JOURNAL OF VIROLOGY, JUIY 1990, p. 3492-3499 Vol. 64, No. 70022-538X/90/073492-08$02.00/0Copyright © 1990, American Society for Microbiology

Envelope Cross-Reactivity between Human Immunodeficiency VirusTypes 1 and 2 Detected by Different Serological Methods:

Correlation between Cross-Neutralization and Reactivity againstthe Main Neutralizing Site

BLENDA BOTTIGER,l* ANDERS KARLSSON,2 PER-AKE ANDREASSON,3 ANDERS NAUCLEIR,3CELESTINO MENDES COSTA,4 ERLING NORRBY,s AND GUNNEL BIBERFELD'

Department ofImmunology, National Bacteriological Laboratory, S-105 21 Stockholm, Sweden'; Sodersjukhuset, Box38100, S-J00 64 Stockholm, Sweden2; National Public Health Laboratory,3 and National Hospital Simao Mendes,

Bissau, Guinea-Bissau; and Department of Virology, Karolinska Institute, S-105 21 Stockholm, Sweden5

Received 31 October 1989/Accepted 16 April 1990

A total of 70 human immunodeficiency virus type 1 (HIV-1) and 42 HIV-2 antibody-positive serum samples,collected from groups of individuals in which only one type of HIV prevails, were tested for cross-reactivityagainst HIV-2 and HIV-1 proteins by Western blot (WB) (immunoblot), radioimmunoprecipitation assay(RIPA), neutralization analysis, and enzyme-linked immunosorbent assay with as antigen synthetic peptidesrepresenting selected parts of the envelope (env) glycoproteins. Cross-reactions against the env glycoproteinswere observed by WB in 10% (7 of 70) and by RIPA in 40% (28 of 70) of the HIV-1 antibody-positive serumsamples and by WB in 29% (12 of 42) and by RIPA in 48% (20 of 42) of the HIV-2 antibody-positive serumsamples. Testing by enzyme-linked immunosorbent assay against a 36-amino-acid peptide (Cys-301-Cys-336)of the external glycoprotein of strain HTLV-IIIB of HIV-1 (HIV-1lTLVIIIB) (known to represent a dominating,linear neutralizing site) showed type-specific reactions in 67% (38 of 57) of HIV-1 antibody-positive serumsamples. Type-specific reactions against a homologous 35-amino-acid peptide of strain SBL-6669 of HIV-2(HIV-2sBL-6669) were found in 75% (30 of 40) of HIV-2 antibody-positive serum samples, and these reactionswere correlated to neutralization against HIV-2SBL6669. Cross-reactions against these peptides were seen in23% (13 of 57) and 33% (13 of 40) of the HIV-1 and HIV-2 antibody-positive serum samples, respectively.These cross-reactions were correlated to cross-neutralization against HIV-1HTLV-111B and HIV-2SBL-6669.Cross-neutralization against one heterotypic virus strain was demonstrated in 16% (9 of 57) of HIV-1antibody-positive serum samples and in 22% (5 of 22) of HIV-2 antibody-positive serum samples, but nocorrelation was found between cross-neutralization and env cross-reactivity in WB or RIPA.

The worldwide epidemic of acquired immunodeficiencysyndrome (AIDS) (22) has so far been caused mainly byinfection with human immunodeficiency virus type 1 (HIV-1)(28). Another, more recently identified retrovirus, humanimmunodeficiency virus type 2 (HIV-2), has also been de-tected in patients with AIDS (9). Infections with HIV-2 haveoccurred principally in West Africa (16, 18), but cases havenow also been identified in central Africa (A. J. Georges,M. C. Georges-Courbot, D. Salaun, P. M. V. Martin, F.Barre-Sinoussi, X. Couland, and E. Chouaib, Lancet i:188-189, 1988), Europe (25; G. Biberfeld, B. Bottiger, U.Bredberg-Ratden, P. Putkonen, L. Eriksson, 0. Berglund, C.Starup, and C. H'akansson, Lancet ii:1330-1331, 1986; G.Brucker, F. Brun-Vezinet, M. Rosenheim, M. A. Rey, C.Katlama, and M. Gentilini, Lancet i:223, 1987), and NorthAmerica (19). The simultaneous presence of HIV-1 andHIV-2 infections will have implications for diagnostic serv-ices.HIV-1 and HIV-2 have similar biological properties and

similar overall genetic organization (8). The gag- and pol-encoded proteins of HIV-1 and HIV-2 show a high conser-vation of the genome, which is reflected in a high degree of

