CA2082806A1 - Immunoassays for anti-hiv-1 antibodies - Google Patents

Abstract

A method of diagnosing an HIV-1 infection in an individual comprising contacting a biological sample from the individual with a first antigen capable of forming immune complexes with antibodies specific for native HIV-1 but substantially incapable of forming immune complexes with antibodies specific for a second antigen which causes production in an individual of HIV-neutralizing antibodies, formation of immune complexes with the first antigen being diagnostic of an HIV infection in the individual.

Description

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The lnvention relate~ generally to immunoassays in which antibodies specif~c for human immunodeficiency YirUS
S type ~ SHIV-l) are detected.
~ IV-l is the proposed causative agent o Acquired I~mune Deflciency ~yndrome (AIDS). Individuals in~ected ~y HI~1 produce ~erum antibodies which bind various viral proteins, par~icularly the core, polymerase, and envelope proteins (Allan et al., 1985, Science 228:1091; Chang et al,, 1985, Science 228:93, and 1985, Nature 315:151; Crowl et alO, 1985, Cell 41:979; Veronese et al., 1985, Science 229:1402, and 1986, Science 231:1289). Immunoa~say which d~tect these ~ntibod~e5 are used to dia~nose HIV-l infection lS and to screen donated blood ~or HIV-1 contamination. For axa~ple, enzyme-linked immunosorbant assays (ELISAs) and radtoimmunoa5~ays (RIAs) o~ various types are used for rapid ~creening and initial diagnosis. Whole viral lysates, which contain both HIV and non-HIV proteins, and puri~ied viral component~ including recombinant proteins and synthetic peptide~, have been used as antibody adsorbants. Western blot. ha~e been used to detect antibodies which rscognize HIV protein~ that havQ been separated by size.
Su~ary o~ the Inventi~n The invention provides diagnostic ~nd detection ~ct~ods wh$ch can be used to distinguish between person~ who ~r~ in~Eected w~th HIV-~ and per~ons who have been vaccinated again~t ~ l wîth ~ept~des correspond~ng to zlll or ~
portion o~ ~he principal neutraliz~ng determinant (PND), ~nd ~l~o o~Per~ a ~or~ ~en~ti~ and less expensiYe diagnostic m~thod, e.y., ~or ~etectlng seroconvers~on.

