CA2050302C - Polypeptide having immunological activity for use as diagnostic reagent - Google Patents

Abstract

A polypeptide having immunological activity for use as a diagnostic reagent for the HIV virus. The polypeptide comprises substantially all of the amino acid sequences of the reverse transcriptase, RNAase H and integrase enzymes coded for by the HIV-pol gene and the amino acid sequences of part of the protease enzyme coded for by the HIV-pol gene omitting the active site responsible for proteolytic activity.

Description

2~5~302 PO~Y~ HAVING TMMIJNOLOGI~AT ACTIVITY FOR I~SE ~q DIAGNOSTIC R~.'T'NT
TEf'~TNI~r, FTT~T,n This invention relates to a polypeptide having 5 immunolqgical activity for use as a diagnostic reagent.
BACKGRQT~D ART
Diagnostic kits for use in screening individuals for inf ection with human immunodef iciency virus (HIV) inf ection frequently include reagents comprising HIV antigens which 10 are used to detect antibodies using known immunological techniques including EI ISA, Western Blot, latex agglutination and immuno - luminescent and immuno- f luorescent techniques .
The eifectiveness of such techniques however depends 15 upon selection of suitable immunological reagents and one particular difficulty which arises is that particular reagents are often specific to individual strains or grQups of strains of HIV. Thus, for example, known diagnostic reagents based upon HIV-1 may fail to detect antibodies 20 resuIting from an infectlon of a patient with HIV-2.
Synthesis and cleavage of the HIV-I pol precursor polyprotein is disclqsed in "Processing Protease and Reverse Transcriptase from Human Immunodeficiency Virus Type I
Polyprotein in Escherichia cQli" by Jan Mous et. al., 25 Journal of Virology, Apr. 1988, p. 1433-1436. T~e process =
disclosed in this reference results in the formation of a 92 kDa polypeptide consisting of protease (18 kDa), reverse transcriptase (64 kDa) and an amino-terminal portion of endonuclease (integrase) (10 kDa). The polyprotein is thus 30 lacking the intact f~nflnn~ l ease sequence (25 kDa), and thus lacks substantial antigenic epitopes representing the ~ntl~nllflease (integrase). Thus, the protein is unlikely to be suited for the preparation of diagnostic tests for HIV-I.
It is an obj ect of the present invention to overcome

3~ such problems.
-s~ ~

-- ~ 20~0302 D I SC~OSURE OF INVENTION
It has now been found that the product of expressing a substantial part of the HIV-pol gene in a suitable host has antigenic properties which allows the above-mentioned 5 problems to be overcome.
Thus according to one aspect of the present invention there iB provided the use of a polypeptide as a reagent in a diagnostic test for XIV infection characterized in that said polypeptide comprises substantially all of the amino acid 10 sequences of the reverse transcriptase, RNAase H and integrase enzymes coded for by the XIV-pol gene and the amino acid sequences of part of the protease enzyme coded for by the HIV-pol gene omitting the active site responsible for proteolytic activity.
1~ Diagnostic kits comprising said polypeptide and the polypeptide itself ~orm further aspects of the present invention .
The HIV-pol gene codes for four enzymes, namely a protease, a reverse transcriptase, a ribonuclease referred 20 to ;as RNAse H and an enzyme referred to as Integrase.
It is believed that during inf ection of a T cell by XIV
a f ull length precursor is expressed which is then cut up into the discrete proteins listed above. These have the foIlowing activities and (it is thought) act in the order 25 indicated:-Protease: ~ Precursor Cleavage Reverse Transcriptase Preparation of viral DNA from viralRNA
RNAse H Destruction of viral RNA leaving 3 o newly synthes~sed DNA
Integrase Insertion of said DNA into host cell genome ~ 2050302 In vivo, the initial product of expressing the HIV-pol gene is cleaved into its individual elements by the - protease. The active site ~or proteolytic activity occurs adjacent the ~2-terminus of the expression product, 5 corresponding to the 5'-end of the protease gene.
In the present invention, the polypeptide omits at least that part of the amino acid sequence of the HIV-pol protease -gene which codes for the active site regponsible for proteolytic activity. By omitting this portion, the 10 integrity of the polypeptide is n-;nt~inPrl and it is less liable to degrade.
BRIEF DESCRIPTION OF DRAWTNGS
Figure 1 is a schematic diagram showing the procedure of Example l;
Figure 2 shows the results of electrophoresis tests carried out in the manner explained i~ Example 2; and Figure 3 is a graph showing the results of the experiments carried out in Example 3.
BEST MODE FOR CARRYING OUT rTT~ I~VENTI~N
The l~IV-pol gene of several strains of XIV-l has been cloned and the corresponding amino acid sequences derived from the determined DNA sequences. The amino acid sequences of ten fitrains appear in the accompanying Table 1 at the end of this disclosure. In Table 1~ the full sequence of strain 25 HIV HXB2 is given, whereas for the other nine strains, only sequence differences are listed. As used herein, the term "constituent protein coded for by the HIV-pol gene" refers to a protein having sufficient amino acid homology with the sequence of HIV ~IXB2 appearing in the ~ ying Table so 3 0 as to result in antibodies raised against the protein cross-reacting with a polypeptide consisting o-f the precise amino acid sequence of HIV HXB2.
The HIV-pol gene can be expresse-d to produce the desired polypeptide by various techniques, e . g . some or all of the 35 baculovirus techniques described in H.S. Patent 4,745,051 to Gale E. Smith et al issued on May 17, 1988; Baculovirus Vectors for Expression of Foreign Genes by C. Yong Kang, ~ 20503~2 - Advances in Virus Research, Vol. 35, pp 177-192, Academic Press Inc., 1988; A Manual of Methods for Baculovirus Vectors and Insect Cell Culture: Prclce~re$, Max D. Summers and Gale E. Smi'ch, May 1987, Texas A&M University; and 5 Baculoviruses as Gene Expression Vectors, Lois K. Miller, Ann. Rev. Microbiol. 42, pp 177-1991. However our rAnA~li An Patent No. 1,330,425 issued June 28, 1994 ~and equivalent US Patent No. 5,194,376 issued March 16, 1993) describes and claims an improved baculovirus expression system capable of 10 producing foreign gene protein$~ at high levels and the use of this expression system is particularly preferred for expressing the polypeptide of the present invention.
The process disclosed in our Canadian patent employs a recombinant baculovirus c~ntA;n;ng at least a major part of 15 a polyhedrin gene promoter region, a tr_nscription termination sequence of a - polyhedrin structural gene, a foreign structural gene (e.g. an HIV-pol gene) ~having a translation start codon followed by coding sequences and a translation stop codon. The foreign gene is located between 20 the promoter region and the termination sequence.
Immediately upstream of the start codon there is a putative insect cell ribosome binding site f or the polyhedrin gene effective for overcoming resistance of susceptible insect cells to express the foreign gene at a high level. The 25 putative ribosome binding site comprises at least the final f our nucleotides of the sequence 5 ' -ACCTATAaAT- 3 ' .
~ xample 3 of the rAnA~-l; An application describes the production of=the pol protein of HIV-1 in a baculovirus expression system based on Autoqrapha califo~:nica 30 nucleopolyhedrosis virus (ACNPV) and specifies that a recombinant baculovirus designated ACNPV-HlV-YK-pol has been deposited at the American Type Culture Collection of 123 01 Parklawn Drive, Rockville MD 20852, USA under Accession No.
ATCC VR 2233 Deposit was made on November 30, 1988.
Utïlising the procedures described in Example 3 of ('AnA~l; An Patent Application Serial No . 591, 908, a polypeptide comprising the protease, RNAse H and Integrase~ enzymes of r .
. .

