral gene is the transactivator gene (tat) of the human; ~ ficiency virus (HIV), which is essential for viral replication. Similar transactivating genes are ~ound in other viruses such as human T cell ly ' _y Lotropic virus (HTLV) I and II, simian '-ficiency virus (SIV), and feline; ~-d-ficiency virus (FIV).
The tat gene of HIV is essential for viral replication; the protein it encodes (TAT) activates transcription of HIV through the HIV long t-rn;n~l repeat (LTR). The TAT protein also activates other host c~ r genes which contribute to cellular activation and sustenance of the virus. For example, there is evidence that the TAT protein activates host cell genes, such as bc1-2, c-myc, IL-6, TGF-B, and TNF [G. Zauli et al, Cancer Res., 53:4481-4485 (1993); J. Laurence et al, Proc. Natl. Acad. Sci.. USA, 88:7635-7639 (1991); G.
Scala et al, J. ~Y-. Med., 179:961-971 (1994); G. Zauli et al, Blood, 80:3036-3043 (1992); and L. Buonaguro et al, J. Virol., 66:7159-7167 (1992) ] .

WO 9~/31999 r~ C ~7 ~

TAT of HIV is also relea6ed extracellularly by infected cells and taken up by other non-infected cells [see, e.g., A. D. Frankel et al, 5~11, 55:1189-1193 (1988); G. Barillari et al, J. Immunol., 149:3727-3734 (1992); B. Ensoli et al, Nature, 345:84-86 (1990) ] .
Picomolar to nanomolar eYtracellular concentrations can be d ~Lated to affect the functions of certain cells.
Furthermore, TAT protein has been shown to selectively depress antigen induced T cell proliferation in vitro [R.
lo P. Viscidi et al, Science, 246:1606-1608 (1989) ] . This latter immunological Ahnnr--l ;ty has been ~ ~Lated in T cells from asymptomatic HIV infected 6ubjects early in the disease.
Uptake of TAT by cells i6 very strong, and appears to be mediated by a short basic sequence of the protein.
S. Fawell et al, Proc. Natl. ~cad. Sci.. USA, 91:664-668 (1994) identified this sequence as Tat 37-72 (CFT~KAT~ ;Y~-~KK~ Y~QC~S~,L~ vbLSKQ) ~SBQ ID No: 15]
and utilized it in fusion proteins to enable the uptake 20 of other proteins into the cells and, more specifically, into the nucleus . If taken up by cells latently inf ected with HIV, TAT protein would likely activate HIV
production. Ir taken up by uninfected cells, TAT would likely activate host genes that would render the cell 25 more receptive to fresh HIV infection, thus ~nAhl ;nq the eYpansion of HIV inf ection in the host .
The t~t gene and its protein have been sequenced and l~YAm;n~-l for involvement in proposed treatments of HIV.
U. S . Patent No . 5 ,158, 877 discloses synthetic DNA coding 3 0 l~or the TAT protein of HIV-l, and provides its cDNA
sequence. U. S. Patent No. 5,238,882 refers to a transformed yeast cell capable of expressing TAT for use in screening for agents which inhibit the function of the protein. U. S . Patent No . 5 ,110, 802 refers to an 35 antiviral agent capable of âttacking the first splice _ _ _ _ _ _ ~wo 95/31999 = = P~~ s/~77 .

acceptor site of tat gene. International Patent Application No. W092/07871, published May 14, 1992 refers to oligopeptide inhibitors o~ HIv r~plication, which operate by direct competition to prevent activation by 5 the TAT protein, while International Patent Application No. W091/10453, published July 25, 1991, refers to inhibition of TAT function by DNA sequences. See, also International Patent Application No. W091/09958, p--hl i sh~d July 11, 1991.
International Patent Application No. W087/02989, published May 21, 1987, provides an E. coli expression vector for producing TAT protein. International Patent Application No. W092/14755, published September 3, 1992, refers to in vitro blocking of TAT uptake by a selected 15 integrin cell surface receptor by fragments of TAT, r., Ls of integrin or antibodies to integrin. Also described in this publication is the i ; 7ation of rabbits with a TAT peptide and Complete Freund's adjuvant to generate antibodies to TAT for experimental work.
20 Despite the growing knowledge about the expression and proposed use of TAT in HIV therapies, there remains a need in the art for the devPl~ L of a sur-P~sful therapeutic or i ~ 7~tion regimen useful in humans (and where cl~L~ iate, other mammals) that utilizes active 25 i i 7ation with TAT protein or peptides in the treatment or prophylaxis of HIV, as well as for similar regimens ut; l i ~ i n~ other An~ 1 o~o~ essential extr2~ 1 Ar proteins of other viruses, or their ; ~ peptides .
ry of the Tnvention In one aspect, the invention provides an immunogen capable of eliciting a humoral and/or antibody ~ .De in a mammal to which the i - J - is administered, the immune response directed against a native extra~ l Ar W095131999 ` r.~ .cs~77 ~
~21gOg72 i~

transactivating protein from a selected chronic viru6, such as HIV. In one ~mho~ the; l~gcn comprises an ~IV TAT protein sequence or peptide fragment thereof including the sequence involved in c~ l Ar uptake of the 5 TAT. Hereafter this latter sequence will be referred to as the "cellular uptake region" t in HIV ~AT, this region spans about AA4 6 to about AA63 of SEQ ID N0: 2 ] as defined by S. Fawell et al, cited above. The; , n itself will be referred to as the TAT; -~ . In 10 other Pmho~; Ls~ the; -, comprises analogous ~;equences of similar extrAc~ r proteins for other viruses, e.g., SIV, I~TLV, and the like. Preferably, this JLnic protein or peptide sequence has the charzlcteristic of inactivated biological function vis-a-15 vi8 the intact TAT protein.
In another aspect, the invention provides a DNAse~u~lce cnl-o~lin~ the TAT; J described above for use as a ' naked DNA ' composition capable of eliciting ~n immune ~e~u,~se in an animal, preferably a mammal, to 20 which it is administered.
In still a further aspect, the invention provides a DNA molecule containing regulatory sequences which control the replication and expression of the inserted DNA 8~ nc o~l; n~ the ~IIV TAT immunogen. Such 25 regulatory sequences may direct the expression of the TAT
o~n in cell cultures for ~L _ h; n~nt production and ~-nir-1l~tion of the DNA. Alternatively, such a vector may also be administered as an i ;_nic CompositiOn, such as a vaccine, for expression of the TAT ;~ o~J. ~
30 vivo in a host mammal and the elicitation of an immune response thereto.
In still a further aspect, the inventiûn provides , n;c compositions, such as vaccine compositions, useful for producing an immune response to, and for 35 i ;~;ng a patient against infection with, a virus ~WO 95/31999 2 ~ g O 9 7 2 P.~ C~077 - . , ~ , . .
.. .

characterized by a transactivating protein or analogous extr~cF-1 1t11Ar protein. One ~mhoA; L of such an ,I:lliC composition comprises the protein or peptide immunogen described herein in a suitable pharmaceutical 5 carrier . Another embodiment of the ; J i c composition comprises a DNA sequence capable of expressing the i - J~ll in vivo upon direct administration of the DNA into a host animal. In still another ~ ; r-nt of such an ; , i c composition, 10 the DNA Cpql~Dnre c~nrotl i n~ the ; ~.. is present in a vector with associated regulatory sequences.
Still a further aspect of this invention is a method for producing an immune response, such as a protective immune response, in a patient against infection with a 15 virus characterized by a transactivating or itn 1lcgo-lc extracellular protein. The method comprises administering to said patient an HIV 'rAT; J or tat nucleic acid sequence as described above in an amount sufficient to evoke production of high titer antibodies 20 capable of specifically binding the native HIV TAT
protein secreted by said virus. Preferably the antibodies would bind to the r~l llll~r uptake region of the 'rAT protein.
In yet a further aspect, the invention contemplates 25 the production of ~n;t1Oslollc methods and compositions designed similarly for other proteins which function in a manner similar to that of the TA~ protein of HIV, ;nrlllA;n~ proteins of other viruses. Thus this method and compositions are anticipated to be useful in the 30 prophylaxis of various chronic viruses including, HIV-1, HTLV-I, HTLV-II, HIV-2, SIV, and FIV.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred: ~o~; Ls thereof.