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W~ 011 P~T/U~91/~021 ~;?;~

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Accordingly, in on9 aspect, the ~nvention feature~ a method of d~agnos~ng HIV-l ~nfection in a hu~an which lncludes contacting a biolog~cal sample from the human with a ~lrst antigen which is capable of forming immune complexe~
5 with antibodies specific for native HIV-l but substantially incapable of ~orming i~mune co~plexes with a~tibodie~
~pecigic for a second antigen whlch causes production ~n an hu~an of HIV-neutraliz$n~ antibodie~, formation of immune complexes w~th the f~rst antigen being diagnostlc of an RIV
~nfectlo~ ~n the indi~dual. (As used herein, 'neutralization' refers to the ability o~ the antibody to inhibit XIV infection o cells by cell-free virions, or fusion o~ infected and uninfected cells, or both; 'immune complex' refers to a non-co~alently bound antibody/antigen pair; ~specific for' and 'reacting with' ~ean ~apable of ~peci~lcally binding to an antigen; an anti~ody that is ~substantially ~ncapable of formin~ immune complexes~ means that ther~ will be les5 than a three~fold di~ference between the binding o~ the antibody to a specifled antigen and the ~inding o~ nor~al hu~an serum to the same antigen.) In preferred embodiments, the biological sample may a bodily fluid, e.g., 6erum; and the first ant~gen is lmmobilized on a solld support, although`any standard immunoassay ~ormat can be used. To determine if a person 2S has ~een vacci~ated, the method ~ay ~urther include the ~tep3 o~ contacting th~ sampl~ with a second antigen which is-immobilized on a second solld support, wherein the second antl~en i8 the lmmunogen. (~s used herein, "firstn and ~s~cond~ do not imply the order in which the screenlng ~tep~
~r~ per~or~ed-i Preferably, th~ first antigen i~ HIV-l gp160 Qnv~lope protein which lacks a portion ol' the princ~pal n~utralizing deter~inan~ (PND) or a portion o~ the PND to WOg~/l9011 PCT/U~91/~40 - 3 o which neutralizin~ antibodies can be raised, i.e., a neutral~z~ portion thereof. (As used herein, th~
~principal neutralizing det~rminant~ eneompas~es the region o~ gp160 located between a~lno ac~d~ 297 and 3~1, inclusive, accordlng to ~he number convention o~ Ratner et al., 1~85, Nature 313:277, hereby incoxporated by reference~) Mo~t preferably, this protein i~ gpl60~135. (As used herein, ~p160~135 means any gpl60 prctein that lacks t~e PND;
gp160~135 can ~2 expressed ~rom a recombinant gene t~at has been engineered to delete the DNA coding f~r the PND or a portion o~ the PND to which neutralizing anitbodies are raised, or may be a protein which has been chemically altered to lack the PND.) Preferably, the second antigen includes the PND of gp160; more preferably, it include~ intact HIV-1 gp160 envelope protein (i.e., which contains the principal neutralizing determinant), ~.g., gpl60MN, or a fragment thereof or a ~ynthetic peptide havlng an amino a~id ~equenc~
corresponding to that oP gp160, any of wh$ch ~ay be the i~munogen. Preferably, the second antigen is a protein fragment or a syntbetic peptide having an amino acid ~eguenc~ corresponding to that o~ the PND of gp160, e.g., the PND o~ gp160MN (i.e., gp160 from the MN isolate) or a neutralizing portion thereof; most preferably, th~ second antigen is a combination of gp160MN and the PND of the im~unogen. (As used herein; an 'immunogen' i5 an antigen which causes th~ production of neutralizing antibodies by th~ i~munQ system o a ~ammal,.2.g., a rabbit on a hu~an.) Thu~, the detection met~od ~n w~lch the anttgen i~
gp160~35 result~ i~ detection of BIV-1-sp~c~fic antibod~eR
whi~h reco~niz~ regions of gp160 ly~ng outside the PND~
where~ the detection ~ethod ~n whlch the antigen i~ gp160 ~and ~ay include PND pepttdes) results in detection of ,, W~ 91/19011 PCT/US91/040ZI

_ ~, antibodie~ specific for ~ny portlon of the gp160 molecule, and will include the detection of PND-speci~ic antlbodie~.
A biological sample ~rom a person infected with HIY-1 would include antibodle~ detectable by both methods, wherea3 a ~ample ~rom a p~rson who had been immunized against HIV-1 with PND peptide~ but who 1~ not in~ected with ~he ~iru~, would include antibodies detectable only in the latter ~ethod, which employs either the complete gpl60 molecule, i . e., that contains the PND, or PND pept~des, or a combination o~ the two. As used herein, an amino acid sequence 'corresponding to' or 'derived from' means the amino acid sequence ~s identical to all or a portion of anot~er amino acid sequence. As used herein, IMNI refers to th~ MN prototype of HIV or a viral variant of the MN
prototype; the MN prototype virus ~s defined by the amino ac~d sub-sequenc~ with~n the PND region o~ the gp160 envelope protein R-I-H-I-G-P-G~R-A-F-Y; MN viral ~arian~s are herein de~ined as variants which exhlbit complete amino acid ~equence homology at re~idue~ I-G-P-G-R, and at least 36~ homology with the remaining 6 amino acids of the MN
s~quence glYen a~o~e.
In other preferred embodiments, the method further includes contacting the sample with an agent ~e.g., an enzymatically or radioactively labeled component) capable of b~nding to the first and second adsorbed antibodies, and detecting the bound agent as an ~ndicatlon of the presence in the ~a~ple o~ ~irst and second antibodies; for example, the agent may b~ protein A or a labeled ant~body capable o~
~inding to the antibod~e3.
The $nvention also ~eatures an HIV-l envelope $n w~c~ the pr~ncipal neutral$~ng deter~inant, i.e., th~ region lying between a~ino ~cid residue3 297-331, .. ~ .