* Corresponding author.

serological cross-reactivity. The envelope (env) glycopro-teins are less well conserved, and cross-reactivity againstthese structures has hitherto been considered to be a rareevent. However, in a recent study, considerable cross-reactivity to the envelope glycoproteins of HIV-1 and HIV-2was demonstrated by Western blot (WB) (immunoblot)analysis (24). We and others have previously shown thatneutralization can be cross-reactive between HIV-1 andHIV-2 (5, 27). This observation led us to study further thepresence of cross-reactions of HIV-1 and HIV-2 antibody-positive sera against the env glycoproteins of the heterolo-gous virus by the use of several serological methods, e.g.,radioimmunoprecipitation assay (RIPA), Western blot (WB)analysis, and enzyme-linked immunosorbent assay (ELISA)with synthetic peptides as antigen. This study shows thatserological cross-reactivity against the env structures can bedemonstrated by all of the test systems used. We also showthat, among HIV-2 antibody-positive sera, type-specific neu-tralization was correlated to positive reactions by ELISAagainst a 35-amino-acid peptide representing a part of theenv glycoprotein of HIV-2 homologous to a region shown tobe the dominant neutralizing site on the external env glyco-protein of HIV-1 (14, 17). Furthermore, the cross-neutral-izing activity of sera was found to correlate with theircapacity to react with the heterotypic peptide.

3492

SEROLOGICAL CROSS-REACTIVITY BETWEEN HIV-1 AND HIV-2 3493

MATERIALS AND METHODS

Sera. A panel of 70 HIV-1 antibody-positive serum sam-ples and 42 HIV-2 antibody-positive serum samples wasused. The sera were collected from groups of individualsexpected to be infected with only a single type of virus. TheHIV-1 antibody-positive sera were collected from homosex-ual men in Sweden at different clinical stages of HIV disease:14 asymptomatic, 22 with persistent generalized lymphade-nopathy, 10 with Kaposi's sarcoma, 14 with AIDS-relatedcomplex, and 10 with AIDS with opportunistic infections.The HIV-2 antibody-positive serum samples were obtainedfrom 19 patients suspected of having AIDS or HIV-relateddisease at Simao Mendes hospital in Bissau, Guinea-Bissau,from 11 presumably healthy individuals (pregnant womenand blood donors) and from 12 outpatients attending theNational Public Health Laboratory because of suspectedtuberculosis. Because of a shortage of some sera, all sampleswere not tested by all serological methods. Sera fromSwedish blood donors and sera from subjects in Guinea-Bissau, attending an outpatient clinic because of suspectedtuberculosis, who were all HIV seronegative, served ascontrols when HIV-1 and HIV-2 antibody-positive sera weretested, respectively.HIV antibody screening. Antibodies against HIV-1 were

determined by ELISA by using two commercially availablekits (Organon-Teknika NV, Turnhout, Belgium, and AbbottDiagnostics, Wiesbaden Delkenheim, Federal Republic ofGermany). Antibodies against HIV-2 were determined byELISA with strain SBL-6669 of HIV-2 (HIV-2sBL-6669) asantigen as previously described (3).WB analysis. All sera were tested by HIV-1 WB analysis

by using a commercially available kit (Du Pont Co., Wilm-ington, Del.) according to the instructions of the manufac-turer. Sera from HIV-1-infected individuals reacted withboth the external and the transmembrane env glycoproteinsof HIV-1, as well as with at least one of the gag- orpol-encoded proteins.

