JOURNAL OF VIROLOGY, JUIY 1990, p. 3492-3499 Vol. 64, No.

7
0022-538X/90/073492-08$02.00/0
Copyright © 1990, American Society for Microbiology

Envelope Cross-Reactivity between Human Immunodeficiency Virus

Types 1 and 2 Detected by Different Serological Methods:
Correlation between Cross-Neutralization and Reactivity against
the Main Neutralizing Site
BLENDA BOTTIGER,l* ANDERS KARLSSON,2 PER-AKE ANDREASSON,3 ANDERS NAUCLEIR,3
CELESTINO MENDES COSTA,4 ERLING NORRBY,s AND GUNNEL BIBERFELD'
Department of Immunology, National Bacteriological Laboratory, S-105 21 Stockholm, Sweden'; Sodersjukhuset, Box
38100, 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-reactivity
against 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 peptides
representing selected parts of the envelope (env) glycoproteins. Cross-reactions against the env glycoproteins
were observed by WB in 10% (7 of 70) and by RIPA in 40% (28 of 70) of the HIV-1 antibody-positive serum
samples and by WB in 29% (12 of 42) and by RIPA in 48% (20 of 42) of the HIV-2 antibody-positive serum
samples. 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 serum
samples. 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 reactions
were correlated to neutralization against HIV-2SBL6669. Cross-reactions against these peptides were seen in
23% (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-1
antibody-positive serum samples and in 22% (5 of 22) of HIV-2 antibody-positive serum samples, but no
correlation was found between cross-neutralization and env cross-reactivity in WB or RIPA.

The worldwide epidemic of acquired immunodeficiency
syndrome (AIDS) (22) has so far been caused mainly by
infection with human immunodeficiency virus type 1 (HIV-1)
(28). Another, more recently identified retrovirus, human
immunodeficiency virus type 2 (HIV-2), has also been de-
tected in patients with AIDS (9). Infections with HIV-2 have
occurred principally in West Africa (16, 18), but cases have
now 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 North
America (19). The simultaneous presence of HIV-1 and
HIV-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 against
these structures has hitherto been considered to be a rare
event. However, in a recent study, considerable cross-
reactivity to the envelope glycoproteins of HIV-1 and HIV-2
was demonstrated by Western blot (WB) (immunoblot)
analysis (24). We and others have previously shown that
neutralization can be cross-reactive between HIV-1 and
HIV-2 (5, 27). This observation led us to study further the
presence 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 that
serological cross-reactivity against the env structures can be
demonstrated by all of the test systems used. We also show
that, among HIV-2 antibody-positive sera, type-specific neu-
tralization was correlated to positive reactions by ELISA
against a 35-amino-acid peptide representing a part of the
env glycoprotein of HIV-2 homologous to a region shown to
be 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 their
capacity to react with the heterotypic peptide.