W091/l90~ P~rt~S~1/~021 - 5 ~ .~J'~J~
~clu6ive, or a region encompasslng all or part o~ the PND, or a neutralizing portlon of ~he PND has hee~ deleted.
In preferred embodiments, this prctein ig gpl60~ 135, and ~ay be immobil~zed on a solid support.
T~s inv~ntion al~o ~eatures a kit for detecting the presence of HIV-l ant~bod~es in a hu~an biological sample, which include~ a flrst antigen capable o~ for~ing immun~
complexes with antibodie~ specific ~or native HIV-l but su~stantially incapable o~ formin~ im~une complexes with antibodies sperific for a second antigen which causes production in ~ human of HIV neutralizing antibodies, and an a~ent capable of reacting with first and second antibodies.
In preferred embodiments, the ~irst antigen is l~obilized on a solid support; preferably, it is HIV-1 gpl60 whlch contains a deletion o~ the PND region lying between residues 297 and 331, or a portion of the PND to which neutralizin~ antibod~e9 are raised; most preferably, the rirst antigen is gp160~135. The detection agent may ~e labeled and ~ay be, ~or ~xa~ple, protein A or a labeled anti-human immunoglobulin, e.g., a labeled ~c-reg~on-~peci ic antibody. T~e ~it may further include the second antiqen immobilized on a ~upport; preferably, t~e second ~ntiq~n is ~ peptide ha~ing an am~no acid sequence corresponding to the amino acid sequence of an immunogen which caus~s production o~ ~IV neutralizing antibodies, i.a., ~h~ ~IV-l PND sequence ~ro~ the ~N variant; mors p~e~erably, gpl60 is immob~li2ed on the 8econd support, and th~ peptid~s have ~equences corresponding to t~e sequence of ~h~ PND 0~ the ~N PND. -In ~noth~r aspeet, ~he invention ~eatures a ~ethod o~ ~etecti~g ~V-l in~ection in ~ person, which includes ~ontacting an ant~body-containing blologi~al sample f~om ~he p~on With HIV-l gp160~N, ~ormat~on o~ immune complexe~

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W~91/19~11 P~T/US91/~0~1 2Y~'~

between the ~ample ant~bodie~ and gpl60MN be~ng diagnostic of HIV infect~ on. 8ecause th~ solate is a co~nonly occllrring HIV isolate, the use o ~p160 ~ontaining the PND
~equence o~ the MN i~olate provides o~ æ more sen~itl~r~
5 a~say and allows for the earlier detection of serocon~ersi~,n than carl be obtained with other gp160 PND sequenc~, e.g., In preferred embodiments, the f~rst antigen ~
~mmobilized on a solid supp~rt. The antigen ~ay further include a peptide having an amino acid sequence corresponding to an HIY-1 gp160 PND or a neutralizin~
portion-of the PND. When a synthetic peptide corresponding to all ~z a porti~n of ~he PND are added to assays based on a gp160 adsorbant, the amount of gp160 used in the assay can be reduced without a loss ~n assay sensitivity.
~he invention also feature5 a kit ~or detecting HIY-1 antibodies in a human biological sample, which ~ontain~
immobilized HIV-1 gp160MN, and an agent capable of reactinq with an antibody.
In preferred embodiments, the ~ample is ~erum and th~ agent ~8 a la~eled ~olecule.
Tha ~nvention also featu~es a kit for diagnosing the presencQ o~ ~IV-l ant~bodies in a hu~an biological sample which lncludes combined ~mmobilized gp160 and a peptide 25 having an amino acld Requence corresponding to ~hat of-the MN ~ or a portlon thereo~, and an agent capable o~
reacting with the antibodies.
~ethods o~ the invention are useful for distinguishing ~etween HI~-l infectlon and lmmunization, ~nd 30 ar~ lnexpensive and ~a~e; gor example, recomb~nant proteln co~ponen~, such as ~ull-length qp160~ wh~ch are expens~Ye to produce, ~y b~ ~upple~ented with synthet~c p~ptlde~ ?
h~ving the ~am~ ant~en~c s~tes. ~ethods o~ the invention WO9111~11 P~T/U~91/~21 - 7 ~
thus reduce expense without sacr~f~c~ s~nsitivity, and enable early detectlon of ~eroconversion.
Other features and advantages of the invention will be apparent from ~he ~ollowing descriptioll of the preferred embodiment~ thereo~, and from the cl~ims~
~e~ç~ lpn Q~ ~referred ~mbodimen~s Be~ore describing preferred embodiments of the i~Yention, t~e drawings will ~e descri~ed.