All sera were tested by HIV-2 WB analysis with HIV-2SBL-6669 as antigen as previously described (1), and somewere also tested by a HIV-2 WB kit (Du Pont Co.). Serafrom HIV-2-infected individuals reacted against at least oneof the env glycoproteins of HIV-2 and, in addition, againstone of the gag- or pol-encoded proteins.RIPA. All HIV-1 antibody-positive sera were tested by

RIPA against HIV-2, and all HIV-2 antibody-positive serawere tested by RIPA against HIV-1. The RIPA was per-formed essentially as described previously (7). HIV-1 anti-gens were prepared both from cell lysates and from cell-freesupernatants of strain HTLV-IIIB of HIV-1 (HIV-lHTLVIIIB)-infected Molt 4 cells. The virus was radioac-tively labeled by incubation in medium containing[35S]cysteine (150 pXCi/ml) for 14 h. Intracellular antigen wasprepared from cells by lysis in RIPA buffer (140 mM NaCl,1 mM dithiothreitol, 10 mM Tris (pH 8.0), 0.035% phenyl-methylsulfonyl fluoride, 0.5% Nonidet P-40) and clarificationby centrifugation (10,000 x g for 15 min). The supernatantsof the cell cultures were clarified and thereafter were centri-fuged at 65,000 x g for 75 min. The pellet, constituting theextracellular antigen, was then lysed in RIPA buffer. Intra-cellular and extracellular antigens of HIV-2SBL-6669 wereprepared in the same way from U937 clone 2 cells persis-tently infected with HIV-2SBL-6669. The antigens were incu-bated with protein A-Sepharose for 2 h prior to usage.

Peptide ELISA. All sera were tested by ELISA againstpeptides representing selected parts of the env glycoproteins

of HIV-1HTLV-IIIB and HIV-2SBL-6669. Microdilution plateswere coated with 1 jig of antigen per well and were blockedwith 1% bovine serum albumin. Sera from Sweden and fromGuinea-Bissau were diluted 1:20 and 1:50, respectively, inphosphate-buffered saline with 20% fetal calf serum and0.5% Tween 20. Two hundred microliters of the serumdilution was added, and the plates were incubated for 1 h at37°C. After the plates were washed thoroughly, 100 RI of arabbit anti-human immunoglobulin peroxidase conjugate(Dakopatts, Glostrup, Denmark), diluted 1:5,000, wasadded. After 45 min of incubation at 37°C, a color reactionwas developed by adding 1,2-phenylenediamine dihydro-chloride as substrate and A405 was read. Sera with an opticaldensity above the maximum optical density of the controlsera were considered positive. All sera were also tested byresearch HIV-1 and HIV-2 peptide ELISA kits (R. W.Johnson Pharmaceutical Research Institute, San Diego, Cal-if.), in which peptides representing the immunodominantpart of the transmembrane env glycoprotein of HIV-1 andHIV-2, respectively, were used as antigens. In the HIV-1peptide ELISA, two partly overlapping peptides were used:the previously described E34 (23) and a longer peptide, E35,representing the amino acids Ala-587 to Ser-618. In theHIV-2 peptide ELISA kit, a peptide homologous to E34 butincluding an additional C-terminal cysteine (20) was used asantigen. The tests were performed according to the instruc-tions of the manufacturer. In some tests, another conjugate,alkaline phosphatase-conjugated rabbit anti-human immuno-globulin (Dakopatts), diluted 1:500, was used in combinationwith serum dilutions of 1:50, and the cut-off value forpositive reactions was then determined as three times themean value of the negative controls.

Neutralization assay. Neutralization was determined by aninfection inhibition assay with MT-4 cells as previouslydescribed (5). Briefly, twofold dilutions of serum sampleswere mixed with an appropriate dilution of stock virus andwere incubated for 1 h in microdilution plates. Thereafter,MT-4 cells were added and the plates were incubated for6 days, at which time the cytopathic effect was visuallyinspected and the cell growth was measured by [3H]thymidine incorporation. Only serum dilutions giving thesame cell growth as cell cultures without virus and with nocytopathic effect visible were considered positive for neu-tralization.

Statistics. Comparison of proportions of serum samples ofdifferent groups was done by chi-square test and Fisher'sexact test.