wo 95/31999 = = ~ c ~77 ~
2190~2 ief Descrii~tion of the Drawinqs Fig. 1 illustrates the cDNA sequence [SEQ ID NO: 1]
and amino acid sequence [SEQ ID NO: 2] of the HIV-1 TAT
protein .
Fig. 2 i6 a graph illustrating the purification of the Ii2i lAhpl l~d SIV-TyrO TAT~590 seqUenCe using reverse phase HPLC . The purif ied label will be used to detect the presence of an~;ho~lipc to TAT following; ; ~ntion.
See, Example 2 below.
DetP~;led De~ccri~tion of the Invention The present invention provides novel ; J~A~C I
,unic compositions, vaccine compositions, and methods of eliciting an immune response in mammals, particularly humans, against viruses that produce proteins that are released extrArP~ Arly, which proteins are in turn taken up by other uninf ected, or latently infected, cells, thereby rendering the uninfected cells susceptible to viral infection. One such viral protein capable of facilitating infectivity from one cell to another uninfected cell is the transactivating TAT protein of HIV (and in the monkey virus , SIV); other such proteins exist in other viruses .
For ease of discussion, the following description relates to the TAT proteins of HIV ana SIV as specific examples of extrA~ ll Ar proteins which perform the abuv. described role of p-:Ly~l-uc~ting viral infection.
However, it should be understood by one of skill in the art that analogous extrA~elllllAr proteins n~cPccAry for the development and spread of the viral infection in HIV, SIV, and other viruses may be ~-n1r-lAted similarly to provide o~her i ngPn; c compositions and methods ~n~ _-C,CP~9 by this invention.
While not wishing to be bound by theory, the inventors have tiPtPrminP~ that the novel method of this ~WO 95131999 2 1 9 0 9 7 2 P ~ n77 invention and the related compositions herein described permit the elicitation of an immune response in a mammal directed against these extraG~ ]l sr or transactivating - proteins. Because infection is p~:L~Ludted either by activation of latently infected cells or by activation of host genes of uninfected cells which render the cells susceptible to viral infection, the -- sni~m underlying this invention is significant.
An important feature of the invention and an advantage over other therapeutic or vaccinal compositions useful for the treatment of viral infections, such as HIV, is that this invention elicits and directs an i 7e~ host ~ s immune response against the extr.9r~ 1.9r TAT protein. Active i i 7ation against a selected virus , e . g ., HIV-l , may be accomplished by eliciting high titer antibodies that will complex with the native secreted TAT protein pl uduced by the viru6 and prevent its uptake by cells. That is, once TAT protein has been expressed by the virus in an infected cell and is separated from the virus itself and is secreted from the infected cell in which it was produced, the immune response produced by the methods and compositions of this invention interdicts the TAT protein extrsc~ l srly bef ore it can be taken up by another cell .
This i i-7stion has a particularly desirable advantage in contrast to other treatments and prophylactic methods employed against such viruses.
Because the i i 7ation i5 not directed against the virus itself, interdiction of the TAT protein 3 o extracellularly does not create a selective pressure on the parent virus itself, which would encourage the dev~ , t of mutant virus variants producing a TAT
protein that is not recognised by the induced antibodies.
EIowever, any viral strain which is not producing the same TAT protein, or an immunologically cross-reactive Wo 95/31999 PCT/US95/06077 219~972 ~

protein, will be unaffected by the u6e of the i ,_.-ic compo6itions of this invention. In this ca~;e treatment with another i j n; c composition reactive with that TAT protein and prepared according to thi6 invention is 5 contemplated. Additionally, it is anticipated that blocking the incc ~ tion of TA~ protein by the patient ' s cells may also reduce the level of viremia.
A. Protein/Peptide T , - of the Invention One pnlhotl~ L of this invention involves the development oi~ a selected TAT; , . By '~ ullullO~
is meant any molecule which elicits an immune response, either cPl1l-l~r or humoral, in an animal expbsed to that molecule i~n vivo. An; , of this invention is 15 desirably an HIV TAT protein or peptide frngment thereof, which comprises cPl 1~I1Ar uptake region of the TAT protein or fragments thereof. T~e cellular uptake region is that region which mediates the binding of TAT protein to the cell to be infected. One variant of the HIV TAT protein 20 is illustrated in SEQ ID NO: 2 and in Fig. l; the cPlllll~r uptake region is identified as spanning amino acids 46 to 63 of that sequence :iy~iK~KKK7KKKAPZGSQ of SEQ
ID NO: 2. Preferably, the cell~ll?r uptake region i n~ P~ a smaller frAI; L, such as amino acids 46 25 through 59 thereof. However it is known that other variants of HIV exist which differ from that sequence at one or more amino acids. Thus, the cellular uptake region as def ined herein can include all or a portion of the sequence identified above, as well as variants 30 thereof.
Preferably, the TAT protein or peptide fragment useful as the i ~ is biologically inactive; i.e., it does not share the hl nl~ function of the intact TAT protein. The i - ~An may be made inactive by 35 deletion of amino acids at the amino tPrminll~ or carboxy ~YO 9~/31999 2 1 9 0 9 7 2 F~IIU~. 5.'C 6'?77 ' ~ ) I;; ;

tPrmimlq, or the deletion or replA~ of native Cysteine residues to interfere with naturally-occurring disulf ide bonds in the protein . One , ` ~ ' i - L of a TAT
- J ~n may consist of a l'AT protein characterized by 5 deletion of at least 3 or more amino acids at the amino tPrmi n-lq .
Additional ~mhorl; S of a TAT protein i include a peptide fragment from a selected region of TAT
protein, which peptide fragment alone is biologically 10 inactive, and may be optionally coupled to a carrier. A
desired size for such an immunogenic peptide may be between about 12 to about 22 amino acids. However, other sizes may be desired, dPpPn~lin~ on the peptide construct, i.e., for a multiple antigenic peptide talso referred to 15 as an octameric lysine core peptide) as described in detail below, the peptide may desirably be about 20 amino acids in length.
A selected region of the TAI' i -, I of this invention may be a ~u~lSeL ~t d rcgion or an i - 3 i ~
20 region of ~AT protein. Preferably, this peptide sequence inrl-lAPI: the relll~lAr uptake region. For example, where the virus selected for use in this invention is HIV-1, the selected ~AT i -, I may include, among other sequences, the following peptide sequences and 25variants:
syl;KKKK,~kKAAPQGSQ [SEQ ID NO: 3]
~iyCiKKKKUQ~kKAP [SEQ ID NO: 4]
:jy~iKKKK~ Kk~AP [SEQ ID NO: 5]
:jy~iKKKKKr,~l~kKAP [SEQ ID NO: 6]
tiyr~cK~p tSEQ ID NO: 7]
SYI.KKKKKQKQRAP [SEQ ID NO: 8]
SY~;KKKKK~KKCAP [SEQ ID NO: 9]
SY~.~KRRRQ17--K~rP tSEQ ID NO: 10]
SYr;KKKKKQkKK~ [SEQ ID NO: 11]
SYc;~KK~s~AH. [SEQ ID NO: 12]
_ _ . _ . . _ _ _ _ . . . . _ WO 95131999 219 0 9 7 i PCT/US95/06077 Larger peptide6 incorporating the above-identif ied se5~uences of amino acids of TAT protein of HIV or smaller fragments and fragments thereof may al60 be employed to form the TAT; ,_.. of this invention. For example, smaller fragmènts of the cel~ll1Ar uptake region, such as QRRRAP [SEQ ID NO: 16] and its variants or GRKKRRQ [SEQ
ID NO: 17 ] and its variants may also be employed alone or repeated in one peptide in multiple antigenic peptides.
Similarly, where the virus is other than HIV-1, e.g., a non-human virus, such as SIV, one of skill in the art is t~ to be able to select analogous regions of the transactivating or analogous extracellular protein of the virus for use as an; ~, in accordance with this invention. For example, where the virus selected for use in this invention is SIV, the selected TAT protein region may include, among other sequences, the cellular uptake region occurring at about amino acids 79 through about amino acids lOO of the SIV TAT protein, as well as the following peptide seguences and variants

2 0 ~hL~'L~ANTSSAsy [ SEQ ID NO :13 ] which spans the SIV
TA~ sequence from amino acids 80 through 95; and YEQS2~KhL~L~ANTSSAS [SEQ ID NO:14], which spans SIV
TAT7695. Other variants in size and amino acid composition may al50 be employed in this invention, such as the peptide motifs PKKTK {SEQ ID NO: 18], PKK~K [SEQ
ID NO: 19] and QRRRTP [SEQ ID NO: 20] alone or repeated in a 6ingle peptide, desirably in multiple antigenic peptides as disclosed below.
In a pref erred ~ '; L, th~ immunogen of the invention is in the form of a multiple antigenic peptide construct comprising a plurality of peptides from a TAT
protein. For example, such a cv--~,LLll~ L may be designed employing the multiple antigenic peptide system described by Tam, Proc. Natl. Acad. Sci. USA, 85:5409-5413 (1988).
This system makes use of a core matrix of lysine residues _ _ _ _ _ _ _ _ ~WO 95/31999 ~ J .,5 î ;~77 onto which multiple copies of an ; , i c peptide are synthesized tD. Posnett et al, J. BiQl. Chem.
~(4) :1719-1725 t1988); J. Tam, ~lrhp~;rAlly DefinedSynthetic T - y~llS and Vaccines by the Multiple 5 Antigen Peptide Approach", vacr-; nP RpcQArch An~
DQvel -ntc, Vol. 1, ed. W. Koff and H. Six, pp. 51-87 (Marcel Deblau, Inc., New York 1992) ] . These ~:v~;YLLIlvLs are rlpci~3npd for use in the present invention, as described in detail in Example 1 below. Other 10 conventional protein/peptide i -, n; c- constructs may be designed by resort to known techniques.
In addition to the amino acid sequences of the specifically-recited HIV-1 TAT proteins described herein, the invention al50 ~Yn~ ccpc; _ ~ ~LepaL~d from 15 other amino acid sequences of viral transactivating or analogous extrarQlllllAr proteins. For example, selected TAT peptides may be developed from allelic or engineered variations of viral genes or gene fragments Pnro~l;nt3 the particular l'AT protein. The ; - 3_ns employed in this 20 invention may also have amino acid se~uences which are analogc or derivatives of TAT or related extrarc~ r protein sequences of a selected virus.
The; ~ns of this invention may also be prepared to reflect variations in the native DNA and 25amino acid 6Pqupnrpc of selected ~AT proteins. For example, such ; , - may typically include ~AT
protein analogs, and the DNA Pnro~l;ng them, that differ by only 1 to about 4 codon changes. Other examples of analogs include polypeptides with minor amino acid 30 variations from the natural amino acid s~qUPnre of TAT
proteins, in particular, conservative amino acid repl AC Ls.
Conservative replA~ LY are those that take place within a f amily of amino acids that are related in their 35 side chains. Genetically encoded amino acids are . . _ . _ _ _ _ _ _ .