3492
VOL. 64, 1990 SEROLOGICAL CROSS-REACTIVITY BETWEEN HIV-1 AND HIV-2
MATERIALS AND METHODS
Sera. A panel of 70 HIV-1 antibody-positive serum sam-
ples and 42 HIV-2 antibody-positive serum samples was
used. The sera were collected from groups of individuals
expected to be infected with only a single type of virus. The
HIV-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-related
complex, and 10 with AIDS with opportunistic infections.
The HIV-2 antibody-positive serum samples were obtained
from 19 patients suspected of having AIDS or HIV-related
disease at Simao Mendes hospital in Bissau, Guinea-Bissau,
from 11 presumably healthy individuals (pregnant women
and blood donors) and from 12 outpatients attending the
National Public Health Laboratory because of suspected
tuberculosis. Because of a shortage of some sera, all samples
were not tested by all serological methods. Sera from
Swedish blood donors and sera from subjects in Guinea-
Bissau, attending an outpatient clinic because of suspected
tuberculosis, who were all HIV seronegative, served as
controls when HIV-1 and HIV-2 antibody-positive sera were
tested, respectively.
HIV antibody screening. Antibodies against HIV-1 were
determined by ELISA by using two commercially available
kits (Organon-Teknika NV, Turnhout, Belgium, and Abbott
Diagnostics, Wiesbaden Delkenheim, Federal Republic of
Germany). Antibodies against HIV-2 were determined by
ELISA with strain SBL-6669 of HIV-2 (HIV-2sBL-6669) as
antigen 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 with
both the external and the transmembrane env glycoproteins
of HIV-1, as well as with at least one of the gag- or
pol-encoded proteins.
All sera were tested by HIV-2 WB analysis with HIV-
2SBL-6669 as antigen as previously described (1), and some
were also tested by a HIV-2 WB kit (Du Pont Co.). Sera
from HIV-2-infected individuals reacted against at least one
of the env glycoproteins of HIV-2 and, in addition, against
one 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 sera
were 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-free
supernatants 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 was
prepared 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 clarification
by centrifugation (10,000 x g for 15 min). The supernatants
of the cell cultures were clarified and thereafter were centri-
fuged at 65,000 x g for 75 min. The pellet, constituting the
extracellular antigen, was then lysed in RIPA buffer. Intra-
cellular and extracellular antigens of HIV-2SBL-6669 were
prepared 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 against
peptides representing selected parts of the env glycoproteins
3493
of HIV-1HTLV-IIIB and HIV-2SBL-6669. Microdilution plates
were coated with 1 jig of antigen per well and were blocked
with 1% bovine serum albumin. Sera from Sweden and from
Guinea-Bissau were diluted 1:20 and 1:50, respectively, in
phosphate-buffered saline with 20% fetal calf serum and
0.5% Tween 20. Two hundred microliters of the serum
dilution was added, and the plates were incubated for 1 h at
37°C. After the plates were washed thoroughly, 100 RI of a
rabbit anti-human immunoglobulin peroxidase conjugate
(Dakopatts, Glostrup, Denmark), diluted 1:5,000, was
added. After 45 min of incubation at 37°C, a color reaction
was developed by adding 1,2-phenylenediamine dihydro-
chloride as substrate and A405 was read. Sera with an optical
density above the maximum optical density of the control
sera were considered positive. All sera were also tested by
research HIV-1 and HIV-2 peptide ELISA kits (R. W.
Johnson Pharmaceutical Research Institute, San Diego, Cal-
if.), in which peptides representing the immunodominant
part of the transmembrane env glycoprotein of HIV-1 and
HIV-2, respectively, were used as antigens. In the HIV-1
peptide 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 the
HIV-2 peptide ELISA kit, a peptide homologous to E34 but
including an additional C-terminal cysteine (20) was used as
antigen. 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 combination
with serum dilutions of 1:50, and the cut-off value for
positive reactions was then determined as three times the
mean value of the negative controls.
Neutralization assay. Neutralization was determined by an
infection inhibition assay with MT-4 cells as previously
described (5). Briefly, twofold dilutions of serum samples
were mixed with an appropriate dilution of stock virus and
were incubated for 1 h in microdilution plates. Thereafter,
MT-4 cells were added and the plates were incubated for
6 days, at which time the cytopathic effect was visually
inspected and the cell growth was measured by [3H]
thymidine incorporation. Only serum dilutions giving the
same cell growth as cell cultures without virus and with no
cytopathic effect visible were considered positive for neu-
tralization.
Statistics. Comparison of proportions of serum samples of
different groups was done by chi-square test and Fisher's
exact test.
RESULTS
Envelope cross-reactions observed by using RIPA and WB.
The proportions of HIV-1 and HIV-2 antibody-positive
serum samples reacting with the heterotypic env glycopro-
teins are shown in Table 1. Positive reactions in RIPA or WB
were 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 been
considered to be positive. By RIPA, only cross-reactions
against the external env glycoprotein gpl20 of HIV-1 and
gp125 of HIV-2 or their precursors (gpl60 of HIV-1 and
gpl40 of HIV-2) or both were observed (Fig. 1 and 2).
Cross-reactions could also be detected by WB against the
transmembrane env gp36 of HIV-2 when WB strips (of our
own 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
3494 BOTTIGER ET AL. J. VIROL.