RESULTS

Envelope cross-reactions observed by using RIPA and WB.The proportions of HIV-1 and HIV-2 antibody-positiveserum samples reacting with the heterotypic env glycopro-teins are shown in Table 1. Positive reactions in RIPA or WBwere classified as strong (++), clear (+), or weak (+W-)(representative reactions can be found in Fig. 1 through 4).Only sera with at least a clear cross-reactivity have beenconsidered to be positive. By RIPA, only cross-reactionsagainst the external env glycoprotein gpl20 of HIV-1 andgp125 of HIV-2 or their precursors (gpl60 of HIV-1 andgpl40 of HIV-2) or both were observed (Fig. 1 and 2).Cross-reactions could also be detected by WB against thetransmembrane env gp36 of HIV-2 when WB strips (of ourown fabrication) with HIV-2SBL6669 antigen were used (Fig.3a) but not when the commercial WB strips were employed(Fig. 3b and 4). HIV-2 antibody-positive sera showed a

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3494 BOTTIGER ET AL.

TABLE 1. Cross-reactivity against envelope glycoproteinsof HIV-1 and HIV-2

Sera positive No. positive/no. tested in:for: Western blot analysisa RIPAb

HIV-1 antibody 7/70 (10%) 28/70 (40%)HIV-2 antibody 12/42 (29%) 20/42 (48%)

a Reactions with heterotypic antigen against the transmembrane env gp3O-36 or the external env gp125 of HIV-2 or both; reactions against the externalenv gp120 or its precursor gpl60 or HIV-1 or both.

b Reactions with heterotypic antigen against the external glycoproteins ortheir precursors (gpl20 and gpl60 of HIV-1; gp125 and gpl40 of HIV-2) orboth.

1 2 3 4 5 6 7 8 9 10 11

..~ i gp 160gp 1 20

higher frequency of cross-reactivity than did HIV-1 anti-body-positive sera both in WB and RIPA, but the differencewas significant (P < 0.01) only for the former test.

Separate identification of the HIV-2 env gp125 and itsprecursor gpl40 was not always possible in all RIPAs. Still,among the 28 HIV-1 antibody-positive serum samples,where cross-reactions against the env glycoproteins could bedetected by RIPA, separate cross-reactions against gp125and gpl40 could be distinguished in 20 serum samples. In allbut 2 of these 20 serum samples, clear cross-reactions togpl40 were seen, and in 13 cases (approximately 25% of allHIV-1 antibody-positive serum samples), reactions to gp125were observed. Among the HIV-2 antibody-positive serumsamples showing cross-reactivity, 19 reacted with the envprecursor gpl60, and 7 (17% of all HIV-2 antibody-positiveserum samples) reacted with the gpl20 of HIV-1.

Correlation of neutralization and RIPA and WB results.The fraction of HIV-1' and HIV-2 antibody-positive serumsamples that could neutralize HIV-lHTLv-IIB and HIV-2SBL-6669 are shown in Table 2. No correlation was found

A B C D EIx i x i x ix jX

j p 1 4@0 ._:ip 1 25 ".

p26-

t....'--200k0C

*.:.' Ii -92.SkD1.OOkD0

-69kD

_0-4 6k D

6^. _ *4 * -30kD

1 2 3 4 5 6 7 8 9 10 11 12 13

FIG. 1. Serum samples from five HIV-1-infected subjects (lanesA through E) reacting by RIPA against HIV-2 intracellular (i) andextracellular (x) antigens. A clear reactivity against gp125 and gpl4Oof the intracellular antigen is shown in lane 3; lanes 1, 5, and 9 showweak reactivity, and tane 7 shows negative reactivity. A clearreactivity against gp125 of the extracellular antigen is shown in lanes4 and 6, and a weak reactivity is shown in lane 10. Lane 11, HIV-2antibody-positive control showing strong reactivity; lane 12, HIVantibody-negative control; molecular size markers are shown in lane13.

m_ 4M1- p24

FIG. 2. Serum samples from nine HIV-2-infected subjects react-ing against intracellular HIV-1 antigen by RIPA. Lanes 3, 5, and 8show a strong reactivity and lane 7 shows a clear reactivity againstgp160. Lanes 2 and 6 show clear reactivity against gp120. Lane 10,HIV antibody-negative serum; lane 11, HIV-1 antibody-positiveserum. Lanes 10 and 11 are from another RIPA tested underidentical conditions.

between cross-neutralization and cross-reactivity in WB orRIPA in either HIV-1 or HIV-2 antibody-positive sera.