WO ss/3~999 ~ ~ -~- r~ r~o77 ~
21~ 0~ 7 ~

generally divided into four families: (1) acidic =
aspartic acid, glutamic acid; (2) basic = lysine, ~rginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenyl ;1 l An; n~ ~ meth; rm; n~
5 tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Pheny3AlAn~n~, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
For example, it is reasonable to expect that an isolated 10 replA~ ~ of a leucine with an isoleucine or valine, an aspartate with a glutamatic acid, a threonine with a serine, or a similar conservative repl ~ ~ of an amino acid with a structurally related amino acid will not have a significant effect on peptide ~L~ ul~LuLe or activity.
Pref erably, the TA~r protein/peptide ; ~ s of this invention are prepared by rhem; C;~l synthesis techniques, such as described by Merrifield, J. Amer.
~hPm. Soc., 85:2149-2154 (1963), and including the multiple antigenic techniques described above.
20 Alternatively, they may be prepared by known recombinant DNA techniques by cloning and expressing within a host microorganism or cell a DNA rL, - t carrying a coding sequence for the s~leetetl TAT i ~,'~_.1. Coding sequences for the TAT protein fragments can be y~ e~aLed 25 synthetically or can be derived from viral RNA by known techniques, or from available cDNA-containing plasmids.
Systems for cloning and expressing the TAT j in various microorganisms and cells, including, for example, E. ~P~i, Bacillus, Stre~tomYCes. Saccharomvces,

3 o ~ n ~ yeast and insect cells, and 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 1~ ;In cell such as Chinese Hamster ovary cells (CIIO) 35 or COS-l cells. These hosts may be used in connection _ _ . . _ . _ . . _ _ . . _ _ _ _ _ _ _ WO 95/31999 r~ s.~ ~Q77 21 g 0,,~17 i~

with poxvirus vectors, such as vaccinia or swinepox. The selection of other suitable host cells and methods for transf ormation, culture, amplif ication, 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, ~, 293:620-625 (1981).
Another preferred system; nr] lldQc the baculovirus expression system and vectors. Bacterial expression may also be desired.
When produced by conventional recombinant means, the TAT immunogens may be isolated either from the cellular contents by conventional lysis t~-hniql~Pc or from cell medium by conventional methods, such as chromatography.
See, e.g., Sambrook et al, MQlPr~llAr ~lrnin~
T~h,,ratorv MAnl-~l., 2d Edit., Cold Spring Harbor Laboratory, New York (1989).
The resulting TAl' protein or peptide i ~PnC, or multiple antigenic peptides may be screened for efficacy as a i -~n by in vivo assays, employing; i 7ation of an animal , e . g., a 6imian, with the ~AT protein or fragment, and evaluation of titer6 of antibody to the native TAT protein of the selected virus. Examples 3 and

4 d;c~loce 6uitable as6ay 6y6tem6 for such evaluation.
B . ' Naked DNA ' Compositions of the Invention Still another aspect of this invention involves nucleic acid seguences which encodes the TAT protein J. described above. The nucleic acid sequences, - 30 such as that described as SEQ ID N0: 1 or allelic variants thereof, or DNA 6equence6 containing certain preference codons or the species of the indicated patient preferably in the form of DNA, may be u6ed a6 60-called ' naked DNA ' to expre66 the protein/peptide ; ~ n in vivo in a patient. See, e.g., J. Cohen, WO9S/31999 r~ 6~77 ~
~ ~ 9 0 g 7 ~

Science, 259:1691-1692 (March l9, 1993); E. Fynan et al, Proc. Natl . Acad. Sci., USA, 90 :11478-11482 (Dec.
1993) ;and J. A. Wolff et al, Biotechniaues. 11:474_485 (l99l), all incorporated by reference herein.
The DNA 6eauences encoding the selected ~r~
immunogen of this invention may also be employed in a plasmid for either direct injection into a host animal or in a vector under 511; t lhl e regulatory sequences f or expression of the protein in vitro. The latter form of lO expression is conventional (see, Sambrook et al, cited Above and the ref erences above to production of the protein). The fsrmer type of plasmid for in vivo expression of the i ~, n may be designed as described in the references above or, e.g., as described in International Patent Application PCT WO94/01139, pl]hl;~hed January 20, 1994. Briefly, the DNA encoding the TAT protein or desired r- L thereof is inserted into a nucleic acid cassette. This cassette is engineered to contain, in addition to the tat sequence to be ~Le:ssed, other optional flanking sequences which enable its insertion into a vector. This cassette is then inserted into an appropriate DNA vector downstream of a promoter, an mRNA leader sequence, an initiation site and other regulatory sequences capable of directing the replication and expression of that sequence in vivo.
This vector permits infection of patient's cells and expression of the TAT i , n in vivo.
C. Immunogenic Compositions of the Invention Thus, the present invention provides; , 1c compositions containing a TAT protein or peptide 3en of the invention or the 'naked DNA' of the invention . These ; , ~ ; c compositions are capable of eliciting in an; ;7ed host mammal, e.g., a human, an ~VO9~!i/31999 r~ l~O... ~n7~
15 2 ~ ~ O ~ 7 2 immune response capable of interdicting extracellular TAT
protein .
In one ~ , such ; , ; c compositions contain at least one such protein/peptide;