Fig. 1 is ~n E~ISA in which gp160~ 135 and gp160IIIB
(i.e. ~ gpl60 from the IIIB isolate~ are compared as antigens for a panel o~ HIV positive human sera.
Fig. 2 i~ an ~ISA in which the response o~ the gp160~ 135-based assay was compared to the response o~ the 15 gpl 6oIIIB-based assay to Yarious dilutions of a single serum.
Fig. 3 1~ an ELISA in which PND-based peptides were u6ed .
Fig. 4 is an E~SA in which the ant~gen consisted of ~yn~hetlc peptides in combinatlon with gp160IIIB.
Fi~. 5 i~ a ~est~rn blot in which gp160~135 and gp160IIIB were probed with normal human serum (NHS), human ~IV po~itiv~ seru~, or anti-RP174C serum.
. Th~ invention pro~des a method o~ detectin~ and di~tinguishing between a~t~bodies which recognize different r~g~on3 o~ the HIV l envelop~ prot~in gp160, e.g., the prlnc~pal neutral~zing determ~nant vs. non-PND reg~ons, without 11 ~ignif icant loss of sensitivity ~or the ant:lbodies. Thi~ i3 accompl$~hed lby usirlg, as an adsorban~c, ~ r~coDb~nant envelop~ protein ~rom which the amino acid~
correspond~ng to only one determinant ar~ deleted; as a r~sult, anti~odl~3 directQd aga~nst the non-deleted , . . ~ ~ ~ . . .

W0 91/1901l PC~/US91/04021 ~ 8 ~
d~terminantt~) are detected, whereas those directed a~alnst the deleted determin~nt ar~ not detected.
,l~r~atis~o~ re~om~inan~el60III~
O~ ~I~L~
gpl60~ ~35 is a gp160 polypeptide which contains a deletion of tbe PN0 region. gp160~ 135 is used herein a~ an exaDlple of a gpl60 polypeptlde having a delet~ on which renders the protein incapabl~ o~ reactirJg with an antibody to the PND; the del tion may encompass one or more amino acids between residues 297 and 331, inclusive. The preparation of gp160~ 135 was described by JaYaherian et al., 1989, Proc. Nat. Aca. Sci. 86: 6768 . gp160~ 135 was derived from the gpl60IIIB envelope gene cloned into the ~aculovirus transfer ves::tor pAc610 (Rusche et al, 1987, ~u~ra). ~he BglII ~ragment of this envelope gene, containing the PND, waq subcloned into an ~13 vector for site-direc~ed mutagene~is. A synthetic oligonucleotide (5'CTACTAATGTTACAATGTGCGGGTCTTGTACAATTAATTT3') was used to direct the deletion of nucleotides encompassing the P~D
resulting in the deletion o~ amino acids 300-328 of the envelope gene. This mutagenized fragment was then cloned back into the baculovirus transfer vector and recombinant .. ba~uloviru~ obta~ned as described in Rusche et al., 1987, sup~a.
In the assays described below, a gp160MN/IIIB hybrid envelope gene, in which sequence from the I~IB isolate ~urrounds PND reglon cons~sting of ~N determina~ts, was used a~ an a~tigen. Although the gp160MN/IIIB ~ybrid was us~d, any MN polypeptidQ or fragment, or any ~N hybrid which retains ~he ~D of the MN isolate ~ay be used, ~.g., gp160~N. ~h~ gpl60~N/IIIB hybrid wa~ constructed by dir~ct r~placement of th~ BglII frag~ent o~ the ~N envelope~ Thi~
re~ulted in a hybrid IIIBIMN envelop~ gene h~ving an ~N P~D