Type-specific reactions and cross-reactions observed bypeptide ELISA. All sera were tested by ELISA againstsynthetic peptides representing selected regions of the envglycoproteins of HIV-lHTLVIIIB and HIV-2sBL-6669, and theresults obtained are presented in Table 3. In this study, wehave focused on the reactions against peptides representingthe dominant neutralizing antigenic site. on the external envglycoprotein of HIV-1 (14, 17) and the homologous region ofHIV-2, presented in the form of a longer (I-l'and 1-2; Table3) and a shorter peptide (IT-1 and II-2; Table 3), and peptidesrepresenting one main type-specific nonneutralizing site ofthe transmembrane glycoproteins of H V-1 and HIV-2.

Positive type-specific reactions against the longer peptidesI-1 and 1-2 were seen in higher proportions of HIV-1 andHIV-2 antibody-positive sera, respectively, than were type-specific reactions against the smaller peptides TI-1 and 11-2,representing the N-terminal parts of the longer peptides(Table 3). The same was true for the cross-reactions ob-served against these peptides.When sera were tested against peptides representing the

immunodominant part of the transmembrane env glycopro-teins of HIV-1 and HIV-2 by using the research ELISA kits,no cross-reactions were detected among the HIV-1 anti-body-positive sera against the HIV-2 peptide. However,48%o (20 of 42) of the HIV-2 antibody-positive serum samplescross-reacted with the HIV-1 peptides (recommended cut-offvalue, 0.15) and 12% (5 of 42) had optical density valuesabove 0.4. By using other test conditions, with sera diluted1:50 and an alkaline phosphatase conjugate diluted 1:500,only 1 of 40 HIV-2 antibody-positive serum samples cross-reacted in the HIV-1 ELISA. All HIV-1 antibody-positiveserum samples were still positive under these conditions,although one of the HIV-1 antibody-positive serum sampleshad as low an optical density value as the cross-reactingHIV-2 antibody-positive serum samples.

J. VIROL.

SEROLOGICAL CROSS-REACTIVITY BETWEEN HIV-1 AND HIV-2 3495

1 2 3 4 5 a 1 2 3 4 5 b

ss.s:...gp 1 40*gp 1 25

U

-p2

$-I

am Om,p26

FIG. 3. Serum samples from three HIV-1-infected subjects (lanes 1 through 3) tested in parallel by HIV-2 WB strips made in our lab (a)and by commercial HIV-2 WB strips (b). All three serum samples show a clear reactivity against the transmembrane glycoprotein gp3O-36 (a)and weak reactivity against gp125 and gpl40 (b). Lane 4, HIV antibody-negative serum; lane 5, HIV-2 antibody-positive serum.

Correlation of neutralization and peptide ELISA results.Among HIV-1 antibody-positive sera, neutralization againstHIV-lHTLV IIIB was correlated to reactivity against thelonger peptide (II-1) representing the dominant neutralizingsite of HIV-lHTLV IIIB (Fig. Sa) (chi-square value, 23.3; P <0.001). The correlation of neutralization against strainHTLV-IIIRF of HIV-1 (HIV-lHTLV IIIRF) and reactivityagainst the same peptide was less pronounced but was alsosignificant (chi-square value, 5.8; P < 0.05) (data not shownin figures).

Furthermore, among HIV-2 antibody-positive sera, a sig-nificant correlation (P < 0.01) was found between type-specific neutralization against HIV-2sBL-6669 and type-spe-cific reactivity against the peptide 1-2 (Fig. 6a). This peptide,derived from the external env glycoprotein of HIV-2SBL-6669,represents the homologous region to the dominant neutrali-zation site of HIV-1.Among both HIV-1 and HIV-2 antibody-positive sera,

cross-reactivity to the heterotypic peptide I, representing theneutralization site, was significantly correlated (P < 0.05) tocross-neutralization against HIV-2sBL-6669 (Fig. Sb) andHIV-lHTLV IIIB (Fig. 6b).