5 directed to a selected native TAT protein according to the invention or a fLCII_ L thereof, together with a carrier suitable for administration as a composition for prophylactic treatment of virus infections.
Alternatively, the compositions may contain more than one 10 ; - ,~n, the amino acid sequences of the; J
differing to ~- ,-C5 allelic variants of the TAT
protein. For cxample, a multiple antigenic peptide of the invention may contain repeats of the same peptide, e.g., for HIV, RMAP [SEQ ID N0: 21]. The composition 15 may preferably contain a numbcr of l~APs, each bearing copies of different TAT peptides as described herein.
Further, where the oc~uLL-~ e of more than one strain of a particular virus is ~iuspected, which strain produces a different TA~ protein, novel TAT; , -20 may be ~Pc;~n~d by resort to this disclosure. Forexample, the HIV strain of a patient not responsive to a composition of this invention may be isolated and the TAT
protein sequence ~ rm;n~d by conventional methods, e. g., polymerase chain reaction. If the sequence is 25 indeed different from that ~ey~.el,ce which wa6 employed in the first composition, another TAT; -, - may be obtained and administered with the f irst ; , in a single; J -;~ composition. Thus, an; 3_.~ic or vaccine composition of this invention may contain several 30 different TAT; , - of this invention, each directed to a different TAT protein produced as described herein.
The TAT; ~, ~s) of the invention or LL s thereof can be employed in a vaccine composition containing a carrier, such as saline, and a selected 35 adjuvant, such as aqueous 5uspen5ion5 of aluminum and WO 95/31999 p~~ ~r~ n77 219~972 magnesium hydroxides, liposomes and others. Such compositions may optimally contain other conventionalvaccine ~ ~-n~ntS. Other suitable carriers and adjuvants are known to the art and the selection 5 thereof is expected to be routine. The preparation of a rhAr~ tically acceptable vaccine composition, having appropriate pH isotonicity, stability and other conventional chzlracteristics is within the skill of the ~rt .
These immunogenic or vaccine compositions according to the present invention can be administered by an appropriate route , e . g ., by the oral , intranasal , subcutaneous, intravenous, intraperitoneal or i~lLl ~r~ r routes. The presently preferred route of 15 administration is il~LL qC~ r.
The amount of the TAT i - j n of the invention present in each vaccine dose is selected with regard to c~nc;cloration of the patient's age, weight, sex, general physical condition and the like. The amount required to 2 0 induce an immune response, pref erably a protective response, in the patient without significant adverse side effects may vary ~rl~n~inq upon the; , employed and the optional presence of an ad~uvant. Generally, it is expected that each dose will comprise between about 50 25 ~g to about 1 mg of TAT protein/peptide i ~oq~ll per mL
of a sterile solution . A more pref erred dosage may be about 200 ,~g of TAT i , I of this invention. Other dosage range5 may also be contemplated by one of skill in the art. Initial doses may be optionally followed by 30 repeated boosts, where desirable.
~ lternatively, ~ nic compositions of this invention may be designed f or dirsct administration of ' naked DNA ' ~nro~l i nq one or more TAT i og~nC of this invention. As ~licCllc5~tl above with reference to the 35 recited art on the administration of DNA vaccines, it is ... . . _ . . .. _ _ . .. .. _ . .. . . _ _ . _ _ _ ~A10 9~/31999 ~ ,C 11 219.Q972 anticipated that nucleic acid seguences ~nror1; n~ one or more desired TAT; ogen~: of this invention may be directly delivered into the patient, alone or as part of a plasmid. Suitable vehicles for direct DNA
administration include, without limitation, saline, or sucrose, protamine, polybrene, polylygine, polycations, proteins, CaP04 or spermidine. See e.g, PCT application W094/01139 and the references cited above. A6 with the protein immunogenic compositions, the amounts of _ ~rl~ntS in the ' naked DNA ' composition and the mode of administration, e.g., injection or intranasal, may be selected and adjusted by one of skill in the art.
Thus, the i J ; r. compositions of this invention are designed to prevent infection by the selected virus of an uninfected mammal, e.g., human. Such; ~ ic compositions thus have utility as vaccines.
D. Therapeutic Uses of the Invention It is also further anticipated that the compositions of this invention may also be employed therapeutically to treat already infected patients. The r- ' on;~- of the present invention as described above may be useful in ^~;n~ 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. In conjunction with other therapeutic regimens for HIV infected patients, for example, the compositions - 3 o of the present invention are anticipated to assist in the reduction of viremia and prevention of clinical deterioration. For such therapeutic uses, the f ormulations and modes of administration are substantially identical to tho5e described specif ically 35 above and may be administered u~ uLL~ ly or wo 95/31999 . ~ x.,_ r ,q77 219~9-72 simult~n~ollsly with other conventional therapeutics for the specific viral infection. For therapeutic use, repeated dosages may be desirable.
5 E. Other Ut; l ;t;-~ of the TAT T J ' of this Invention As with other peptides, proteins, and nucleic acid setauences in general, the TAT ; - ~ S of this invention may have a number of other utilities. For 10 example, the TAT peptide or protein; , n5 of thi6 invention may be employed in conventional assays , e . g ., Western blots, ELISA, and other protein binding assays, for use in identifying antibodies which are capable of detecting HIV strains with different extr~cnl l~ r TAT
15 proteins.
Similarly, the DNA seyut:llces ~nrofl~n~ the TAT
3 n5 or complementary strands thereto may be used in nucleic acid assays, such as Northern and Southern blots, and other nucleic acid hybridization assays for 20 the same purpose. The nucleic acid sequences ~nro-l;nrJ
the TAT; ~ ~ ~ of the invention may be employed as polymerase chain reaction 5~ nc to identify HIV
strains on the basis of the 5~qn~nre of the tat gene.
Other uses f or the proteins are in the development 25 of antibodies which are directed to the c~ l Ar uptake regions .
Polyclonal ant i hgtl; PS: may be produced by conventional methods, i.e., by injecting a laboratory animals with sufficient quantities of the TAT protein or 30 fragments thereof and collecting the ant;h~A;~ from the animal ' s tissue.
A " ~ n:~l antibody" refers to h~ , nr~us populations of i -~loh~ll;ns which are capable of spnr;f;c~l1y binding to the TAT protein of this 35 invention. It is understood that the TAT protein may _ _ _ _ _ 0 95131999 ~ J,.,5. -(~77 ~ 2190972 have one or more antigenic det-~rminAnts. The an~ho~1iPC
o~ the invention may be directed again5t one or more of these det~rm; nAnts.
~Iybridomas capable of secreting the6e - orln~
5 an~ i ho~ q are produced by conventional technigues by employing as antigen, the TAT protein described herein.
Generally, the hybridoma proce6s involves generating a B-lymphocyte which produces a desired polyclonal antibody.
Techniques for obtaining the ~ u~liate lymphocytes from lO mammals injected with the target antigen are well known.
Generally, the pcripheral blood lymphocytes (PBLs) are used if cells of human origin are desired. If non-human sources are de6ired, spleen cells or lymph nodes from other - l; An sources are u5ed. A host animal, e . g . a 15 rabbit, is injected with repeated doses of the purified antigen, and the mammal is permitted to generate the desired polyclonal antibody producing cells.
Thereafter the B-lymphocytes are harvested for fu~3ion with the immortalizing cell line. Immor~ i 20 cell lines are usually transformed l i~n cells, particularly cells of rodent, bovine and human origin.
Mo~t frequently, rat or mouse myeloma cells are employed.
Techniques for fusion are also well known in the art and generally involve mixing the cells with a fusing agents, 25 e . g . polyethylene glycol .
Immortalized hybridoma cell lines are selected by standard ~Locedules, such as HAT selection. From among these hybridomas, those secreting the desired monoclonal antibody are selected by as~qaying the culture medium by 30 standard i ccayS~ such as Western blotting, ELISA, or RIA. Antibodies are re~,~,v~L~ad from the medium using standard purification techniques. See, generally, Kohler et al, ~a~, 256:495 (1975) for antibody production techniques. Alternatively, non-fusion technigues for WO 95/31999 ~ 77 ~
. .
0~7~

generating an immortal antibody-producing hybridoma cell line is possible, e.g. virally induced transformation.
Recombinant techniques, such as IlPqrr;hPrl by Huse et al, Science, ~46:1275-1281 ~l988), or any other modif ications thereof known to the art may also be employed to produce re: ` ;nlnt ant;ho~l;pq and chimeric antibodies, where desired.
These ant; ho~ q are likewise useful in diagnostic methods for detecting and identifying different EIIV
strains based on the ~l;ffPrPnrPC in their extracellular ta~ gene6 . These antibodies may perf orm such detection in conventional assays known to one of skill in the art.
Other Ut;litiPq are conventional and may include use of the TAT protein and peptide ;~lln~Pnq as molecular weight markers.
The followlng examples illustrate preferred methods f or preparing TAT ; ~ ~ S of the invention and utilizing these; , to induce immune response to the virus in an; ; 7efl ho8t. These examples are illustrative only and do not limit the scope of the invention .
E 1 -- 5Y~ S OF ~ lL~ES OF THE INVENTION -SIV TAT ANr~ HIV-1 TAT ~5VULI~ S
The peptides of the SIV TAT and HIV-1 TAI' were synthesized on an Applied Biosystems Model 430A peptidQ
synthesizer by the solid-phase method developed by Merrif ield, cited above . Peptide assembly was carried out starting with the p-methylbenzhydrylamine resin 3 o andthe Boc-protected amino acid5 u5ing a symmetrical anhydride activation procedure. All the amino acids were double coupled to ensure complete coupling. Side chain protecting groups were as follows: benzyl ether (Bzl) for the hydroxyl group of threonine and serine; benzyl ester W095131999 r~l,u.,.h.~Q77 for the carboxyl group of aspartic and glutamic acids;
tosyl (Tos) for the g~ ni-7inr~ of arginine;
benzyloxymethyl (Bom) for the imirln701e of histidine; 2-- chlorobenzyloxycarbonyl (C1-Z) for the ~-amine of lysine;
2-bL. IrlbAn7yloxycarbonyl (Br-Z) for the phenolic hydroxyl of tyrosine. Asparagine, glutamine, and arginine were coupled as their 1-hydL ~Lybenzotriazole esters .
A. SYnfhAsis of Tvr-Ara-Ara-Ara-'rhr-Pro-LYs-L
rrhr-Lvs-A1 A -A~n-rrhr-ser-ser-A1 A -Ser-N~b: rTYr~-SIV ~ 9,1 N~ rs~s Tr~ N0: 221 The protected peptide, Boc-Tyr(Br-Z)-Arg(Tos)-Arg (Tos) -Arg (To6) -Thr (Bzl) -Pro-Lys (Cl-Z) -Lys (Cl-Z) -Thr (Bzl) -Lys (Cl-Z) -Ala-Asn-Thr (Bzl) -Ser (Bzl) -Ser (Bzl) -Ala-Ser (Bzl) - (Me-BHA) -re6in was synthe6ized by the symmetrical anhydride coupling t~A~hnirrlp~ ut71;7ing standard coupling protocols (Std 1 cycle, version 1. 40) on the ABI 430A peptide synthAq; ~Ar. [Note that the initial Tyr is referred to as TyrO, since the first SIV
t~t amino acid is Arg (SIV TAT protein amino acid 80).
The Tyr is added to permit ligand att~ ` L. ~ The synthesis was initiated with p-methylbenzllydLylamine re~3in (0.6 mmole, 780 mg, 0.77 meq/g). The N-tArmin Boc ~1 ~u~ was removed by the end-NHI program (version 1.40). The resin was then washed with DMF (5 X 10 mL) and CHICl2 (5 X 10 mL), and dried in a vacuum oven at 30C
(2 . 96 g) .
The peptide was cleaved from the resin support (1.48 g) by stirring in liquid HF (50 mL), p-cresol (1.2 mL), p-thiocresol ( 1 . 2 mL), aDd dimethyl sulf ide ( 1 . 2 mL) 30 for 1 hour at 0C. After the removal of excess HF under reduced pressure, the resin-peptide mixture was extracted with anhydrous diethyl ether (3 x 200 mL). The ether extracts were discarded. The cleaved peptide was then extracted with 309~ aqueous acetic acid (3 X 70 mL).
35 After removal of solvents under reduced ~)L~S::~ULd, the WO 95/3 1999 2 1 9 g 7 2 P~ "1 s ~ 77 residue obtained was dissolved in water and freeze-dried.
The crude peptide was dis~olved in 3096 acetic acid and passed through an Amberlite IRA-68 (acetate form) ion exchange column (60 g, 1.6 meq/mL, 2.73 cm i.d. X 18 cm S length) in 30% aqueous HOAc at a flow rate of 60 mL/h.
The appropriate fractions from two such experiments were combined and freeze-dried (1. 59 g) .
The crude peptide was dissolved in 0.1% TFA/H2O
and puri~ied by preparative RP-HPLC using a Vydac 218TP1022 column (22 X 250 mm). The mobile phases employed were as shown below:
A = 0.1% TFA/H2O
B = 0.1% TFA/CH3CN -- H20, 4:1, v/v A linear gradient of 0~6 B to 8~6 B over 80 15 minutes at a f low rate of 8 mL/minute was used . The rractions were analyzed by HPLC and those containing pure peptide were combined and the organic solvents were removed under reduced pL.as~uL~. The residue was dissolved in water, converted into the acetate f orm by 20 passing through an ion-exchange column, and freeze-dried (327 mg).
Thin layer chromatography (TLC) was per~ormed on Merck F-254 plates (5 X 10 cm) in the following solvQnt systems (v/v):
Rf~l) = 0.65 (l-BuOH:Pyr:HOAc:H2O, 5:4:8:2) R~2) = 0.25 (l-BuOH:Pyr:HOAc:H2O, 5:4:4:2) Amino acid analy6is (AAA):
Ala 2.16 (2), Arg 3.23 (3), Asx 1.23 (1), Lys 3.21 (3), Pro 1.07 (1), Ser 2.27 (3), Thr 2.77 (3), Tyr 1. 09 (1) . During the hydrolysis of the peptide, Ser undergoe~; clr oq~tion resulting in low L C~JV~::L~'.