TABLE 1. Cross-reactivity against envelope glycoproteins 1 2 3 4 5 6 7 8 9 10 11

of HIV-1 and HIV-2
Sera positive No. positive/no. tested in: ..~ i gp 160
for: Western blot analysisa RIPAb gp 1 20
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 external
env gp120 or its precursor gpl60 or HIV-1 or both.
b Reactions with heterotypic antigen against the external glycoproteins or
their precursors (gpl20 and gpl60 of HIV-1; gp125 and gpl40 of HIV-2) or
both.

higher frequency of cross-reactivity than did HIV-1 anti-
body-positive sera both in WB and RIPA, but the difference
was significant (P < 0.01) only for the former test.
Separate identification of the HIV-2 env gp125 and its
precursor 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 be
detected by RIPA, separate cross-reactions against gp125
and gpl40 could be distinguished in 20 serum samples. In all
but 2 of these 20 serum samples, clear cross-reactions to
gpl40 were seen, and in 13 cases (approximately 25% of all
HIV-1 antibody-positive serum samples), reactions to gp125
were observed. Among the HIV-2 antibody-positive serum
samples showing cross-reactivity, 19 reacted with the env
precursor gpl60, and 7 (17% of all HIV-2 antibody-positive
serum 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 serum
samples that could neutralize HIV-lHTLv-IIB and HIV-
2SBL-6669 are shown in Table 2. No correlation was found
A B C D E the dominant neutralizing antigenic site. on the external env
Ix i x i x ix jX
t....'-
j p 1 4@
0 ._:
ip 1 25 ". *.:.'
_0
p26- 6^. _
*4
1 2 3 4 5 6 7 8 9 10 11 12 13
FIG. 1. Serum samples from five HIV-1-infected subjects (lanes
A through E) reacting by RIPA against HIV-2 intracellular (i) and
extracellular (x) antigens. A clear reactivity against gp125 and gpl4O
of the intracellular antigen is shown in lane 3; lanes 1, 5, and 9 show
weak reactivity, and tane 7 shows negative reactivity. A clear
reactivity against gp125 of the extracellular antigen is shown in lanes
4 and 6, and a weak reactivity is shown in lane 10. Lane 11, HIV-2
antibody-positive control showing strong reactivity; lane 12, HIV
antibody-negative control; molecular size markers are shown in lane
13.
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 8
show a strong reactivity and lane 7 shows a clear reactivity against
gp160. Lanes 2 and 6 show clear reactivity against gp120. Lane 10,
HIV antibody-negative serum; lane 11, HIV-1 antibody-positive
serum. Lanes 10 and 11 are from another RIPA tested under
identical conditions.
between cross-neutralization and cross-reactivity in WB or
RIPA in either HIV-1 or HIV-2 antibody-positive sera.
Type-specific reactions and cross-reactions observed by
peptide ELISA. All sera were tested by ELISA against
synthetic peptides representing selected regions of the env
glycoproteins of HIV-lHTLVIIIB and HIV-2sBL-6669, and the
results obtained are presented in Table 3. In this study, we
have focused on the reactions against peptides representing
glycoprotein of HIV-1 (14, 17) and the homologous region of
HIV-2, presented in the form of a longer (I-l'and 1-2; Table
3) and a shorter peptide (IT-1 and II-2; Table 3), and peptides
-2 00 k0C representing one main type-specific nonneutralizing site of
Ii -92.SkD
1.OOkD the transmembrane glycoproteins of H V-1 and HIV-2.
0 Positive type-specific reactions against the longer peptides
-69kD I-1 and 1-2 were seen in higher proportions of HIV-1 and
HIV-2 antibody-positive sera, respectively, than were type-
specific reactions against the smaller peptides TI-1 and 11-2,
-4 6k D 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
* -30kD 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 samples
cross-reacted with the HIV-1 peptides (recommended cut-off
value, 0.15) and 12% (5 of 42) had optical density values
above 0.4. By using other test conditions, with sera diluted
1:50 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-positive
serum samples were still positive under these conditions,
although one of the HIV-1 antibody-positive serum samples
had as low an optical density value as the cross-reacting
HIV-2 antibody-positive serum samples.
VOL. 64, 1990 SEROLOGICAL CROSS-REACTIVITY BETWEEN HIV-1 AND HIV-2 3495