20~0302 . ~ 5 BIV strain HIV-XB2 may be produced.
The polypeptide can be used as a diagnostic reagent in ways known to persons skilled in the art, e . g . by the techniques indicated in the publication entitled 5 Clinica, Testing for HIV and AIDS, The Next Five Years, George Street Publications Ltd., Richmond, Surrey, UK.
The invention is illustrated in more detail by the following Examples. Example 1 illustrates the production of a modified recombinant plasmid pUC18-Dpol3 having a 10 273 bp deletion at the 5 ' -terminus and its expression as polypeptide lacking the first 91 amino acids at the NHz-terminus of the HIV-pol protease. Examples 2 and 3 relate to the expression of the polypeptide and its use as a diagnostic reagent .
15 EXArlP~E 1 Construction of baclll ovirl~ transfe~ vector cont~ininq HIV-1 ool qene with 273 b~ deletioll ~t 5 ' terminUs As illustrated in Figure 1, the BglII and SalI fragment of plasmid pHXB-2D containing the HIV-1 pol coding region 20 was isolated and inserted into BamHI and SalI sites of pUC18. The resulting re~ ;n~nt plasmid ~pUC18-Dpol 1) was cut with Sstl and dephosphorylated. A synthetic double-stranded crossover linker ,-~nt~ining a Sstl cohesive end, a BamHI site, the putative insect S~odootera fruqi~erda ~SF9) 25 ceIl ribosome binding site ~P) and 15 nucleotides of the homology searching sequences which overlaps with the 5 ' term~Lus of the pol gene was ligated at the Sstl site and transformed. The recombinant plasmid, ~pUC18-Dpol 2) was isolated, digested with sPH1, dephosphorylated and ligated 30 with another crossover linker DNA containing SphI cohesive end at the 3' tt~rminl~, BamH1 site and 15 nucleotides of the homology searching sequences which recognise the 3' t~rminllq of the pol gene.~The resulting recombinant plasmid WO 90/1~1230 ~ _ PCr/C~90/00062 ~ 6 ~ 3f[~
(pUC18-Dpol 3 ) contains the putative SF9 cell ribosome binding site (P) followed with pol open reading frame starting with the first ATG (TI) codon tmap unit 2357-2359) in the pol gene and the translation termination (TT) codon 5 TAG (map unit 5093-5095). This whole cassette was flanked with BamH1 sites. The BamHl rL~I' L was isolated and inserted into the 3amHl site of the pAcYM1 baculovirus transfer vector (pAcYM1-Dpol). The pAcYM1-Dpol transfer vector DNA was used to co-transfect SF9 cells with wild type 10 AcNPV DNA to isolated recombinant AcNPV HIV-YK pol virus.
E~AMPr F 2 ~nression of ~ol qene ~roducts bv re~~ ; nAnt; barlll ov; ruses Recombinant AcNPV-HIVWHpol contains an insert comprising essentially the whole DNA sequence of the HIV-pol gene (see 15 Table 2 at the end of the present disclosure). ~hen e.sl,L~,ed, the resulting full length gene product of the HIV-pol gene is ''l.Lu~essed'', i.e. the proteolytic active site of the HIV pol protease gene cleaves the protein into 66 kD, 51 kD and 32 kD ~L, Ls.
By way of comparison, recombinant AcNPV-HIVYKpol (see Table 3 at the end of the present disclosure) omits NH2-tF~minAl amino acid sequences containing the proteolytic active site of the HIV-pol protease. When ~ LeSSed, the re6ulting gene product is not "processed", i . e. the - 95 kD
25 protein remains intact.
The following experiments illustrate this.
Uninfected S. flu~iPerda (SF9) cells, or SF9 cell infected with recombinant baculoviruses AcNPV-HIVWHpol, AcNPV-HIVYKpol or with wild-type AcNPV, were harvested after 30 72 hours of infection. Lysates of the infected or uninfected cells were electrophoresed in a 12% polyacrylamide Laemmli gel and proteins are identified by either Coomassie blue staining (S) or Western blot analyses tw) using the standard HIH HIV positive immunoglobulin. As shown in Figure 2, lanes 35 1, 2 and 3 represents the lysates of AcNPV-HIVYHpol ~ inAnt virus intected c~ , lan 4, 5 aDd 6 represent ., .~ .
, I"
- the lysates of AcNPV-EIVYKpol recombinant virus inf ected cells, lane 7 shows the wil~-type AcNPV infected cell lysate, lane 8 shows uninfected cell lysate and lane 9 shows molecular weight markers. Lane l and 6 show the whole cell 5 lysate, lanes 2 and 5 show proteins in the infected cell nuclei and lanes 1 and 4 show proteins in the infected cell cytoplaæm. P denotes polyhedrin protein and arrows show 95K
Dal uncleaved pol gene product representing 91 amino acid deletion of protease produced by AcNPV-HIVYKpol virus and 10 66K Dal, 51 K Dal and 33K Dal processed pol gene products in AcNPV-HIVYHpol virus infected cells.

A. PrDduction of Pol qene Product Recombinant ACNPV-HIVYKpol virus infected Spodoptera 15 fruqiPerda (SF9) cells were harvested 4 days after infection. Nucl~i of infected cells containing most of the pol gene product were isolated by treating the infected cells with O.1~ Triton~ X-lOQ and O.5g6 NP40* on ice for 20 minutes followed by centrifugation at 750 g for 10 minutes.
20 The pelletea nuclei were denatured with 19~ SDS in TRIS-HCl pH 8.0 at room temperature for-30 minutes. The cf~ r DNAs were re~oved by ethanol precipitation using 2 volumes of 1005~ ethanol. The SDS in the solution were removed by add-ition of 25 mM KCL incubated at 40C for 3d minutes followed 25 by centrifugation at 12, 700 g for 15 minutes. The pol gene product in the supernatant was used for anti-pol ELISA.
B . Detection of HIV ant; hodies b~,r ELIS:~
The pol antigen was diluted in PBS and dispensed in a microtite~ plate (Nunc cat 269620). The concentration of pol 30 to co~t plates was determined empirically on the strength of bands on polyacrylamide gels.
The concentration of pol necessary to coat one well was between 1 and 10 ~Lg.
The plate was covered and incubated at 4 C . The time of 35 incubation varied between 12 and 24 hrs without no apparent differences in reactivity.
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. ~ 8 The plates were then washed three times in PBS-Tween~ 20 employing a Skatron plate washer.
Various standards , NIH HIV+ immunoglobulin ~NIE STD), pool HIV+ plasma (PAT STD) and plasma ~rom non-infected 5 individuals (NS) were employed. The standards were diluted beginning at 1: 2 0 0 f or NIH STD, and 1 :10 f or PAT STD and NS .
Unknowns were tested usually at 1: 50 but dilutions as high as 1:10 can be employed.
All samples were inactivated before testing. Normal sera 10 were processed in the same fashion~as sera from AIDS
patients. The inactivation was performed with