WO 95/319g9 r~ ,r.'C ;q7 Liquid Chromatography - Mass Spe~;~L LLY (LC-NS):
[NH+] at m/z = 651. 9 a.m.u., charge 3+;
977.1 a.m.u., charge 2+. Observed mol. wt. 1952 5 (Theoretical mol. wt. 1951.2).
B. Svnthesic of Ser-Tvr-GlY-Arq-Lys-Lys-Ar~-Ara Gln-Ara-Arq-~ra-Al A-Pro-Gln-GlY-Ser-Gl~-NH?: rE~IV-l T
1--NE~2 r SEO In NO: 3 l This peptide was synthesized as described 10 aboYe, using the appropriate protected amino acid derivatives. The characteristics of the peptide are as f ollows .
TLC: Rf (1) = 0.24 (1-BuOH:Pyr:HOAc:HzO, 5:4:8:2, v/v) - Rf t 2 ) - 0 . 09 ( 1-8uOH: Pyr: HOAc: H2O, 5:4:4:2, v/v) AAA: Ala 1.71 (1), Arg 5.85 (6), Glx 2.97 (3), Gly 1.87 (2), Lys 2.03 (2), Pro 1.37 (1), Ser 1.30 (2), Tyr 0.93 (1).
LC-MS: [MH+] at m/z = 1108 . 2 a.m.u., charge 2+. Observed mol . wt. 2214 . 4 (Theoretical mol . wt.
2214 . 5) .
~AMPr,F 2 ~ ~ 1S OF MT1T,TTPL~ ANTI~NTC ~ :.' OF
TM~ SIV TAT AND T~Tv--l TAT SEOU~N~ q Traditionally, small peptides have been conjugated to larger carrier proteins or self-polymerized to render them; ngen;C~ Both of these techniques suffer from significant limitations that reduce their utility for the production of highly specific ant;ho~ c. These obstacles were OVt:L ~ ~ by using the multiple antigen peptide system of D. N. Posnett et al, J. Biol. eho7a 263:1719-1725 (1988). The peptide was synthAsized directly onto a branching lysine core with 8 copies of the peptide sequence linked to the core by the carboxy _ _ _ _ .

. . :
g 7 2 tP~;n~l amino acid. The octameric multiple antigenic peptide was highly t ~r_niC in mice and rabbits, allowing production of ~cl~nll and polyclonal antibodies of high titer. The resultant antlbodies were 5 capable of recognizing the specific peptide sequence within a larger synthetic peptide LL, L or natural SIV
TAT protein.
A. SYnthesis o} MultiPle ~tiaPn; c Pe~tide:
r (Boc~ -Lvs(Boc) l~-LYs2-LYs-GlY-OCH1-PAM Resin The synthesis o~ a multiple antigenic peptide was accomplished manually by a stepwise solid-phase procedure on Boc-Gly-OCH2-PAM resin. The synthesis was initiated with 1.5 mmoles (1.97 g, 0.76 mmol/g) of the resin . The synthesis of the f irst and eYery subsequent 15 level of the carrier core was achieved using a 3 molar excess of Boc-Lys (Boc) -OH. All the couplings were done by the DCC-HOBt method following the protocol given below:
,Ste~ Rea~ent Time (minllte 20 1. CH2Cl2 wash 3 X 1 min.
2. 40% TFA - 10% anisole in CH2Cl2 1 X 5 min.
40% TFA - 10% anisole in CH2Cl2 1 X 25 min.
4. CH2Cl2 wash 3 X 1 min.
5. 10% NNM in CH2Cl2 2 X 5 min.
3 0 6 . CH2Cl2 wash 3 X 1 min .
7. DMF wash 2 X 1 min.
8 . Boc-Lys (Boc) -OH (4 . 5 mmol) and 1 X 3 min.
HOBt (1.5 mmol) in DMF
9. DIC (4 . 5 mmol) added to the 1 X 180 min.
above and shaken 10. RPco~lr] P, iî nPcPaa~y by repeating steps 4-9 11. D~F wash 3 X 1 min.

W0 95/319~9 r~ G~77 25 2 1~0g 7~
After three successive coupling8 of Boc-Ly6 (Boc) -OH, the resin was washed with DMF (3 X 50 mL) and CH2Cl2 (3 X 50 mL) and dried in a vacuum oven at 30C
(3 . 71 g) -B. Synthe~i ~ Df rArq--Arq--Ara-Thr--Pro-Lvs--Lys--~rhr Lys-Al ~-ARn-Thr-Sc~r-Ser-Al ~-SPrl~-LY6~-LYs~-LY6-Glv-oH:
r ~SIV TAT~oq~ rqulti~le Antiq~n;c Pe~tidel rSEO ID NO: 231 The protected pL~- UL_OL of (SIV T~8~9~) multiple antigenic peptide, tBoc-Arg(Tos)-Arg(Tos)-10 Arg(Tos)-Thr(Bzl)-Pro-Lys(Cl-Z)-Lys(Cl-Z)-Thr(Bzl)-Lys (Cl-Z) -Ala-Asn-Thr (Bzl) -Ser (Bzl) -Ser (Bzl) -Ala-Ser(Bzl) ]8-Lys4-Lys2-Lys-Gly-OCH~-PAM resin was synth~ scl by growth from both the ~ and ~ amino groups using the :,y L~ical anhydride coupling t~rhn;que, ut;l;7;n~ the 15 standard coupling protocols (std 1 cycle, software version 1. 40) on the ABI 430A peptide synthesizer. All the amino acids were double coupled to ensure complete coupling. The synthesis was initiated with [Boc-Lys(Boc) ]4-Lys2-Lys-Gly-OCH2-PAM resin (0.22 mmol, 94 mg) 20 prepared as described in A. above. The N-t~rm;n;~l Boc group was removed by the end-NH2 program (version 1. 40) .
The resin was then washed with DMF (5 X 10 mL) and CH2C12 (5 X 10 mL), and dried in a vacuum oven at 30C (1.9 g).
The protected peptide-resin was treated with 25 liquid IIYdL~ I fluoride, in the presence of p-cresol, p-thiocresol and dimethylsulfide as sLclv~nye~s, at 0C for 1 hour with constant stirring. Excess HF was removed by vacuum and the residue treated with ether to remove scavenger p~ luL L:,. The peptide wa6 extracted with 50%
30 acetic acid (3 X 50 mL), the solvents evaporated ln vacuo, and the product freeze-dried.
The crude pep~ide was initially purif ied on an Amberlite IRA-68 ion-exchange column; further purification was accomplished by RP-HPLC on a preparative 35 C18 column. The solvents used were: water containing 0.1%

WO 95/31999 r~ .c-,^77 2~9~972-TFA (Buf~er A) and CH3CN - H20 (4 :1) containing 0.1% TFA
(Buffer B). A linear gradient of 0-20% B over 100 minutes was u6ed. The appropriate fractions containing the peptide were pooled, the solvents evaporated i12 5 vacuo, and the product freeze-dried (644 mg). The purified peptide gave satisfactory amino acid analysis.
AAA: Ala 18.42 (16), Arg 23.74 (24), Asx 10.41 (8), Gly 1.47 (1), Lys 25.91 (31), Pro 8.14 (8), Ser 23.68 (24), Thr 23.99 (24).
C. SYnthesis of rser-TYr-GlY-Arq-LYs-~Ys-~rq-Arq-.'1 n-~rq-Ara-Arq-Ala-Pro-t'.l n-Gly-ser-Glnl ~-LYsA-LYS~-LYS-GlY-OH: r rHIV-l TAT,~7~ Multiple Antiqon;c Pel~tidel rSEO
Tn NO: 24]
This peptide was synthPc; 70A as described above, using the a~luuLiate protected amino acid derivatives. The purified peptide gave sati6factory àmino acid analysis.
AAA: Ala 9.74 (8), Arg 46.5 (48), Glu 26.55 (24), Lys 23.21 (23), Pro 8.56 (8), Ser 10.95 (16), Tyr 7 . 52 (8) . During the hydrolysis of the peptide, Ser undergoes decomposition resulting in low recoverie8.
D. Other MultiDle Antiqon;c PePtides r~aking use of repQating Lr__ Ls of the SIV
TAT protein, the following multiple antigenic peptides can be produced using the techniques described above.
( Gln-Arg-Arg-Arg-Thr -Pro-Gln-Arg-Arg-Arg-Thr -Pro-Gln-Arg-Arg-Arg-Thr-Pro-G:Ln) 8-MaP [ SEQ ID
NO: 25].
( Pro-Lys -Lys-Thr-Lys-Thr-His-Pro-Lys-Lys-Thr-3 0 Lys-Thr-His-Pro-Lys-Lys-Thr-Lys-Thr-Hi6 ) 8-MAP
[SEQ ID NO: 26].