1 2 3 4 5 a 1 2 3 4 5 b
:...gp 1 40
ss.s

*gp 1 25
U

$-I

-p2

S 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. DISCUSSION

Among HIV-1 antibody-positive sera, neutralization against
HIV-lHTLV IIIB was correlated to reactivity against the
longer peptide (II-1) representing the dominant neutralizing
site of HIV-lHTLV IIIB (Fig. Sa) (chi-square value, 23.3; P <
0.001). The correlation of neutralization against strain
HTLV-IIIRF of HIV-1 (HIV-lHTLV IIIRF) and reactivity
against the same peptide was less pronounced but was also
significant (chi-square value, 5.8; P < 0.05) (data not shown
in 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 the
neutralization site, was significantly correlated (P < 0.05) to
cross-neutralization against HIV-2sBL-6669 (Fig. Sb) and
HIV-lHTLV IIIB (Fig. 6b).
We have shown that sera from HIV-1- or HIV-2-infected
patients can react with the env glycoproteins of the heterol-
ogous virus and that these cross-reactions can be detected
by several different methods: RIPA, WB, neutralization
assays, and ELISA with synthetic peptides as antigens.
It is very unlikely that any of the patients in our study
were infected with both HIV-1 and HIV-2. The HIV-1
antibody-positive sera were from patients in Sweden, where
only four cases of HIV-2 infection, all of them in individuals
from West Africa, are known. The HIV-2 antibody-positive
sera 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%)
3496 BOTTIGER ET AL. J. VIROL.

TABLE 3. Proportions of HIV-1 and HIV-2 antibody-positive

1 2 3 4 5 6 7 sera with reactivity against synthetic peptides representing
parts of the envelope glycoprotein of HIV-1 and HIV-2
.....~~~~~~~~~~~~~~~~~~~
...t
1,f
_,gp 1 60 No. positive/no. tested in:
-~~~~~n_,
_p 120 Peptide
number
Amino acid sequence of
peptides and their origin
HIV-1 anti-
body-posi-
HIV-2 anti-
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.
gp4l b Strain SBL-6669 of HIV-2.

prevalence of HIV-2 infection is high and infection with

is _ :.p24
:I-
FIG. 4. Serum samples from five HIV-2-infected subjects tested
against HIV-1 antigens by WB. Lanes 1 through 4 show a clear
reactivity and lane 5 shows a weak reactivity to gpl60. Lane 5, HIV
antibody-negative serum; lane 6, HIV-1 antibody-positive serum.
HIV-1 is rare (18). The findings of double reactivity against
HIV-1 and HIV-2 among the sera we tested, therefore, most
probably represent cross-reactivity between HIV-1 and
HIV-2. ELISA, with synthetic peptides derived from an
immunodominant site on the transmembrane glycoprotein as
antigen, has proved to be a highly type-specific test to
discriminate 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 than
were the cross-reactions. In our hands, site-directed serol-
ogy by ELISA was a simple and objective method for
discrimination between infection with HIV-1 and HIV-2 in
subjects with double serological reactivity in WB or RIPA. It
has to be further studied whether a peptide ELISA can be
made sensitive enough to detect all sera with type-specific
antibodies without detecting any cross-reacting sera. How-
ever, sera with high and equal reactivities by ELISA against
both HIV-1 and HIV-2 type-specific peptides have been