DISCUSSION

We have shown that sera from HIV-1- or HIV-2-infectedpatients can react with the env glycoproteins of the heterol-ogous virus and that these cross-reactions can be detectedby several different methods: RIPA, WB, neutralizationassays, and ELISA with synthetic peptides as antigens.

It is very unlikely that any of the patients in our studywere infected with both HIV-1 and HIV-2. The HIV-1antibody-positive sera were from patients in Sweden, whereonly four cases of HIV-2 infection, all of them in individualsfrom West Africa, are known. The HIV-2 antibody-positivesera were collected in Guinea-Bissau, a country in which the

TABLE 2. Neutralization against HIV-1 (strain HTLV-IIIB)and HIV-2 (strain SBL-6669) of HIV-1 and HIV-2

antibody-positive sera

Sera positive No. positive/no. tested in:for: HIV-lHTLV IIIB HIV-2SBL-6.69

HIV-1 antibody 38/57 (67%) 9/57 (16%)HIV-2 antibody 5/22 (22%) 17/22 (77%)

S

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3496 BOTTIGER ET AL.

1 2 3 4 5 6 7

_,gp 1 60.....~~~~~~~~~~~~~~~~~~~...t1,f -~~~~~n_,_p120

gp4l

:I-:.p24is _

FIG. 4. Serum samples from five HIV-2-infected subjects testedagainst HIV-1 antigens by WB. Lanes 1 through 4 show a clearreactivity and lane 5 shows a weak reactivity to gpl60. Lane 5, HIVantibody-negative serum; lane 6, HIV-1 antibody-positive serum.

a X

2.o -

1.5-

1.0 -

0.5 -

*-

0

*00050*00*

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:--@5

TABLE 3. Proportions of HIV-1 and HIV-2 antibody-positivesera with reactivity against synthetic peptides representingparts of the envelope glycoprotein of HIV-1 and HIV-2

No. positive/no. tested in:Peptide Amino acid sequence of HIV-1 anti- HIV-2 anti-number peptides and their origin body-posi- body-posi-

tive sera tive sera

I-1 Cys-301-Cys-336 of HIV-1a 38/57 (67%) 13/40 (33%)1-2 Cys-301-Cys-335 of HIV-2b 13/57 (23%) 30/40 (75%)II-1 Cys-301-Ala-321 of HIV-1 20/56 (36%) 8/40 (20%)11-2 Cys-301-His-321 of HIV-2 5/56 (9%) 27/40 (68%)a Strain HTLV-IIIB of HIV-1.b Strain SBL-6669 of HIV-2.

prevalence of HIV-2 infection is high and infection withHIV-1 is rare (18). The findings of double reactivity againstHIV-1 and HIV-2 among the sera we tested, therefore, mostprobably represent cross-reactivity between HIV-1 andHIV-2. ELISA, with synthetic peptides derived from animmunodominant site on the transmembrane glycoprotein asantigen, has proved to be a highly type-specific test todiscriminate between infection with HIV-1 and HIV-2 (12,20). In this study, and also in the report by Norrby et al. (20),a few double-reactive serum samples were found. However,the type-specific reactions were considerably stronger thanwere the cross-reactions. In our hands, site-directed serol-ogy by ELISA was a simple and objective method fordiscrimination between infection with HIV-1 and HIV-2 insubjects with double serological reactivity in WB or RIPA. Ithas to be further studied whether a peptide ELISA can bemade sensitive enough to detect all sera with type-specificantibodies without detecting any cross-reacting sera. How-ever, sera with high and equal reactivities by ELISA againstboth HIV-1 and HIV-2 type-specific peptides have been

A405b t

2.o -000

0

00

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0

00.