~ ' ~
_Wo 95/31999 P~
21gO972 (Pro-Lys-Lys-Ala-Lys-Thr-His-Pro-Lys-Lys-Ala-Lys-Thr-Hi5-Pro-Lys-Ly8-Ala-Ly8-Thr-His ) 8-MAP
[SEQ ID NO: 27].
Similarly, making use of repeating fragments of 5 the HIV TAT protein, the following MAPs can be ~lo-luced using methods analogous to those described above.
(Gln-Arg-Arg-Arg-Ala-Pro-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Arg-Arg-Arg-Ala-Pro-Gln) 8-MAP [SEQ ID
NO: 28].
(Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Gly-Arg-Lys-1y8-Arg-Arg-Gln-Gly-Arg-Ly8-Ly8-Arg-Arg-Gln~ 8-MAP [ SEQ ID NO: 2 9 ] .
EXAMPT ~ 3 ~ UltIZATION o~ ANTMAT ~ WITH ANTI--TA~r 15 ~LTTPT ~ ANTIG~NIC PEPTIDES
The following experiment is performed to evaluate whether; i ~ation with a multiple antigenic peptide of this invention in alum adjuvant induces a high titer antibody response and whether the presence of such 20 antibodies will prevent or ameliorate acute viremia and subsequent disease p~vyLas-iion in rhesus monkeys inoculated with infectious SIV.
Young adult, male, colony born rhesus monkeys (Naraca mulatta) older than 2 years of age, but weighing 25 less than 10 kg (a species commonly used in experimental models of SIV infection) are employed. The animals are ~ed a commercial primate diet, optionally suppl I ed with fresh fruit. Water is provided ad libitum. Animals are rAn~ mi 7Pd into two treatment groups (peptide or 3 0 placebo) .
Prior to challenge infection with SIV, the animals are; i s~Pd intrAmllcclllArly (by thigh injection) on day 0, day 10 and monthly as nPc~cFAry with either 200 mi~ LvyLCllll~ of test peptide mixed with alum as an adjuvant .

WO 4~/31999 2 1 9 0 9 7 2 ~ !6^77 ;~ .

or ~ saline-ad~uvant placebo, until suitable antibody titers are attained.

Peptide GroU~ ~ T ~ n Route Dose Vol.
placebol i.m. 0 ~g 400 ,uL
10 2 10 test peptide i.m. 200~g2 400 ~L
s~line ~d~uvAnt only (~ri i70d control) ' Disnolved in saline l~nd mixed 1:1 $n an alum Dlla~onair, Serum for antibody testing is collected prior to initial i ; 7Ation, on day 7 and on day 17 post ; 7ation and then 7 days after each monthly injection until suitable antibody titers are attained. SIV
20 antibody in serum is determined by both radio; --CCAy and ELISA and is confirmed by Western Blot analysis.
After suitable antibody titers are attained in the peptide; ;z~-l animals, all animals are rhAll~n~ed intravenously with 50 AID50SIV delta B670 virus by way o~
25 the sArh~n~Uc vein using a 23 gauge butterfly needle.
Needles are flushed with PBS before and after virus to a6sure accurate delivery of material. The B670 SIV
strain is a well-characterized viral isolate originally isolated at Tulane Regional Primate Research Center 3 0 ( TRPRC) .
At selected times after infection the clinical, immunological and virological status of the animals are evaluated. For example, a lymph node biopsy is performed at 2 and 4 weeks after rhAl 1 on~e~ and a sample of blood 35 is collected for the doto~m;nAtion of antibody titers at week 4 after challenge and then monthly until death or completion of study. For all mea~ Ls of antibody, : `
~0 95/31999 r~ 77 , .
219T~I972 the mean titer level, expressed on a log~O scale is computed at each time point.
Results of this protocol are anticipated to reveal significant protection of the animals by use of the 5 peptide; ~_115 of this invention.
T~AT~IPrT~ 4: EvATTTA~rIo~ OF ANTIBODY TITET~ OF IN~UNT~Tn M~NT~T~YS WITH T.~T IT~MTTT~OCT~N FOR SIV
Serum from monkeys i i 7Pt~ with the SIV TAT8095 10 multiple antigenic peptide are tested f or the presence of anti-SIV TAT binding activity. This activity is A~=';
by two i n~lPron.1P~t methods.
First [TyrO]-SIV TAT8095 is radiolabeled with l25I by the pulse ~ Ah-ol i n~ method of Culler et al, P~oc. Soc.
EXT~. BiQl, Med., 173:264-268 (1985) and purified by partition chromatography [QUSo, Degussa Corporation, Dublin, Ohio] and subsequent high ~LI:S~UL- liquid chromatography. The HPLC elution profile of [l25I-TYRo]-SIV TAT8095 is illustrated in Fig. l. Serial dilutions of the monkey serum are incubated with the [l25I-TYRo]-SIV
TAT8095 for 18-24 hours at room temperature. The antibodies present in the samples are precipitated, along with any bound [l2~I-TYRo]-SIV TAT809s, by subsequent incubation with goat anti-monkey serum followed by centrifugation. The supernatant is removed and the r^--- i n i n~ pellet is counted in a gamma counter to ~PtPrminp the amount of [125I-TYRo]-sIV TAT20g5 bound by the monkey serum dilutions. For comparison, titer is defined as the dilution of monkey serum required to bind 5096 of the added [l25I-TYRo]-SIV TAT8095 tracer.
Second, the monkey serum is titrated by enzyme-linked; -~say (ELISA) on polystyrene microtiter plates that have previously been coated with both the SIV
TAT8095 multiple antigenic peptide to determine the total binding activity, and with ovalbumin to determine amount , . . .

Wo 95/31999 r~ c -077 ' 30 ~ g ~ 2 of the binding that is not 6pecif ic f or SIV I'A~B~95 -Briefly, the monkey serum is serially diluted using 0. OlM
phosphate buffered saline containing 0 . 01% Tween 20 and 0. 5% BSA (PBS/Tween 20) . lO0 ~Ll of each serial dilution of monkey serum is added in trlplicate to the wells of a 96-well microtiter plate coated with either SIV TAT8C95 multiple antigenic peptide or ovalbumin. The plates are incubated for 2 hours at 37C with rocking. Following incubation, the plates are washed 5 times with the PBStTween 20 buffer. Goat anti-monkey IgG, labeled with horse-radish peroxidase (HRP0), is added to each well at the appropriate dilution in PBS/Tween 20 buffer.
Following an incubation at 37C for l hour, the plates are again wa6hed 5 times. The specific HRP0 substrate, Ultrablue (Transgenic Sciences, Inc., Milford, MA), is added and incubated in the dark f or 3 0 minutes at room temperature. The reaction is terminated by adding 2 . 5N
HCl to each well and the optical density of each well is measured at 450 nm. For comparison, the titer i5 defined as the cùllcenL~ ~tion of monkey serum which results in an optical density reading of l . 0 . The titers from each monkey are followed over time to determine empirically when the plateau antibody response against the SIV TATao95 multiple antigenic peptide is attained.
Nl u.,s modif ications and variations of the present invention are i n~ in the above-identif ied specif ication and are expected to be obvious to one of skill in the art . Such modif ications and alterations to the compositions and processes of the present invention are believed to be ~n~ ,~##~d in the scope of the claims ~rP''n'l~ hereto.