4 ' -aminoethyltrioxsalen- hydrochloride (AMT) from Lee Biomolecular- Research Inc. ~San Diego, California cat 231) and an ultra violet light trans-illuminator (Spectroline 15 model TC-365, Fisher Scientific Ottawa Ont. ) . The AMT was reconstituted in 50~ ethanol at 1 ~g/ml. The sera was aliquoted in Eppendorf tubes and for every 100 ~Ll of serum or plasma, 10 ~Ll of AMT was added to the sample. The samples were layed in the transilluminator and irradiated 20 for 5 minutes. An additional 10 /11 of AMT was added to the~
sample and the samples were irradiated for a further 5 minutes. The samples were inactivated by this procedure.
The incubation time o~ the human-anti-pol was 30 to 40 minutes at room temperature (23C) (the time of ;n~llh~lon 25 found to be quite critical). Therefore, all dilutions of standards (negative and positive) and unknowns was performed in a separate plate. Once all dilutions were done, the dilutions (100 111) were transferred to the ELISA plate coated with pol employing a multichannel pipettor. All dilutions were with PBS-Tween* 20 (0.19~) .
The state of the serum or plasma sample was found to be important. Samples repeatedly frozen and thawed usually gave higher backgrounds. This was especially evident with samples from normal individuals.
The plates were washed three times in PBS-Tween* 20 after the 30 minute incubation with the first antibody.
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. ~

.- ~ g . A Skatron II plate washer was employed for this purpose.
The second antibody used ~goat anti-human Ig linked to horse radish peroxidaæe) was an a~finity purified reagent obtained from Tago Diagnostics (Inter Medico To DNT cat

5 2393) . An dyy, ~Liate dilution was determined experimen-tally (approximately 1:2,000) is made in PBS-Tween* 20 (0.1~). 100 ~l was dispensed into the wells except for one which will be employed as a blank for the plate reader.
The plate was incubated f or 1 hour at room temperature .
The plates were washed three times with PBS-Tween* 20 employing the Skatron II plate washer.
Freshly prepared substrate (100 ~l) was added to the wells and after 20 minutes the reaction stopped with the additio~ of 100 ~Ll of n O7M H2SD4 ~5 The plate was read at 450 nm in the BIOTEK BL/310 ELISA
plate reader. A hard copy of the data was obtained ~rom the reader and the data also stored directly onto computer diskette ior further procesæing by the AnPl; ~ r program.
Additionally, controls were also performed on each 20 plate. In two or three wells no serum or plasma was added.
In one well no primary or secondary antibodies were added but substrate was. This well was employed to blank the ELISA plate reader. ~he r~m~;nin~-~ells were employed to determine the extent of binding of the ~ n~l~ry antibody 25 (Goat anti-HIg-~PO) to POL. Thus, these wells received no primary antibody but secondary antibody and substrate with the ayy~yliate washes in between each incubation. Usually the value of this latter control is below 0.1000 OD.
The results are shown in Figure 3.
The following materials were use for the anti-pol ELISA
procedure Buf f ers Phosphate Buffered S;~l ;nP (PBS) - -Na2HPO4 ~dibasic anhydrous) 13 . 6 g Na~2PO4 (monobasic) 2.4 g NaCl go . 0 g * TRADE M~REC

~ 2050302 Salts are dissolved in 8 litres of distilled deionized water and pH is adjusted to 7 . 2 with NaOH or HCl . This buffer is employed as coating buffer, diluent and washing buffer. The latter two buffers are modified as indicated 5 below.
Diluent for ~rimarY and secnn~3ry a~tjhn~;es and w;lch;n~
PBS + ~.I96 Tween*20 (Sigma, St. Louis MO) (0.1 ml Tween*
20 + 100 ml PBS). The diluent bufier is made up daily.
10 Substra~e buffer _.
Equal volumes of 0.1M Na2HP04 (0.709 g/50 ml) and 0.1M
citric acid (0.960 g/5û ml). The pH~=is adjusted to 4.0 with NaOH or HCl. The substrate buffer is made up weekly.
Substrate A tablet (2 mg) of o-phenylf~nP~ m; n~ (Sigma cat . P6787) is dissolved into 10 ml of substrate buffer. Hydrogen peroxide (4 ~l of 30~6) is added to the solution just prior to plating. The solution should be kept in the dark as much as posaible.
2 0 Sto~inq reacrent The enzymatic reaction is stopped with 0 . 07M HzSO~ .
It is a particularly advantageous feature of the polypeptides, the use of which is described herein, that they cross-react with ~nt;hnrlies against diverse strains of 25 HIV. Thus, for example, the polypeptides described herein based on HIV-1 can cross-react with antibodies raised against various strains of HIV-1 and HIV-2. Thus they may be used in diagnostic kits for detecting either virus category.
Similarly, in vaccinea they can provide broad-spectrum 30 protection : :
Industr; ~1 A~7~licabilitY
As will be apparent ~rom the above, the present invention can be used in the medical field for testing for HIV infection, as well as for other_diagnostic or prognostic 35 purposes.
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205~302 T~LE 1 ~IV-1 pol protein se~uence of ~IVHXB2 viruS
Data from Human Retroviruses and AIDS 1988 Los Al a;~s ~ati~nal La:~oratory AcNPV-HIVWHpol HIvl!xBz~ph-ArrJr~ Aer~u~nr: ~ur~ yLysAloAr9Gluph~sorse-rGlu.~. 19 HIYBH102 Cln 2~
H~VBHS Cln 2a HIYPY22 Cln 20 HIV8RU Cln 2a HIVMN ......................................................... ----- 0 HIVRF '- r. ~, Lou 19 HIVMAL A r, _ r, _ 19 HIYEL~ A rl v Cly------L--u------PrrLys----- 19 HIVHXB2 .... : .. GlnThrArgA]~-e~' F, _Thr~rg 28 HIVBH102 ThrAraA1r.e_~- r, _ThrIl-S-rS-rClu 42 HIVBH5 ThrArgAl ~ - P, ~ThrTl ~~ S_ ,.lu 4D
HIVPV22 ThrArllAl-'~ P~vThrT~- S~ Glu 4a HIVBRU ThrArg~r^--~ r, _ThrT~ r.~, 4a HIVMN .......................... , a HIYELI Ser 28 HIVHXB2 Arr~,r~ n~rl nvslTrpGlyAr~AerAe--~-e r. ~SerGluAloGlyAloAspArg 48 HIYBH1 02 6a HIYBH5 ---6a HIVPV22 6a HIVBRU I - 6a HIVMN .......................... ,.. 0 HIVSF2 ClyGlu L~_ 48 HIYRF ... __l- C~ -- 4~
HIVMAL ~, ~I Cly----- . . .Ly~ThrL-u------------Thr----------Glu----- 47 HIVEL~ A. u . . .ProL-u---LysThr------Glu--- 47 HIVHXB2 GlnGlyTh~Vn1 ~ r ~ heProGlnVolThrL-uTrpGlnArr,ProL-uVolThr b8 HIVBH102 Ilo 80 HIVBH5 Ilo sa - HIVPV22 Il~ Ba HIVBRU Ile 8a HIVMN ............................................................ a HIVSF2 Ilc 68 HIVr~F s- Il~ Ile------ 67 HIVMAL ~ l------ 67 HIVELI Il- Alo 67 -WO 90/10230 ~ Pcr/cA9O/0006 1.-- 12 l~a~le 1 cont'd ~- rco ~ds end HIVHXB2 }leLysIloGlyGlyGlnLeuLye*ll~^1n~ r-u~\SrThrGlyAlo~SpAspThrVcl 88 HIYBH102 1 oa H}VPY22 1 00 HIVBRU 1 a0 HIVMN ............................................................ 0 HIVSF2 ----A. ~ 88 HIVRF Vol 87 HIVMAL VolAr3Vol 87 ~-HIVYKpol st.lrts HIVHXB2 L-uGluGlL~;rLouPrrGlyAroTrpLy_ProLysMe~IleGlyGlyIloGlyGly 1 08 HIvaH5 120 HIVPV22 1 2a HIVMN ......... ----A:.~. A. ~ 17 HIVSF2 ^ Lr_ 1a8 HIVMAL ------_ Tl r ~ LY:- 197 HIVELI J LyC- 107 HIVHXB2 PheIl-LysVolAr3GlnTyr/~ernl nTl~ Tl~~1~ITloCysGlyHisLysAlo~lr~ 128 HIVBH5 14a HIVMN Thr---Gly ~7 HIVSF2 r. u~'ol - 12 HIVMAL Lr~ 127 H~VELI r. ~ Cln 127 HIVHXa2 GlyThrVolLeuVolGlyProThrProVclAsnIleIl~GlyArg~ o~ThrGln 14B