WO 91/1~011 PCr/US91/04021 2~

and c:ontained in a IIIB gp160 framework. Antibodle~
generated to th~ result~ng PND region of the hybrid molecule are ~pec~f~c ~or MN. Thi~ hybrid en~elope gene contained in the baculoviru~ transfer vector pAc610, was then used to generat~ reco~nant baculovirus, as described in Rus~he et al., 1987 " upr~.
Product~on of recombinant envelope proteins by infectlon o~ ~nsect cell5 (S . fru~iperda3 with recombinant baculoYiru5 Wa~ performed as described by Rusche et al., 1987, ~
prePar~tion o~ Svnthetic Peptides Synthetic peptides corresponding to the principal neutralizing determinant were prepared by the Merrifield ~olid-phase synthe is procedure ~Merrifield, 1963, J. Amer.
Che~ . Soc. 85:2149). Peptides were purified by reverse-phase HP~C and characterized by amino acid composition analysl8, according to procedures well-known in the art.
The deslgnation of the peptides, the isolate to which they correspond, and ~heir seqUences are as follows:
RPl35 (III~ RKSIRIQRGPGRAFVTIGXIGC
RP1~4c ~III9) CNll~RXSIRIQRGPGRAiV~IGKIGC
RPl42 (MN) YNKRKRI~IGPGRAFY5TXNIIGC
RP70c tMN) INCTRPNYNXRKRI~GPGRAFYTTXNIIGTIRQAHCNIs Peptides RP174c and RP70c each contain an intramo~ecular disulfide bond between the two cysteine ~esidue~. A mQthod ~or creating such a bond is described in Zhang ot al ., 1988 , Blochemistry 27:3785. The existence of thes~ ~onds was con~ir~ed by mass spectrometric analysis.

ThQ adsorbed antibod~e~ ~re detected ~y any of ~vsral wQll-known ~ethod~, one example o~ which ls ~n ~nzy~e-linked ~mmunosorbant ~38ay ~E~S~ he ant$gen ~gplS0~135, gp160IIIB, gpl60MN/IIIB, synthetic peptides, or .. j :

WO91/1~11 pcr/us91~2l 2~

a combination o~ these reagents) ls diluted in 6uitable bu~fer ~uch as a sodlum carbonate buff~r ~p~ 9.6~ to a concentration o~ >0.l25 u~/ml (preferably l ug/ml) and 50 ul i8 added to each well o~ a polystyrene 96-well microtlter plate (Immunodynamic5, Chantilly, VA). After incubation for 16-20 hours at room temperature (RT), the wells are emptied, refllled w~th a Sloc~ing solution such as lO mg/~l bovine ~erum albumin ~BSA) in phosphate-bufered ~aline ~PBS) containing 0.02% sodium azide, and allowed to incubate > 60 ~n at ~ he wells are then emptied and washed three ti~es with PBS containing 0.1% Tween-20 (PBS-T). Serum ~amples to be tested for the pr~sence of anti-gpl60 antibodi~s are diluted in PBS-T and added to the plate (50 ul per well~. Several serum dilutions may be selected, lS After incubation for 90 mln at room temperature (RT), the wells are empt$ed ~nd again washed t~ree times with PBS-T.
In order ko detect human antibodies adsorbed to the gp160 antigen, a labeled antibody-detecting agent solution l~ added to the well~ and allowed to incubate for 30-90 min ~0 ~t RT. Exam~le5 of agents which may be used are goat anti-hu~an IgG:horseradish peroxidase con~ugate, goat anti-human IgG:alkaline phosphatase con~ugate, Staphylococcal protein A:hor~eradish peroxidase con~ugate, or any conventional . con~ugate. Following incubation with the probe, the wells are washed three time9 with PBS-T and treated with a ~uitable sub~trate solution such as the horseradi~h peroxidasQ substrate 3,3'-azlno-bis(3-ethyl benzthiazoline-6-sul~onic ~cid~(AB~S~ and hydrogen perox~de. The ~olutlon~ are allowed to lncubate 15-20 ~n and the ab~orba~ce o~ the ~olution in each well (at 410 nm gor ABTS~
~8 det~r~ined. The absorbance Or each ~olution i~
proportional to the a~ount o~ antibody adsor~ed by the antiqen.