95/31999 PCTIUS951060~7 ~w ~Igo.9,72 SEQUENCE LISTING
~1 ) GENERAL INFORMATION:
(i) APPLICANT: T - iology Research, Institute Inc.
(ii) TITLE OF INVENTION: Vaccine Interdiction of Extrac~ 1 Ar Transactivating Proteins of Human T - '^f iciency Virus and Other Chronically Infecting Viruses Employing Similar Interc~ 1 Ar Transactivating Strategies (iii) NUMBER OF SEQUENCES: 38 (iv) Cu~Kk;~C)..L)L~.CE AûDRESS:
'A'I Anm7T ~ r Howson and Howson (BJ STREET: Spring House Corporate Cntr, PO Box 457 ' C j CITY: Spring House (D; STATE: Pennsylvania 'E I COUNTRY: USA
F;I ZIP: 19477 (V) '_ ~IL~iK RT'An~RT r FORM:
(A) MEDIUN TYPE- Floppy disk (B) CO.~UL~;K: IBM PC compatible (C) OPERATING SYSTEN: PC--DOS/NS--DOS
(D) SOFTWARE: PatentIn Release #l.o~ Version ~1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
( B ) FILING DATE:
(C) CLASSIFICATION:
(vii~ PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/Z47,991 (B) FILING DATE: 23--MAY--1994 (viii) ATTORNEY/AGENT I
(A) NANE: Bak, Mary E.
(B) REGISTRATION NUNBER: 31,215 ( C ) REFERENCE/DOCKET NUNBER . IRI4 4PCT
(iX) TrA~rlT~c~AlMMrrNTcATIoN INFORMATION:
(A) TELEPHONE: 215--5~0--9200 (B) TELEFAX: 215--540--5818 WO 95l3 1999 p ~ 77 ~

t2) INFORMATION FOR SEQ ID NO:1:
( i) SEQUENCE rTTAR~C~RT~TICS:
(A) LENGTH: 261 base pairs (B) TYPE: nucleic acid (C) S~RANr~EnNT.~: double ( D ) TOPOLOGY: unknown (ii) M~-T.T~CtTT.T. TYPE: cDNA
( ix ) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..258 (Xi) ~ UL.._J:; DESCRIPTION: SEQ ID NO:l:
ATG GAG CCA GTA GAT CCT AGA CTA GAG CCC TGG ~AG CAT CCA 4 2 Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gln Val Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr His Gln Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Ser Arg Gly Asp Pro Thr Gly Pro Lys Glu ( 2 ) INFORMATION FOR SEQ ID NO: 2:
U~SNC:~: rTTARArTT~RT~ TIcs (A) LENGTH: 86 amino acids (B) TYPE: amino acid tD) TOPOLOGY: linear 0 95/31999 r~ c m7 ~w 2190972 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DE~ lON: SEQ ID NO:2:
et Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly er Gln Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys ys Phe His Cys Gln Val Cys Phe~ Ile Thr Lys Ala Leu Gly Ile er Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln ly Ser Gln Thr His Gln Val Ser Leu Ser Lys Gln Pro Thr Ser ln Ser Arg Gly Asp Pro Thr Gly Pro Lys Glu 2 ) INFORMATION FOR SEQ ID NO: 3:
;yur,._~; rr~ARAcTT~RT~c:TIcs (A) LENGTH: 18 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: unknown (ii) MnT.T..rTrT.T.~ TYPE peptide (xi) S~;yu~;Nc:~: DESCRIPTION: SEQ ID NO:3:
Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Gly Ser Gln (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE t~r~ARAC`~T~'RT.~TICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: unknown (ii) Mr~T,T.~CrTT.T~ TYPE: peptide W095/31999 - - r~ll1.,._.C6q77 ., ,~,, ,.
219~9~2 (Xi) ~;15~U~;NC~; DESCRIPTION: SEQ ID NO:4:
Ser Tyr Gly Arg Lys Ly6 Arg Arg Gln Arg Arg Arg Ala Pro (2) IN~ORMATION FOR SEQ ID NO:5:
(i) SEQUENCE ~'~TARACTF.RT~TICS
(A) LENGTH: 14 amino acids (8) TYPE: amino acid tD) TOPOLOGY: unknown ~ii) Mnr.T~TTT.T~ TYPE: peptide (Xi) ~;UU~;NC~!; DESCRIPTION: SEQ ID NO:5:
Ser Tyr Gly Lys Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro ( 2 ) INFnRMATTnN FOR SEQ ID NO: 6:
(i) ~itil~lJ~;N~ TARAt~TT~RT~STIC5 (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MnT,T~clTT.T~ TYPE: peptide (Xi) ~:yU~NC~: DESCRIPTION: SEQ ID NO:6:
S~r Tyr Gly Arg Lys Lys Arg Lys Gln Arg Arg Arg Ala Pro ( 2 ) INFORMATION FOR SEQ ID NO: 7:
!;yU~;N(:~; CHARACTERISTICS:
(A) LENGTE~: 14 amino acid~:
(B) TYPE: amino acid (D) TOPOLOGY: unlcnown tii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ser Tyr Gly Arg Lys Ly~ Arg Arg Pro Arg Arg Arg Ala Pro O 95/31999 r~ .q77 ~w 2190972 (2) INFORMATION FOR SEQ ID NO:8:
(i) ~I~;yU~NC,~; CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: unknown (ii) M~lT.T.~f~TrT.T.~ TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Ser Tyr Gly Arg Lys Ly6 Arg Arg Gln Arg Gln Arg Ala Pro ( 2 ) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE ~r-TARAt~T~lRT~TIcs (A) LENGTH: 14 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: unknown (ii) Mf)r.T.~rTTT.T.~ TYPE: peptide (Xi) ~;yu~ DES~:R~ li: SEQ ID NO:9:
Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Gly Ala Pro (2) INFORMATION FOR SEQ ID NO:10:
(i) ~$yu~~ ; ~TAR~ r~RT~TIcs (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) M~ T.T~CrTT.r~ TYPE: peptide (xi) SEQUENCE DES~:Kl~ll( N: SEQ ID NO:10:
Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Thr Pro (2) INFORMATION FOR SEQ ID NO:ll:
yU~ ; rr-T ARAt~'rT~'RT~TICS
(A) LENGTH: 14 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: unknown WO9~/31999 ~ r~ r- 77 ~!190972 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro ( 2 ) INFORMATION FOR SEQ ID NO :12:
(i) SEQUENCE r~ARAc~T<~pT~sTIcs (A) LENGTH: 14 a~ino acids (B) TYPE: amino acid ( D ) TOPO~OGY: unknown (ii) MOLECULE TYPE: peptide (Xi) ~ih~UL ._~; DESCRIPTION: SEQ ID NO: 12:
Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala His ( 2 ) INFORMATION FOR SEQ ID NO :13:
(i) SEQUENCE rT~RA-'T~RT~TICS:
(A) LENGTH: 17 a~ino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) Mf-T.T.'t'TTT.~ TYPE: peptide (Xi) ~i~;yUhN(:~: DESCRIPTION: SEQ ID NO:13:
Arg Arg Arg Thr Pro Ly6 Lys Thr Lys Ala Asn Thr Ser Ser Ala Ser Tyr ( 2 ) INFORMATION FOR SEQ ID NO: 14:
ihS,IUL.._h rT~ARArTT~RT.~TIcs:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: unknown (ii) M~T.T.CUT.T~. TYPE: ~peptide ~WO95/31999 ~,~9~9~ r~ c-.~77 . ;

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Tyr Glu Gln Gln Arg Arg Arg Thr Pro Lys Lys Thr Lys Ala Asn Thr Ser Ser Ala Ser (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: unknown (ii) Mf~T.R~ITT.R TYPE: protein (xi) SEQUENCE DESCRIPl`ION: SEQ ID NO:15:
Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gly mr His Gln Val Ser Leu Ser Lys Gln (2) INFORMATION FOR SEQ ID NO:16:
hs2~ ; rTlz~R~ TRRT~sTIcs (A) LENGTH: 6 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: unknown (ii) Mt)T.RC'UT.R TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Gln Arg Arg Arg Ala Pro (2) INFORNATION FOR SEQ ID NO:17:
hQulsNc~; CH~RACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown WO9513l999 ~ r~l~u.. sr -n77 ~ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Gly Arg Lys Lys Arg Arg Gln (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE ~'r~ARA~TT~RT~sTIcs:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) M~T.T~rrTT.T.. TYPE: peptide (xi) SEQUENCE DES~Kl~lu~: SEQ ID NO:18:
ro Lys Lys Thr Lys (2) INFORM~ATION FOR SEQ ID NO:l9:
(i) S~;QUL..~ r~ARACTT~RT~TICS
(A) LENGTH: 5 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: unknown (ii) ,r.~rTTT.T.~ TYPE: peptide (Xi) ~;YUL.._~; L~ES~:Kl~lON: SEQ ID NO:19:
ro Lys Lys Ala Lys ( 2 ) INFOKMATION FOR. SEQ ID NO: 2 0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: unknown (ii) M ~T.T~crTT.T~ TYPE: peptide (Yi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Gln Arg Arg Arg Thr Pro ~wo 9s/31sgg F~ ';177 t2) INFORMATION FOR SEQ ID NO:21:
; t;UU~;NC:~ CHARA~l ~ lCS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: unknown (ii) MOT.T~'C'TTT,T~' TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID No:21:
Arg Arg Arg Ala Pro (2) INFORMATION FOR SEQ ID NO:22:
(i) S~!;UU~;N~:~' rTTARAcTTi~RT~TIcs tA) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) ~!U~ E DESCRIPTION: SEQ ID NO:22:
Tyr Arg Arg Arg Thr Pro Lys Lys Thr Lys Ala Asn Thr Ser Ser Ala Ser ~2) INFoRMAT~o~ FOR SEQ ID NO:23:
(i) ~i~;S~L _~; r~TAR~r~l~T~RT.~TIcs:
(A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOT.T'rrTT,T' TYPE: peptide (Xi) ~i~;Uu~ E bESL:Kl~llON: SEQ ID NO:23:
Arg Arg Arg Thr Pro Lys Lys Thr Lys Ala Asn Thr Ser Ser Ala Ser Lys Lys Lys Lys Lys Lys Lys Gl WO9~/31999 ~ r~l~u.,. '~6~77 219n972 : ~