HIVBH5 1 6a HIVBRU 1 6a HIVMAL ~ l r1_ ~ -- 147 H~VEL~ 1 47 WO 90/lOZ30 PCI /CA90~00062 13 _ _ l'a~le 1 con~ ' d \/ p6b, F51 HIVHXB2 IleGlvCysThrLouAsnPheProIloSerProIloGluThrVolProVclLysLeuLys lbB
HIVBH102 18a HIVBH5 1 Ba H IVP Y22 1 8a HIVBRU 18a HIVMN I ~ 7~
HIVSF2 1 b8 HIVRF 1 b7 HIVELI 1 b7 HIVHXB2 ProGlyMetAspGlyProLysVclLysGlnTrpProL~uThrGluGluLy~IleLysAlo 18B
HIVBH12 20a HIVBH5 20a HIVPV22 - 2Da HIVBRU 2ao HIVMAL A. ~ lB7 HIVHXB2 LeuVclGluIloCysThrGlurletGluLysGluGlyLysIloSorLysIloGlyProGlu 208 HIVBH1 a2 22a HIVBH5 22a HIVBRU 22a HIVMN ---Ilo 117 HIVSF2 2~8 HIVRF 2a7 HIVMAL ------Tl.. Ly~A, L~ 207 HIVELI ----Tl... - A, A. ~ 2a7 HIVHXB2 AsnProTyrAsnThrProVolPhRAloIleLysLysLysAspSerThrLysTrpArgLys 22B
HIVBH1 a2 240 HIYEH5 24a HIVPV22 24a HIVBRU 24a HIVMAL
HIVELI . Ile 2277 = ,=.. --~ = .

WO 90/10230 PCr~CA90/00062 ~ S~3~

Tab~e 1 colLt ' d HIVHX32 LeuValAspPhoAr~ ysArgThrGlnAspPheTrpGluVclGlnLouGly 248 HIVBHT02 26a H~VBH5 A, y 260 HIV8RU 26a HIYMN Lys 157 H~VMAL ~ = 247 HIVHXB2 Ilr~ProH~sProAloGlyLeuLysLysLysLysSerVolThrValLeuAspVolGlvAsp 268 HIVBH1 a2 ~ 28a HIVBH5 28a HIVPV22 - 28a HIVBRU 28a HIVHXB2 AloTyrPheSerVolPrr~ ^er~ Aer~heArsLysTyrThrAloPheThrIlePro 288HIV8HlD2 3aa HIV8H5 3aa HIVPV22 - 30a HIV3RU . 30a HIVMN Lys- 197 HIVSF2 Ly~- 288 HIVRF LysGlu - 287 HIVELI Ser 287 HIVHX82 SerT~ ThrProGlyIleArgTyrGlnTyrAsnV~lLeuProGlnGlyTrp 3a8 HIVaH1 ~2 32a HIV3H5 SerGly 32a HIVPV22 ~2a HIVMN 32a HIVSF2 ~ = 32D78 HIVRF Ar ~I 307 HIVMAL 3a7 HIVELI 3a7 ..

WO 90/10230 PCI'/CA90/00062 2~5031~2 _ _ lra~le 1 cont'd HIVHXB2 LysGlyserproAloIlérl ~ln~ SerMetThrLysTle~ r~ propheArgLys 320HIVBH1 a2 Lys----- 340 HIvaRu 340 HIVRF Lys------ 327 HIVMAL rhr 327 HIVHXB2 GlnAsnProAspIloV31IleTyrGlnTyrMetAspAspLeuTyrVolGlySerAspLeu 348 HIVRF Clu 347 HIVMAL Lys-----------Glu 347 HIVELI CluMet 347 HIVHXB2 GluIl~GlyGlnHis~r~ThrLy~Tl e~l . ~ Ar n~ ArgTrpGly 363 HIVBH102 D _ 330 HIVBRU 38a HIVMN Alo A, ~ 277 HIVRF Ilt Clu Lys------~ 367 HIVMAL Clu Lys-------- 36i HIVELI Lys----Glu 367 HIVHXB2 LeuThrThrProAspLysLysHisGlnLysGl~r. ~r. ~.rl,~LeuTrpMetGlyTyrGlu 338 HIVBH1 02 40a HIVBH5 F~._ 400 HIV3RU 4a0 HIVMN r~ 297 HIVSF2 r- 388 HIVRF r 387 HIVMAL rh- 337 HIVELI Phe---A. ~ 387 , - -WO 90/10230 ~ PCT/CA90/00062 1~
T~ble 1 cont~d .
HIVHXB2 LeuHisProAspLysTrpThrVclGlnProIleYclLeuProGluLysAspSerTrpThr 408 HIveHl 02 420 HIV8H5 Ilo 420 HIVSF2 ~1~ L 408 HIVRF - 4a7 HIVMAL Cln------Asp---Glu------- 407 HIVELI ' L~_ Glu 4~7 HIVHXB2 VnlAe~AerTl~ ysLeuVolGlyLysLeuAsnTrpAloSerGlnIleTyrProGly 42BHIVBH1 02 440 HIVMN Alc----- ~7 HIVSF2 Ala--- 428 HIVRF Alc---- 427 HIVELI ' CluA, y 427 HIVHXB2 IleLysVclArgGlnL~uCysLysLeuLeuArgGlvThrLysAloLeuThrGluVclIle 448 HIVBH1 a2 4G0 HIVBH5 46~
HIVPV22 4~o HIVBRU 46~
HIYMN ----------~Ly: ~57 HIVSF2 ---- Lys 448 HIVRF ---------Lvs Vol 447 HIVMAL ---------- Lyc Alo AspIloVcl 447 HIVHXB2 ProLeuThrrl~ A1nr~ A~nrl~ ^rgr~ T1~ ~ ~.-l vrGluPro 468 HIVBH1 ~2 480 HIVRF Gln-------Lys 4~7 HIYMAL ---------Alo 467 -_= :
_~