l .5

1.0

0.5 -

2*

so

00@0

*-

:-

S

0*S..*-

2

00

0

Controls Neutr.- Neutr. + Controls Cross neutr.- Cross neutr.+FIG. 5. Reactivity by ELISA against the HIV-1 peptide Cys-301-Cys-336 among sera from HIV-1-infected subjects positive (Neutr. +)

or negative (Neutr. -) for type-specific neutralization against HIV-lHTLV IIIB and among HIV antibody-negative sera from Swedish blooddonors (Controls) (a) and reactivity against the HIV-2 peptide Cys-301-Cys-335 among the same sera positive (Cross neutr. +) or negative(Cross neutr. -) for cross-neutralization against HIV-2SBL-6669 (b).

J. VIROL.

A405

SEROLOGICAL CROSS-REACTIVITY BETWEEN HIV-1 AND HIV-2 3497

a

2.o-

1.5 -

1.0 -

0.5 -

A4054 A4054

2.o

0 1.5 -

1.0-0so

00

U.I.

0.5-0*0

.S

6

S

S.S

.!$s_

_ _0"_"_*

Controls Neutr. - Neutr +Controls Cross neutr- Cross neutr. +

FIG. 6. Reactivity by ELISA against the HIV-2 peptide Cys-301-Cys-335 among sera from HIV-2-infected subjects positive (Neutr. +)or negative (Neutr. -) for type-specific neutralization against HIV-2SBL-6669 and among HIV antibody-negative sera from Guinea-Bissau(Controls) (a) and reactivity against the HIV-1 peptide Cys-301-Cys-336 among the same sera positive (Cross neutr. +) or negative (Crossneutr. -) for cross-neutralization against HIV-lHTLV-IIIB (b).

described (M. C. Cot, M. Poulain, J. F. Delagneau, M.Peeters, and F. Brun-Vezinet, AIDS Res. Hum. Retrovi-ruses 4:239-241, 1988), supporting the existence of truedouble infection or infection with variants of HIV-1 orHIV-2.By RIPA, we found cross-reactions against env glycopro-

teins, in addition to those observed against gag-encodedproteins. Cross-reactions were seen mainly against the envprecursors of HIV-1 and HIV-2 (in 48 and 40% of the serumsamples tested, respectively), but also, in a lower propor-tion, against the external env glycoproteins. In an earlierstudy, Kanki et al. (15) have described similar patterns ofcross-reactivity in RIPA against the env glycoproteins ofHIV-1 and simian immunodeficiency virus (SIV), previouslycalled simian T-lymphotropic virus type III and closelyrelated to HIV-2 (3, 6, 11). In the study by Kanki et al. (15),5 of 23 U.S. HIV-1 antibody-positive serum samples cross-reacted with the env gpl20/gpl6O of SIV and 10 of 16 WestAfrican SIV antibody-positive serum samples reacted withthe env gp160 of HIV-1; of these serum samples, 4 alsoreacted with the env gpl20. The cross-reactions were rela-tively faint as compared with the type-specific reactions.Another more recent study has also shown a weak butreproducible cross-reactivity by RIPA of HIV-1 antibody-positive sera against HIV-2 and SIV antigens (S. Butt6, P.Verani, F. Titti, G. Rezza, L. Sernicola, and G. B. Rossi,AIDS 2:139-140, 1988).When we tested HIV-1 and HIV-2 antibody-positive sera

by using commercial WB kits, cross-reactions were seenonly against the external env glycoprotein and its precursorof HIV-2 and HIV-1, respectively. By using WB strips madein our laboratory with HIV-2SBL-6669 as antigen, cross-reactions against the transmembrane gp3O-36 of HIV-2 wereclearly visualized and, in a few cases, reactions to theenvelope gp125 were also seen. In a recent report, the HIV-1viral antigens, migrating with mobilities of 160 and 120