a X
A405
b t
A405
2.o - 00 2.o -
0

1.5- l .5
0
0

1.0 - 1.0 0
0
S.
*-
0
2*
0.
0 so 0

0.5 - S 0.5 - 0*-

S

0*
0 0@ S..
*-

*0
:--
@5 :-
0

0050
*00*
2

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 blood
donors (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).
VOL. 64, 1990 SEROLOGICAL CROSS-REACTIVITY BETWEEN HIV-1 AND HIV-2
A405
a 4
2.o-
1.5 - 0
1.0 - so
0
0.5 - U.
0 0.5-
I. 0 _*
*0
Controls Neutr. - 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 (Cross
neutr. -) 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 true
double infection or infection with variants of HIV-1 or
HIV-2.
By RIPA, we found cross-reactions against env glycopro-
teins, in addition to those observed against gag-encoded
proteins. Cross-reactions were seen mainly against the env
precursors of HIV-1 and HIV-2 (in 48 and 40% of the serum
samples tested, respectively), but also, in a lower propor-
tion, against the external env glycoproteins. In an earlier
study, Kanki et al. (15) have described similar patterns of
cross-reactivity in RIPA against the env glycoproteins of
HIV-1 and simian immunodeficiency virus (SIV), previously
called simian T-lymphotropic virus type III and closely
related 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 West
African SIV antibody-positive serum samples reacted with
the env gp160 of HIV-1; of these serum samples, 4 also
reacted 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 but
reproducible 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 seen
only against the external env glycoprotein and its precursor
of HIV-2 and HIV-1, respectively. By using WB strips made
in our laboratory with HIV-2SBL-6669 as antigen, cross-
reactions against the transmembrane gp3O-36 of HIV-2 were
clearly visualized and, in a few cases, reactions to the
envelope gp125 were also seen. In a recent report, the HIV-1
viral antigens, migrating with mobilities of 160 and 120
3497
A405
4
2.o
.
1.5 - S
1.0- S
S S.
_"_0"
_
.
!$s_
Controls Cross neutr- Cross neutr. +
kilodaltons on commercial WB strips, were shown to be
primarily multimers of the HIV-1 transmembrane gp4l and
to 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 be
interpreted according to these findings, the cross-reactions
against the high-molecular-size env glycoproteins observed
by WB could be directed partly against the transmembrane
glycoproteins. In a recent study in which cross-reactions
were studied by WB by using commercial WB kits, Tedder
et al. (24) showed that at least 6 out of 17 HIV-2 antibody-
positive serum samples reacted clearly with the external env
glycoproteins of HIV-1 whereas 2 out of 17 HIV-1 antibody-
positive serum samples reacted with the env glycoproteins of
HIV-2. Our findings are in good agreement with these
results. Cross-reactivity of HIV-1 antibody-positive sera
against the transmembrane glycoprotein of HIV-2 have been
reported 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 linear
region of the high-molecular-size env glycoprotein of HIV-1
was demonstrated in several studies (13, 14, 17, 21). A
monoclonal antibody, preventing infection of cell-free virus
and cell fusion of HIV-1-infected cells, has been shown to
bind to amino acids Asn-308 to Gly-331 of the external
glycoprotein 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 in
sera that could inhibit HIV-lHTLV IIIB cell fusion. In this
study, we have also demonstrated a strong correlation
between neutralization against HIV-lHTLV IIIB and positive
reactions by ELISA against a 36-amino-acid peptide repre-
senting the same neutralization site of the external env
3498 BOTTIGER ET AL.
glycoprotein. We found a higher proportion of sera positive
by ELISA (67%) than did Goudsmit et al. (8 to 44%). This
difference might be explained by the use of slightly different
peptides and different test conditions for ELISA.
This study further demonstrated a correlation among
HIV-2 antibody-positive sera between neutralization of
HIV-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 this
region, defined by amino acids Cys-301 to Cys-335, is
possibly also a dominating neutralizing site for HIV-2.
However, further experiments demonstrating whether pep-
tides representing the selected region can block or adsorb
out neutralizing activity of postinfection sera and evaluating
the capacity of peptides to induce the production of neutral-
izing antibodies are required for the eventual identification of
the proposed neutralizing site. In addition, the need to
search for other linear as well as discontinuous neutralizing
env antigenic sites in HIV-2 remains.
Cross-neutralization of HIV-1 or HIV-2 antibody-positive
sera against HIV-2SBL-6669 or HIV-lHTLVIIIB was correlated
to positive reactions by ELISA against peptides representing
the neutralization site of the heterotypic virus. This obser-
vation may indicate that the main neutralizing site harbors
not 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, Modern
approaches 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 representing
this site or by evaluation of immunogenic activities of the
corresponding peptides.
ACKNOWLEDGMENTS
This work was supported by grants from the Swedish Medical
Research Council (projects 16H-2380-23A and 16H-7786-03A).
The skilled technical assistance of Lena Vetterli, Nasrin Bavand,
and Marianne Thuresson is gratefully acknowledged.
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