WO Q0/10230 2 ~ 5 3 Q ? P~,CA90,00062 ~ble l cont'd HIVHXE2 VolHisGlyVclTyrTyrAspProSerLysAspLeuIleAloGluIleGlnLysGlnGly 488 HIVPV22 SD~

HIVMN Vcl ~97 HIVSF2 ~ -Glu Val 488 HIVMAL 4~7 HIVHXB2 GlnGlyGlnTrpThrTyrGlnIl-TyrGlnGluProPheLysAsnLouLysThrGlyLys S0 HIVBHS S2a HIVBRU 52a HIVRF S~7 HIVMAL ClnTy.- 5~7 HIVEL~ Hi~- S07 HIVHXB2 TyrAloArgMetArsGlyAlcHisThrAsnAspYolLysGlnLnuThrGluAloVolGln S28 HIVBH1 ~2 S40 HIVBRU ---~ ----Tl.. Sl-O
H IVMN 4 ~7 HIVMAL ---- --~IleLy~' - 527 HIVELI Alo S27 HIVHXB2 LysIleThrThrGluSerIleValIleTrpGlyLysThrProLysPheLysLeuProIle S48 HIVBH102 ~ S60 HIVBHS S~
HIVPV22 S6a HIVMN ~----Ala A. ~ 4S7 HIVSF2 _----Vnl S~ S48 HIVRF -----VolAlo S47 HIVMAL -------AloGln A, y S47 HIVELI Ars----~ A. !~ A. ~ S47 WO 90/10230 Pcl'~CA90/00062 ~o~03-0~

lleble 1 cont~d HIvHxa2 GlnLysGluThrTrpGluThrTrpTrpThrGluTyrTrpGlnAlaThrTrpIleProGlu SG8 H~VBH1 02 580 HIV8HS s8a HIVGRU ssa HIVMN Tll. I I 1 477 HIVSF2 ~lo---- Met 568 HIVRF Alo 557 HIVMAL ~lc 567 HIVELI Alo 567 HIVHXB2 TrpGluPheVolAsnThrProProLeuVolLysLeuTrpTyrrl r~ ysGluPro 588 HIVPV22 6a0 HIVMN--------Vol 497 HIVMAL Thr----- - 587 HIVHXB2 IleVolGlyAloGluThrPheTyrVolAspGly~1n4~ n'irgGluThrLysLeuGly 608 HIVBHS S~ 62a HIYPV22 Arg---- - 620 HIVBRU Ser- 620 HIVMN Lys---_ 517 HIVRF ~ lc 607 HIVMAL Lys------ 607 HIVELI ---Ilr 6~7 HIVHXB2 LysAlrGlyTyrVolThrAsn~rsGlyArgGlnLysYclVolThrLeuThrAspThrThr 628 HIV8HlD2 Ly~ r, ~ ~r- - 640 H~VBHS ll~s--------- 640 HIVPV22 - Leu------LyL- r, ~. Asn-~ - 64a H~VBRU 64a HIVMN ~ 5~7 H}VSF2 A, S~rIleAlo----_ 628 HIVRFA, '`~ - 627 HIVMAL ~j S~ - Clu---- -- 627 .HIVELI ~p r, ~ 627 WO 90/tO230 _ PCI~/CA90/00062 20~03a2 1 9 ~
Table l cont'd H~VHXB2 AsnGlnLysThrr~ nTloryrl~uAlnl~l~L~lnAer~- GlyLeuGluVcl b48 HIYBH1 ~2 b6a HIVBHS llis ~ b6a HIVPV22 b b a HIVBRU llis. b6a HIVMN llis HIVSF2 llis 6 8 HIVRF ~ llis b447 HIVMAL llis- Se~r-------- 647 HIVELI A~/~ B47 HIVHXB2 AsnIloVclThrAspS~rGlnTyrAloLouGlyT1 oTl ~"1 L~Al ,.r1 nPro~`erG1 n-., bb8 HIVBH102 ~ Lys----- bS~a HIVBHS Lys----- 68a HIVPV22 b8a HIVBRU Lys----- b8a HIVMN
HIl~RF Ly~___ bb687 HIVMAL Lys~-- b6bb7 HIYHXB2 GluSerGluLeuVnl ~ '1 nTlnTl ~rl 1 ''l nl ~.~ITl ~ysLysGluLysVclT rL
HIVBH102 Y cu 78a8a HIVBHS 7aO
HIVPV22 Cln 7aa HIVBRU 7~a HIVMN S6.-HIVRF .~ b88 HIVMAL -----~-Ilo Cln ', - b87 HIVELI ~ ~ b87 HIVHXB2 AlcTroVclProAl~lHisLysGlyIleGlyGly~snGluGlnVclA~pLysLeuVclsor 7a8 HIVBHS 72a HIVPV22 72a HIVMAL C,.. A, ~ 7a7 HIVELI 77oa77 WO 9O/10230 _, _ P.Cr/CA9O/00062 . = =~

T~ble l Cont ' d 2 Q ~ 0 3 0 2 HIYHXB2 AloGlyIloArgLysVclL.~.ri l ,A~rr- yIleAspLysAlnr:1 r A ',nGl H; er~1 u 728 HIVBH102 Ilc 740 HIYBH5 nO Clu -- 74a HIVPV22 Ilc 74D
HIYaRU 740 HIVMN rl ,.A~r - 6'i HIVSF2 ,en Clu 728 HIVRF Tl~. 727 HIVMAL ' , Clu 727 HIVELI Gln Clu -727 HIYHXB2 LysTyrHisSerAsnTrpArrAlnMetAlaSerA~r; ,A~ vr. .,VGlVclAlc 748 HIVBH102 . - 7bC