kilodaltons on commercial WB strips, were shown to beprimarily multimers of the HIV-1 transmembrane gp4l andto react with monoclonal antibodies to gp41 (S. Zolla-Pazner, M. K. Gorny, W. J. Honnen, and A. Pinter, N.Engl. J. Med. 320:1280-1281, 1989). If our results were to beinterpreted according to these findings, the cross-reactionsagainst the high-molecular-size env glycoproteins observedby WB could be directed partly against the transmembraneglycoproteins. In a recent study in which cross-reactionswere studied by WB by using commercial WB kits, Tedderet al. (24) showed that at least 6 out of 17 HIV-2 antibody-positive serum samples reacted clearly with the external envglycoproteins of HIV-1 whereas 2 out of 17 HIV-1 antibody-positive serum samples reacted with the env glycoproteins ofHIV-2. Our findings are in good agreement with theseresults. Cross-reactivity of HIV-1 antibody-positive seraagainst the transmembrane glycoprotein of HIV-2 have beenreported earlier by us (4) and others (2, 26).

Several neutralizing linear as well as discontinuous anti-genic sites have been identified on the external env glyco-protein and the transmembrane glycoprotein of HIV-1 (10,13, 14, 17, 21). Among these sites, one dominant linearregion of the high-molecular-size env glycoprotein of HIV-1was demonstrated in several studies (13, 14, 17, 21). Amonoclonal antibody, preventing infection of cell-free virusand cell fusion of HIV-1-infected cells, has been shown tobind to amino acids Asn-308 to Gly-331 of the externalglycoprotein of HIV-lHTLV-IIIB (17). Furthermore, Goud-smit et al. (13) have reported that antibodies to this site,detected by ELISA by using a similar synthetic peptide(amino acids Lys-305 to Gly-321) as antigen, were found insera that could inhibit HIV-lHTLV IIIB cell fusion. In thisstudy, we have also demonstrated a strong correlationbetween neutralization against HIV-lHTLV IIIB and positivereactions by ELISA against a 36-amino-acid peptide repre-senting the same neutralization site of the external env

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3498 BOTTIGER ET AL.

glycoprotein. We found a higher proportion of sera positiveby ELISA (67%) than did Goudsmit et al. (8 to 44%). Thisdifference might be explained by the use of slightly differentpeptides and different test conditions for ELISA.

This study further demonstrated a correlation amongHIV-2 antibody-positive sera between neutralization ofHIV-2sBL6669 and reactivity to a 35-amino-acid peptide,representing the homologous region of the external env

glycoprotein of HIV-2SBL-6669 as one already known neutral-izing site of HIV-1. This finding indirectly suggests that thisregion, defined by amino acids Cys-301 to Cys-335, ispossibly also a dominating neutralizing site for HIV-2.However, further experiments demonstrating whether pep-tides representing the selected region can block or adsorbout neutralizing activity of postinfection sera and evaluatingthe capacity of peptides to induce the production of neutral-izing antibodies are required for the eventual identification ofthe proposed neutralizing site. In addition, the need tosearch for other linear as well as discontinuous neutralizingenv antigenic sites in HIV-2 remains.

Cross-neutralization of HIV-1 or HIV-2 antibody-positivesera against HIV-2SBL-6669 or HIV-lHTLVIIIB was correlatedto positive reactions by ELISA against peptides representingthe neutralization site of the heterotypic virus. This obser-vation may indicate that the main neutralizing site harborsnot only strain-specific antigenic determinants (17) but pos-sibly also harbors type-specific (S. D. Putney, G. LaRosa, R.Grimaila, B. Clark, K. Javaherian, E. Emini, M. Robert-Guroff, R. Gallo, D. Bolognesi, and T. Matthews, Modernapproaches to new vaccines including prevention of AIDS,Abstract, p. 43, 1989) and probably even intertype cross-reactive determinants. The exact position of the latter reac-tions remains to be directly identified by neutralization-blocking experiments with selected peptides representingthis site or by evaluation of immunogenic activities of thecorresponding peptides.

ACKNOWLEDGMENTS

This work was supported by grants from the Swedish MedicalResearch Council (projects 16H-2380-23A and 16H-7786-03A).The skilled technical assistance of Lena Vetterli, Nasrin Bavand,

and Marianne Thuresson is gratefully acknowledged.

LITERATURE CITED

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VOL. 64, 1990