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WO 91~ 11 P~/US91/~4021 ~Cçs~ina Q~ Se~um ~a~Pple~ Uslnc~ Q ~n~ia~
The procedure described above was used to test a panel o~ fourteen HIV-1-posit1ve human sera ~or bir~ding to gp160~135 or gp160IIIB (each coated at lug/ml in carbonat~
~uf~er). serum ~amples, which were obtained from the Interstate Bloodbank, Me~phi~, TN, and shown to test pos~tlve ln th~.HTLVIII EIA (Abbott Laboratories, Chicago, I~), wer~ tested at a 1:200 dilution. A goat anti-human IgG:horseradish peroxidase con~ugate was used as the lo detection agent. Signal-to-background ratios for assay of each serum ar~ shown in Fig. 1. The data demonstrates that i~unoa5says which employ ~p160~ 135 and gp160IIIB are equally effectiYe ~or detectlng anti-HIV antibodies in all Of t~e human serum samples tested.
A single HIV-positive serum was tested at varying dilutions using tlle above procedure. D~lut~ o~s ran~ed from 1:1 to 1: 8192 . As ~hown in Flg~ 2, the response o~ the gp160~135-based assay was ~dentical to the response of the gpl60IIIB-based assay at each dllution, within experimental 2 0 error .
~es~ir~LI~e-8çrum Samele~ UsinqLq~lQ
an~NI:~ Pe~?tides ~S antiaen~
~n order to show that antisera elicited by PND-based ~ynthetic peptides do not cause a response ~n the immunoassay based on gp160~135, the following experiments w~ra per~ormed. Animals werQ immuni~ed with the ~ollowing reagents, prepared a~ described above:
~P174c (gu~nea piq 596, guinea pig ~9~) gp160~135 (goat 1116) gpl60III~ ~rabbit ~
Anti~ra wer~ prepared ~ccording to conYentional ~ethods ~y i~munizing the de31gnated an~al with the corresponding peptld~ or prot~in. Each 6erum was tested in im~unoassays , - ~ .

W~91/1901~ PCT/USgl/~21 2 ~ 2 -u~ g each 0~ ~our di~erent antigens: RP135, R~142 ~
gpl60~ 135 and gp160IIIB~ Antigens ,were coated at 1 ug/ml in carbonate buffer~ and bound antibodies were detected using goat antl-human IgG~hor5eradish peroxida~e conjugate. The r~sulting absorbance valueg are ~hown in Flg. ~. Antisera ~rom tho ~uinea p~gs immunized with RP174c ~corresponding to the I~IB PND) produced ~nif~cant absorbance when RP135 and gp160IIIB were u ed ~s antigens, but produced no respon~e when gp160~135 or RP142 (a peptide from the MN PND) were used. Ant~serum el~cited by gp160~135 or gp160IIIB produced a s~milar response whe~ gp160~135 o~ gpl60IIIB were used as antigens.
~ he results of these E~ISAs show that an enzyme ~mmunoassay based on gpl~0~135 is as effectiYe as an assay ~ed on gp160IIIB in respondin~ to HIV positive human 8erum, bUt that the ~ormer assay, in contrast to the lat~er, doe~ not respond to anti~era rai5ed against peptides eorre~ponding to the PND.
~eço~bina. ~
Recombinant gp160MN/IIIB was compared with gp160IIIB
for u~ a~ an antigen in the enzyme ~mmunoassay descrlbe~
above. Well~ oS the microtiter plate were coated with ~olutlons of gp160 (in carbonate bu~er) at concentrat~ons o~ 0.125, 0.25, 0.50, 1.0, and 2.0 ug/ml. A pool of four H~V-positiva human ~era w~s assayed on each antigen at dilutions o~ l:lO0, l:1000, and l:lO,000. In add~tion, a pool o~ nor~l hu~an sera was aQsayed at a dilution of 1:100. Goat ~nti-human IgG:horseradlsh peroxidase con~ugate w~ us~d ~ prob~ and ABTS was used as ~he ~ubstrate.
~easurQd absorbancQ value~ (at 410 nm) are shown ln Tabl~ 1.
ThQ E~sult~ preaented in Tabl~ 1 show that the response o~
tha a~s~y when gp160MN/IIIB wa~ u~ed as the antigen was greater. Table 1 al~o shows that ~h~ respons~ o~ the W091/19011 PCT/U~91/~21 , .