HIVBRU 76a HIVMN Ile----~ 657 HIVMAL :llo----------- 747 HIVELI ~ 747 HIVHX82 LysGluIle~vnl Al. '`- Cr,AspLysCysGlnLeuLysGlyGluAlci~letHisGlyGln 768 HIVaH5 780 HIVSF2 ~ 76B

HIVHXB2 VclAspcysserrrc6lyIleTrpr~ rCysThrHisLeuGluGlyLysVclIle 788 HIVa H5 80 0 HIVBRU - 88Oo HIVSF2 Ile-~ 788 HI VRF I l n ---- 787 HIVMAL Iln--- 787 - . ~

WO 90/10~30 PCI/CA90/00062 2~503~2 21 ~ ~ _ ~ahle 1 cont ' d HIVHXa2 LeuVelAleVclHisVclAleSerGlyTyrT~ -'`1 Al ~r,l uVclIloProAloGluThr 8a8 HIVBHl ~2 - 82a HIVaHS 82~
HIVPV22 82a HIVBRU 82a HIVMN 7~7 HIVSF2 8a8 HIVRF 8a7 HIVMAL Ile 8a7 HIVELI 8a7 HIVHXB2 GlyGlnGluThrAloTy. r~ ysLeuAloGlyArgTrpProVclLysThrIle 82 HIVBHla2 ` s4a HIVBHS 84a HIVPV22 84a HIVBRU 84a HIVRF Ilo Vcl--- 827 HIVMAL Ilc VelVcl 827 HIVELI Y.. lVcl 827 HIVHXB2 HisThrAspAsnGlySerAsnPheThrGlyAlcThrVolArgAleAleCysTrpTrpAle 8'-8 HIVBH1a2 '~ - Lys B6a HIVBHS S~ - Lys 86a HIVFV22 S Lys- 86a HIYBRU SHrThr--------Lys 8ba HIVMN r. ~, SerThr------Lyr, Thr 757 HIVSF2 S~rThr--------Ly 848 HIVRF ~ Thr--------Ly~ 847 HIVMAL S~r----Ale---Lys- 847 HIVELI Ser---Alo---Lys 847 HIVHXB2 GlyIleLysGlnGluPheGlyIlePreTyrAsnProGlnSerGlnGlyVolVelGluSer 8b8 HIVBHla2 ssa HIVBHS 88a HIVFV22 - 88a HIVBRU 88a HIVMN Il e------ 777 HIVRF ~ 867 HIVELI 8b7 - ' ~
~Q~ z ~able 1 cont ' d .
HIYHXB2 MetAsnLysGluLeuLys~vsIleIloGlyGlnVclArg~rr1 rAl rrl ~HisLeuLys ~88 HI~i3RU - - 900 HIVSF2 ----------A~.. 888 HIVRF Cln------Gln 887 HIVMAL Clu 1187 HIVHXB2 ThrAloVolGlnMetAloVolPhorleHisAsnPheLysArgLysGlyGlyIloGlyGly 908 HIVMN A, y 817 HIYELI ~ ArgA. y 907 HIYHX92 TyrSerAlrGlyGluArgIloYclAspIleIloAloThrA5pIleGlnThrLysGluLeu 928 HIVMN Cl~ 837 HIVMAL Ile---Met 927 HIYELI Ilc 927 HIYHX82 GlnLysGlnIleThrLyeTl~ A~npheArgvr~lTyrTyrArgAspserArgAsnser 948HIVBH102 - r~O 9~Q
HIVBH5 r. v 9bQ
HIVPV22 r. v 9~0 HIVBRU AspPro 9~Q
HIVMN AspPro 857 HIVSF2 A.. ,LysAspPro 948 HIVRF AspPro 947 HIVMAL ~.sn---AspPro 947 HIVEL~ Ilc AspPro 947 -20~03~