(2) INFOR~ATION FOR SEQ ID NO:24:
(i) SEQUENCE rTT~R~r~FRT~TIcs (A) LENGTH: 26 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOT,Ff`TTT.T~ TYPE: peptide (Xi) ~ U~:N~:~; DESCRIPTION: SEQ ID NO:24:
Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Gly Ser Gln Lys Ly6 Lys Lys Lys Lys Ly6 Gly (2) INFl~RM~Tt~ FOR SEQ ID NO:25:
(i) SEQUENCE r~R~rTFRT.~TICS:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) Mr~TT~ UTT~ TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Gln Arg Arg Arg Thr Pro Gln Arg Arg Arg Thr Pro Gln Arg Arg Arg Thr Pro Gln (2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 21 amino acid6 (B) TYPE: amino acid tD) TOPOLOG~: unknown (ii) M-IT-T~'CUTT~' TYPE: peptide (xi) SEQUENCE D~S~~ ON: SEQ ID NO:26:
Pro Ly6 Ly6 Thr Ly6 Thr Hi6 Pro Lys Ly6 Thr Lys Thr His Pro Lys Lys Thr Lys Thr His ~o95131999 P~,llIJ.~_S. q~7 2~ 9~972 (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE f~TTAR~l'~T~RT~TIcs (~) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MnT.T.`CTTT.T~ TYPE: peptide (xi) ~iyu~l~c~; DESCRIPTION: SEQ ID NO:27:
Pro Lys Lys Ala Lys Thr His Pro Lys Lys Ala Lys Thr His Pro Lys Lys Ala Lys Thr His (2) INFORMATION FOR SEQ ID NO:28:
( i ) SEQUENCE ~TT~R~T~'RT ~TICS:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: unknown (ii) Mnr.T.~TTT.T.~ TYPE: peptide (xi) ~;yU _~; DESCRIPTION: SEQ ID NO:28:
Gln Arg Arg Arg Ala Pro Gln Arg Arg Arg Ala Pro Gln Ar Arg Arg Ala Pro Gln (2) INFORMATION FOR SEQ ID NO:29:
(i) S~;QUI!;NC~ 'T~PIR~r'l'T.'RT.CTIcs (A) LENGTH: 22 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MnT.T~ TTT.T~. TYPE peptide (xi) ~;yu~iNc~; DESCRIPTION: SEQ ID NO:29:
Tylr Gly Arg Lys Lys Arg Arg Gln Gly Arg Lys Lys Arg Arg Gln Gly Arg Lys Lys Arg Arg Gln WO 95/31999 2 1 9 ~ 9 7 2; P~ ~ n77 ,~

(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: unknown (ii) Mor,T~TlT.R TYPE: peptide (Xi) ~hylJhNC~; DESCRIPTION: SEQ ID NO:30:
Arg Arg Arg Ala Pro (2) INFORMATION FOR SEQ ID NO:31:
(i) ~;hyUhl~ch r~T~ARAC'rF~RT~STIC5 (A) LENGTH: 5 amino acids (B) TYPE: amino zlcid ( D ) TOPOLOGY: unknown ( i i ) Mt T .T; ('TIT ~T~ TYPE: pept ide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Arg Gln Arg Ala Pro (2) INFORMATION FOR SEQ ID NO:32:
ihyUL.._h rT~ARA~'rr'F~RT.sTIC5 (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MnT.T~rTTr.T<~ TYPE: peptide (Xi) ~ihyU~;NCh I~ES~ lo..: SEQ ID NO:32:
Arg Arg Gly Ala Pro (2) INFORMATION FOR SEQ ID NO:33:
h~luhNch ~TTARA~TF~RT~sTIcs (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown ~WO 95131999 r~ n77 21~09~2`

(ii) M~TT~'c~rT~` TYPE: peptide (Xi) ~;hyU~;NC.t; DESCRIPTION: SEQ ID NO: 33:
Arg Arg Arg Thr Pro (2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE '~ARA~''l'T~'RT.':TICS:
(A) LENGTH: 5 amino acid6 (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) M~T~T~CTlT~ TYPE: peptide (Xi) :~iyU~;NC~: DESCRIPTION: SEQ ID NO:34:
Arg Arg Arg Pro Pro (2) INFORMATION FOR SEQ ID NO:35:
(i) ~;y~ CHARACTERISTICS:
tA) LENGTH: 5 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: unknown ( ii) M~-T-T~rrTT-~ TYPE: peptide (Xi) b~iyl _~; DESCRIPTION: SEQ ID NO:35:
Arg Arg Arg Ala His ( 2 ) INFORNATION FOR SEQ ID NO: 3 6:
(i) ~i~;yU~ ARAC'IIFRT~::TIcs (A) LENGTH- 5 amino acids (B) TYPE: amino acid (D) TOPO~OGY: unknown (ii) MnT.T.~TTT.T.. TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Arg Lys Lys Arg Ar W0 95131999 . P~ 'r~q77 (2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: unknown ( ii) M~T~T~'rUT~T~' TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Lys Lys Lys Arg Ar (2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE rT~ARA~ RT.~TICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) M~lT.T"CTJT.T.' TYPE: peptide (xi) ~i~;yu~;Nc~; DESCRIPTION: SEQ ID NO:38:
Arg Lys Lys Arg Lys

Claims (23)

WHAT IS CLAIMED IS:

1. An isolated immunogen comprising an amino acid sequence comprising the cellular uptake region or a fragment thereof of a native transactivating (TAT) protein from a selected virus, said immunogen capable of eliciting antibody against said native protein.

2. The immunogen according to claim 1 wherein said protein is a TAT protein of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV).

3. The immunogen according to claim 2 wherein said sequence comprises a TAT protein having amino acid deletions at the amino terminus.

4. The immunogen according to claim 2 wherein said sequence comprises a TAT protein sequence having amino acid deletions or substitutions at one or more cysteine residues which normally participate in disulfide bonding.

5. The immunogen according to claim 3 wherein said deletion comprises about 3 to about 10 amino acids at the amino terminus.

6. The immunogen according to claim 1 wherein said cellular uptake region is an HIV TAT sequence selected from the group consisting of SYGRKKRRQRRRAPQGSQ SEQ ID NO: 3, SYGRKKRRQRRRAP SEQ ID NO: 4, SYGKKKRRQRRRAP SEQ ID NO: 5, SYGRKKRKQRRRAP SEQ ID NO: 6, SYGRKKRRPRRRAP SEQ ID NO: 7, SYGRKKRRQRQRAP SEQ ID NO: 8, SYGRKKRRQRRGAP SEQ ID NO: 9, SYGRKKRRQRRRTP SEQ ID NO: 10, SYGRKKRRQRRRPP SEQ ID NO: 11, SYGRKKRRQRRRAH, and SEQ ID NO: 12, fragments and allelic variants thereof.

7. The immunogen according to claim 6 wherein said fragment is selected from the group consisting of RRRAP, RQRAP, RRGAP, RRRTP, RRRPP, RRRAH, RKKRR, KKKRR, and RKKRK (SEQ ID NOS: 30-38).

8. The immunogen according to claim 1 wherein said cellular uptake region is an SIV TAT sequence selected from the group consisting of RRRTPKKTKANTSSASY (SEQ ID NO: 13), YEQQRRRTPKKTKANTSSAS (SEQ ID NO: 14), and fragments and allelic variants thereof.

9. The immunogen according to claim 8 wherein said fragment is selected from the group consisting of PKKAK (SEQ ID NO:
19), PKKTK (SEQ ID No: 18) and RRRTP (SEQ ID No: 33).

10. The immunogen according to claim 1 comprising a synthetic peptide from a selected region of said protein coupled to a carrier.

11. The immunogen according to claim 10 comprising a construct comprising a plurality of synthetic peptides from said protein in the form of a multiple antigenic peptide.

12. The immunogen according to claim 1 wherein said sequence does not share the biological activity of the native protein.

13. A composition useful for inducing an immune response in a patient protective against infection with a virus characterized by a transactivating protein, the composition comprising an immunogen capable of eliciting antibody against a native transactivating protein from said virus, said immunogen comprising an amino acid sequence comprising the cellular uptake region of said protein or a fragment thereof in a selected pharmaceutical carrier.

14. The composition according to claim 13 wherein said carrier comprises an adjuvant selected from the group consisting of alum, liposomes, and magnesium hydroxide.

15. The composition according to claim 13 comprising an immunogen of any of claims 2-12.

16. The composition according to claim 13 comprising at least two different immunogens.

17. An isolated nucleic acid sequence encoding the immunogen of any of claims 1 to 12.

18. The sequence according to claim 17, said sequence suitable for direct administration into a mammal and in vivo expression of said immunogen.

19. A nucleic acid molecule comprising the sequence of claim 17 in association with nucleic acid sequences capable of regulating the replication and expression of said TAT
sequence in vivo in said mammal or in vitro in a host cell culture.

20. An antibody capable of identifying a virus strain having a selected extracellular TAT protein, wherein said antibody is capable of binding to an immunogen of any of claims 1-12.

21. A method of producing an antibody capable of identifying a virus strain having an extracellular TAT protein, said method comprising immunizing an animal with an immunogen of any of claims 1-12 and isolating said antibody from the tissue of said animal.

22. The use of an immunogen of any of claim 1 to 12 or a nucleic acid sequence of claim 17 in preparing a medicament for immunizing a patient against infection with a virus characterized by a transactivating protein.

23. The use of an immunogen of any of claim 1 to 12 or a nucleic acid sequence of claim 17 in preparing a medicament for reducing viremia in a mammal infected with a virus characterized by a transactivating protein.