~pl60~N/IIIB-ba~ed a6say to the noxmal human ~erum pool wa~
~l~o greater than the response of ths gp160IIIB-based assay~
but i~ these backgrDund values are subtracted, the respons8 to th~ ~IV-positive serum pool remain5 greater when gpl60MN/I~IB i~ used as the antigen, particularly at lower concentrat~on~ o~ gpl60~
PND Pç~ es ~n Com~ina~ion wl~h ~ 0 ~g An~iaen ~ynthetic peptide RP142, corresponding t~ the principal neutralizing determinant of the MN isolate, was used ln combination with gp160IIIB as a antigen. Wells of a microtiter plate were coated with varying concentrations of gp160IIIB (in carbonate buffer), either with or without peptide RP142. The response of each assay to an HIV-positive human serum (at 1:200 dilution) is shown in Fig. 4.T~ data in Fig. 4 ~how~ t~at a combination of the synthetic peptide and gp160 results in higher sensitivity in the ETI~
and allow~ for use o~ ~ower concentrations of gp160 than whe~ gp160 i~ used alone as an antigen.
~estern Blot analvsis usinq q~l~Q~~13~
Gpl60~135 and gp160I~B were individually ~ub~ect~d to SDS polyacrylam~de gel electrophoresis and blotted onto nitrocellulose ~llters (Towbin et al., 1979, Proc. Nat. Aca.
Scl. 76.4350). Blots were treated with an HIV-positive hu~an sQrum ~ample, antisera raised against RP174c in gui~ea pi~8, a~d nor~al human serum. Adsorbed antibodies were visualized by trea~ent o~ the blots with goat anti-human IgG:hor3~radi~h peroxidase con~ugate or goat anti-mouse IgG:hors2rad~ h peroxldasa con~ugate, ~ollowed by a dla~inobenzid~nR ~olution and hydrogen peroxlde. Fi~. 5 ~how~ ~hat ~he HI~ pos~t~Y8 human ~erum recogni~ed ~oth o~
~e ~lotted gp160~, where~5 ~ha anti-RP174c seru~ recognized .
'. ~ ..

WO 91/19~1~1 PCI`/US91/1~4021 gp160II~B, b~t not gpl60~ 135. Normal human ~erum did not recogn~ze eithe~ protein.
Other embodiments are wit~in the f ~llowing clai~ .
What i Glai~ed i~:

: -' , ' ~ ' ..

Claims

Claims 1. A method of diagnosing an HIV-1 infection in a human comprising contacting a biological sample from said human with a first antigen capable of forming immune complexes with antibodies specific for native HIV-1 but substantially incapable of forming immune complexes with antibodies specific for a second antigen which causes the production in humans of HIV-neutralizing antibodies, formation of immune complexes with said first antigen being diagnostic of an HIV infection in said human.

2. The method of claim 1 wherein said first antigen is immobilized on a solid support.

3. The method of claim 2, further comprising the steps of contacting said sample with said second antigen immobilized on a solid support and then detecting immune complexes with said second antigen as an indication of the presence of antibodies capable of binding to said second antigen.

4. The method of claim 1, said first antigen comprising an HIV-1 gp160 envelope protein which lacks the principal neutralizing determinant or a neutralizing portion thereof.

5. The method of claim 4, said first antigen comprising gp160 having a deletion of one or more amino acid between residues 297 and 331, inclusive.

6. The method of claim 5, said first antigen comprising gp160.DELTA.135.

7. The method of claim 3 wherein said second antigen comprises the principal neutralizing determinant of the HIV gp160 envelope protein.

8. The method of claim 6 wherein said second antigen comprises the HIV-1 gp160 envelope protein.

9. The method of claim 8, said second antigen comprising the HIV-1 gp160MN envelope protein.

10. The method of claim 3, said second antigen comprising a peptide having an amino acid sequence corresponding to an amino acid sequence of the principal neutralizing determinant of gp160 or a neutralizing portion thereof.