Te~le 1 ~ont'd HIVHX82 L~uT~pLysGly~ro~lcLysL~uLeuTrpLysG~yGluGlyAloVclVclIleGlnAsp 968 HIVPV22 98a HIVRF llis 967 HIVMAL Il~ 967 HIVELI Il~ 967 ror cds stcrt -~
HIVHXB2 AsnS~rAspIluLysVclVclProArsArgLysAl~Ly~IleIl~lArsAspTyrGlyLys 988 HIVBH5 = 1 OOC
HIVPV22 -- 1 ODa HIVMN ___A '~'1.l 897 HIVELI Lys V.. l 987 HIVHXB2 GlnMetAlcGlyAspAspCysVclAlcserAr3r~1nAerr~1. Aerf+~ 1a04 HIVBRU - 101~
HIVMN -----T' - 913 HIVRF 1 O0~
HIVMAL - ClyGly 1 ac3 - "~Z,~
' WO 90/10230 Pcl'~CA90/00062 ~ f Q~ 24 T.~i LF
HIV-l pol gene HIVHXB2 Sequence Data from Human Retroviruses and AIDS, 1988 Los Alamos National La~oratory AcNPV-~lIW~laol Virus - --- RBS
Bam R I t i S~ - GG~TrrTATAAATATG tttttto uY
pol cds tort (NH2-t~rminus unc~lrtoin) ->
2101 ggccttcCto c__uuy_uyy ~'''9~Dnntt LL~L~_yuy r~ 'U''U '~' ''''' ''U' 2161 ccc~ogco30 y ~ LL ~uu t.LyyyyL_y J L~ L__y J yy ~y 2221 csotcgocco ggooCt9tot cctttooctt ~_~L~uyyL~ ~_L~L~yy_ u~
2281 cgtcccoCTA Ac9ctc3Q99 uY ~ yy__y~Lu L~uy_Lu__u a_y,_y_~uu <- gcg cds end 2341 ~ nttn goc3ccctgo uL~Ly_,uuu n.. ~,.l y y.. ~ .. ".1 ~J~I L~.~yyyyy__L
2401 tggoggtttt uL.___uLuu y__uyLuLyu tcogotoctc uLuy___L~L gtggocotoo 2461 ogctotoggt u~_yLuLLuy toggocctoc G__LyL~uu~ uLuuLLuy_u y_uuL~LyLL
2521 goctcogctt ggtt3coctt tooottttcc cottogccct uLLy_y_~ Ly Lu~__yLu__ 2581 ottooogcco Dy__LyyuLy gcccoooogt Lu_~ Luy ~ Li J y __y___uuuL
2641 cooogcotto yLuy___LLL gtocogogot y~ -- -- ~uu ~ Yyy--------LLL c_____LL_u 2701 gcctgococt c__Lu_uuLu ctccogtott Ly__uLuu_y ~ ~, yLu_LuuuLy 27bl gogcoocttc gLuy_LLL~u yGyu__LL__ tccgogcoct L__y__LL~L gggcogttco 2821 cttcggoctc _____L___y __yyyLL___ c__yu_____ tcegtoccog Lu_LyyuLyL
2881 gggtgotgco LuLLLLL__y ttcccttcgc ~y__yu ~LC ~ uu__yLu~C ctgcctttcc 2941 cctccctcgt _L______Ly _y__~___yy gcttogotct __yLu_uuLy Ly_LLCCU - -3D01 gggctggooo yy_L___~,uy ~__LuLL._u a_yL_y~_Lg acoocootct L_y_y~LLL
3061 tcgoooococ ~c~ J ~, LuyLLuL~Lu L.__L___Lu D-Ly_LLLyL otgtoggotc 3121 Ly__LL_y__ UI~u~u~J~ uLuy~_____ __L_y_yy_y ~ Ly_y_____ otctgttgog 3181 gtggggoctt c~ ~ u ocoooccoco ~_y___yuu c,L._uLLcc LLLyyuLhyy 3241 ttotgocctc __L__Ly_Lu cctggoccgt ___u__L_Lu YLY--LYC----y ~ u 3301 ctggcctgtc __Ly___Lu~ ogoogttogt yyyy___LLy uuLLyyy___ gtcogctttc 3361 cccogggott y~ ~y~ u_L~_~yLuu u_L~LLuy_ ~ u ~_~Lu_~_y_ 3421 cgtcctoccc ~Luu__y__y ccgccgogct _yu__Lyyu~ J -- y y _y_LL.L___ WO 90/10230 2 ~ 5 0 3 ~ 2 PCI/CA90/00062 2s T~ble 2 cont~d 348~ Uy~ Uy-U UU~yy_y~y~ ottotgac~c Ul'''' '' J" ~ I J _ aaatacagaa 3541 y~_yyyy~__ yy~_uuLyyu cotatceoot ~U~_Uyuy ~` ''I I 1'' ''''' ctctgoGcac 3601 2ggeaoatat 9UU_yuU~y_ ggggtgccc3 ~ yu~ y~ .. U.. IU~
3661 uy~y~ _y aea6catayt __~u~yyyyu _uy_~ a U_-~LU__~
3721 ~ _yy___~_~ gggaaacetg gLyy_._y_y LuLLyy~__y ~ Lyy_L
3781 tcctgegtgg b--y~y~ etecccctcc u~LuyLy__u LLuLyy~_~ _yL~_yuy__ 3841 agoacccata y~_yy_y~_y aaeccttcte ~y~_y_~yyy g~._y~ a yyy_y_~
3901 etteggeeee y~_yy_~u~y ttecteeteg -,IJ J. u__y~y~C~ y_ 3961 ceceeceeet ~-Y--Y-~h egtteceegc eatttatcto y~ y~_yy u~c~yy_L~
4021 uy__y~ uy~uu~uy ectceceete ~y~_~L_yyu uL~uLL~__y 4a81 tceeegtgee t~uy_yL~_u tcaatceaat ~ Y ~u_~l~____ _yy____yy~
4141 ctetctggce tyyy ~c,c.uy cececeeegg r . ~ ~ ~JJ J-J. __ Ly__~__y ~uyu Lu__ L~
42al ogtcegtgct u~ JU- eegtectett ~uyu~y!~ _y_~u_yy cc~__y_Lyu 4261 G~u~y_yuu_ Lu~ uy-u ettggagasc __~yy~uy~ 5uLL~ y~ LuL
4321 _yLuy~____ y___~_yLuy ccagctgtga u__Ly~ y ~____yy_y __y~hLy~u 4381 ~yyU~__y~a y_~y~_y~C caggaatatg y~ uy_~ tgtacocott ~nDnn9!Jnnn 4441 _Y~ U Y~UY~UY~ etgtog~ceg ~yyu~ ' '-J ' J ttettccegc . 4501 UYU__.__YYY ' JJ Y cetettttct ~ u y~_yyu_y_~ yy.._y~u__ 4561 nn~ nrnt ~ yu.__~y gcegceettt ~_~yyy.,~ e.yy~uyyy ..y..Ly~--y 4621 9 ~yyy~uyyu ~ y eetttggeet tcc. ~ CC~C~C~_y~ __yy_y~_y~
4681 _y_u~Lu~y n l ~....~ teaageooet ~u~uyy_~_y steogegetc uyy~Lyuu~_ 4741 t.LLuuyu._ y~.uy~u~uuu tggCOgtCtt ..uL..u.uu~ ~LLu___y__ u_yyyyyyuL
4801 Lyyyyyy~ul, Gy~y~_yyyu eeegeetegt ~yu~u~ J u Lu~___~_u 4861 u ~ uuu~uu_--~ coeeeettce G__LL~yy gtttottecA rlJ~....u..~J
/~ 3'sj 4921 eeettcecet ~ UJ -~ JUV~ cegceeogct ..~.-yy___ gGTgoogggg ~uyLuyLu_~
5'sj /~
4981 oceaygataat ~y~y_~u_ oegtagtgcc ----u J ----' D~ nt~n ttagggattA
sor 23 hD ~ds .start ->
B~m 1~1 5a41 Tûgaaaaceg ~uy~ayy~y otgettgtgt yy~__y~uy_ c_y__~y_yy etTAGGATCC-3' l~nker ~oquenc~
,~_.