11. The method of claim 10 wherein said principal neutralizing determinant is derived from the principal neutralizing determinant of the MN variant of HIV.

12. The method of claim 9 wherein said second antigen further comprises a peptide having an amino acid sequence corresponding to the amino acid sequence of an immunogen which causes production of HIV neutralizing antibodies.

13. The method of claim 3, further comprising contacting said sample with an agent capable of binding to said first and second antibodies, and detecting said bound agent as an indication of the presence in said sample of said first and second antibodies.

14. The method of claim 13, said agent being protein A.

15. The method of claim 13, said agent being a labeled antibody capable of binding to both of said antibodies.

16. An HIV envelope protein containing a deletion of the principal neutralizing determinant or a neutralizing portion thereof.

17. The HIV-1 envelope protein of claim 16, containing a deletion of one or more amino acids between residues 297 and 331, inclusive.

18. The protein of claim 17, said protein being gp160.DELTA.135.

19. The protein of claim 18, said protein being immobilized on a solid support.

20. A kit for detecting the presence of HIV-1 antibodies in a human biological sample, comprising a first antigen capable of forming immune complexes With antibodies specific for native HIV-1 but substantially incapable of forming immune complexes with antibodies specific for a second antigen which causes production in an individual of HIV-neutralizing antibodies, and an agent capable of reacting with said first and second antibodies.

21. The kit of claim 20, said agent being protein A.

22. The kit of claim 20 wherein said agent is a labeled antibody.

23. The kit of claim 19, said first antigen being immobilized on a solid support.

24. The kit of claim 20, said first antigen comprising an HIV-1 gp160 envelope protein which lacks the principal neutralizing determinant or a neutralizing portion thereof.

25. The kit of claim 24 wherein said first antigen is HIV-1 gp160 envelope protein having a deletion of the principal neutralizing determinant, which lies between residues 297 and 331.

26. The kit of claim 25, said antigen being gp160.DELTA.135.

27. The kit of claim 20, further comprising said antigen immobilized on a support.

28. The kit of claim 27, said second antigen comprising HIV-1 gp160.

29. The kit of claim 28, said second antigen comprising gp160MN.

30. The kit of claim 28 wherein said second antigen further comprises a peptide having an amino acid sequence corresponding to that of an immunogen which causes the production mammals of HIV neutralizing antibodies.

31. The kit of claim 20 further comprising said second antigen immobilized on a support, wherein said second antigen comprises a peptide having an amino acid sequence corresponding to the amino acid sequence of an immunogen which causes production in mammals of HIV neutralizing antibodies 320 The kit of claim 31, said immunogen being the principal neutralizing determinant of gp160MN, or a portion thereof.

33. A method of diagnosing HIV-1 infection in a human comprising contacting an antibody-containing biological sample from said human with an antigen, said antigen being gp160MN, formation of immune complexes between said sample antibodies and said gp160MN being diagnostic of an HIV infection in said human.

34. The method of claim 33 wherein said gp160MN
antigen is immobilized on a solid support.

35. The method of claim 33, said antigen further comprising a peptide having an amino acid sequence corresponding to an amino acid sequence of a PND of HIV-1 gp160, or a neutralizing portion thereof.

36. A method for diagnosing HIV infection in a human comprising contacting a biological sample from said human with an antigen comprising a combination of gp160 and a peptide having an amino acid sequence which correspond to the amino acid sequence of principal neutralizing determinant of HIV-1 gp160MN or a neutralizing portion thereof.

37. A kit for diagnosing the presence of HIV-1 antibodies in a human biological sample, comprising immobilized gp160MN, and an agent capable of reacting with said antibodies.

38. Useful kit of claim 37 wherein said sample is serum and said agent is a labeled molecule.

39. A kit for detecting the presence of HIV-1 antibodies in a human biological sample, comprising combined immobilized gp160 and peptides having amino acid sequences corresponding to the amino acid sequence of the principal neutralizing determinant of HIV-1 gp160MN, and an agent capable of reacting with said antibodies.