WO90/10230 ~ 26 PCr/CA90/00062 HIV-l pol gène HIVHXB2 Sequence - -Data from Human Retroviruses and AIDS, 1988 Los Alamos National Laboratory AcNPV-}~IVWllDol Vir~us PDS
Bam l'I ti (AcNPV-H~VWllpol start) S ' -GvATC-TATA~TATG tttttto gggocgot-t pol ~ds ~tort (NH2-t-rminus unc~rtcin) ->
2101 yy,~LL~L_ cocgggccgg cccggg3c:t LL.LL~ y_y J y J ~ y~,, 2161 J yn gogctteogg tct3gggtc3 n~nrnn~nn~ Lc~L~_~ ~ vJJ y 2221 Cy_L_yy._h ggccctgtct cctttccctt ccctccggtc _~L~LL~yy~ G_~y_~
2281 cgtcoccoTA AogCtCgQgg g9COCCtOOC yy__~L~L_ LL_y_L_~_y h_y~_u_Ly~
AcNPV-YIVYKool sa~ l I P~s ti Virtls ~ -~,~AT~rT~T~ TG (.ti;AcNPV--}~TVY}Cpol ~tart) 2341 L~_yL_LLq~ gccgocotgc gtttgccogg ' y ~ U ,~ Y thyyyyy__L
2401 ~yy_yyLL~ L otcooogtoo goccgtctgc tcogctnctc _~_yy__L~L yLyy_~_Ly_ 2461 ogctotoggt o~ùgtottog toggoc~toc h~.LyL.__. ntoottggoo y___L~Ly~L
2521 goctcogott ggttgcoctt ~_y_LLL~.~ cottogccct _L~y_y__Ly toccogtoao 25B1 _LLu__y..h ggootggoty gcceoooogt L_v_~_~Lyy ccottgocog h_y______L
2641 oooogcotto gtogooattt gtocogogot yy____yy__ yyy____LLL ccccoottgg 2701 y~Ly____L Ccctocooto ctccogtott Ly.._L~__y ''''" " J''' gtcctccctg 2761 y_y____~h gtogotttco y_y___LL__ tcogogooct coogocttct gggoogttcc 2B21 _LL_yy__Ly CcocotCccg _yyyLL~ n J t._yL_h._y ~ ~yy_Ly~
2BBl yyyLy_Ly,_ totttttcog ttcccttogo tgoo30cttc yhy__yLY~u ctgcotttcc 2941 C_L~ yyL otc2cCc2tg ogocoCcogg gcttcgotot ~Yy~_,__Ly Ly~LL~y~_ 3001 yyy_Lyy___ ggotcoccog cootottccc y_y--hy._Ly y_~____L.L tcgcgccttt 3~61 tyy ___._y ootcCogoco togttotctc t~cotocotg g2tgotttgt hLyL_yy_L.
3121 ~y_~LL~y__ otogggCogc ~Lyy_~ J yy~ y ~Ly_y~ a~LgLLy_y 3181 ~Lyy~y_~LL occcCùCCog n~____n_~_ t.:._y_~_y__ ~_L~_LL~ LL~yy_Lyyy 3241 LL_Ly__.~c cctcctgctc qotgg2ccgt occgcctctc y~y~Ly~_y ~ , y ~
3301 ,Lyy_~LyLc qctgocotoc 2920gtt2gt yyyyY__LLy L~ gtc2g2ttt2 3361 ccc2gggctt ocogtooggc _yLLoLyL.,2 _.LC.L._y_ yy ~ y Ch.L__._y_ 3421 _yLy_L_~_ ctoocoooog qogcogogct hy__.Lyy.h ~ ~ y J cgcttctcco ,,_, .. y, ~

..

I-J ~ n ~ ~ l~ tn n ~ ~ a ~ n a r t ~ ~ n tn 0I r a ~ r .1 1~ a ~ ~ e t~ r ~ D~ r D~ tr ~ D a-tr' j . r e-r . i ~ r r r! ~ t O D~
r ~ I
r " ¦ ~ ~
P , I I . ~
k . , . r~ O
11 r r I r ~7 C'' ' I ~ c ~! ~ r~ ~ D7 ~ ~ t~ I r ~ ~ ,(~
r r r t~ t~ I I I r O
1 ~ 1 0 , WO 9O/10230 ~ PCI~/CA90/00062 3~ 28 ~r~ble 3 eont~d . ~
4981 C___yuL__~ ogtgocotoe ---y-_y~y~,C ~ u gcocogotcc ttogggottA
Bam h'~
5041 TGgoooocog ctggcoggtg u~y_~b~y~ yy.._~.y-uy_ coggotgogg atTAG.~TCc ~ph I
C.^A G-3 ' <- pol end 5101 yy_~__y~-- ogtcocococ C~ Y~ _yyy__ ogctoggggo ~yy~ uL_ 5161 ~ tgocogccct ~ u~ ~~~Y~--Y-- ~Y~ C ~ ~YYYY
5221 uLy~.Luy_L~ ggt3cteccc _~.uLu~Lyyy yL-Ly~._Lu~. cggogoccgo y~ yy~u~
5281 ~yyy ~_y_y cgtctc~ctc 9 ~ LIJ 'JU' ' G___y_y~ --Y'--'~ ---- Y L--U--'~ LY
341 u '~ u :~ ccccctcctt C--.-y-_-L ~LlU. I tttttcAGcc ~Ly~_-__ /~ 3 ' sj 401 ~ ottcggcccc u~oy~uy~ uyy~y~y_ ctctcccgcc yy_~_~u_.._ 5461 cgGTcggotc tctococtcc ~-yy~__-uy ~,_yu_~u~ c_y_~_u-yc 5'sj /\
5521 LL~ u~ C tcgtgttocg ~J ''d cggctcgATG y. J. '' C -J ~.~'~G
R orf cds stort ->
558l ~YuJ~ , cgggogccoc c_c_~y__~y gccccTAGog cttttcgcgg _y~__y__ <- scr 23 hD cds end 5641 tgccgctgtt cgccottttc ~.~_yy_~y y~ yy~ ~_yyy~__~_ ototctctgG
57~ yyy gotccttggg .,_yy_y~yyu uy~_-__--~i Gy__LLuLyu G_~__~y~
5761 y~i~uL-~uL tttcAGoctt yyyLy~yu~, cTAGcogcct cgg~gttoct ~,p /\ 3'sj <- R orf cds end 5821 Dng~r~D~ TGgcgccogt _y_-..-_"u ~.~uy_y.,~ yy__yu_~, _yy__y~_y tct cds stcrt ->
5881 ~ ~ u cttgtoccco ~y~u~yL G_u~ _yLyLL u ,-----u~y ~C_y~y 5541 LL._L__.__ oogccttogg cotctcctAT GD~ u ~J~ J~ ' u u I u trs/ort cds stort -> /\ 3'sj 6DDl u.,L-_L~_y_ ocogtcogoc ' l J' ~ L.-.~u~.__ ogcoGToogt ogtocotgto (tct, trs/crt, 27 hD) 5's; r\
6061 ~,rG~ ~t- toccootogt Gy~ uyLu y~_~L~yLDy t~y,~ cotogcorto U orf -->
6121 _LLyL.y~_yL c--=tcgtcot e.. ~uy__~u~ ~yyu___~uL ~u y_~ y uuu__~uDu~
~ '

Claims (5)

1. Claims:
   l. The use of a polypeptide as a reagent in a diagnostic test for HIV infection characterized in that said polypeptide comprises substantially all of the amino acid sequences of the reverse transcriptase, RNAase H and integrase enzymes coded for by the HIV-pol gene and the amino acid sequences of part of the protease enzyme coded for by the HIV-pol gene omitting the active site responsible for proteolytic activity.
2. 2. The use claimed in Claim 1 characterized in that said polypeptide has an amino acid sequence substantially as shown in Table 3 beginning with the amino acid Met marked "AcNPV-HIVYKpol starts".
3. 3. A diagnostic kit for detecting antibodies to HIV
   antigens characterized in that said kit contains, as a test reagent, a polypeptide as defined in Claim 1 or Claim 2.
4. 4. A polypeptide comprising a proportion of the amino acid sequences coded for by the HIV-pol gene characterized in that said polypeptide comprises substantially all of the amino acid sequences of the mature reverse transcriptase, RNAase H and integrase enzymes coded for by the HIV-pol gene and the amino acid sequences of part of the protease enzyme coded for by the HIV-pol gene omitting the active site responsible for proteolytic activity.
5. 5. A polypeptide as claimed in Claim 5 characterized in that said polypeptide has an amino acid sequence substantially as shown in Table 3 beginning with the amino acid Met marked "AcNPV-HIVYKpol starts".