AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 6, 1992 Mary Ann Liebert, Inc., Publishers
Neutralizing Domain in the External Envelope Glycoprotein of Simian Immunodeficiency Virus
Identification of a
BENICHOU,1 ROGER LEGRAND,3 NAOKO NAKAGAWA,4 THÉRÈSE FAURE,4 GUILLAUME VOGT,3 DOMINIQUE DORMONT,3 PIERRE TIOLLAIS,1 TRAINCARD,2 FRANÇOIS MARIE-PAULE KIENY ,4 and PASCAL MADAULE1 SERGE
ABSTRACT Two murine monoclonal antibodies (MAbs), designated MATG2014 and MATG2033, were generated. They are reactive with the external envelope glycoprotein gpl30 of the simian immunodeficiency virus of macaque monkey (SIVmac2Si), and display a cell-free virus neutralizing activity in vitro. In addition, MATG2014 cross-reacts with HIV-2Rod gpl40. Epitope mapping of these MAbs was performed by screening an SIVmac peptide library expressed in yeast and confirmed using synthetic peptides. MATG2014 and MATG2033 recognize two overlapping epitopes localized in an 18 residue domain between amino acid 171 and 188 of the SIV-ac2s1 gpl30. Sera from experimentally SIV-infected macaques are immunoreactive with this neutralizing domain. Sequence comparison with related SIV and HIV-2 viral strains indicates a low variability of this region, consistent with the cross-reactivity of MATG2014 with HIV-2Rod gpl40. This domain should then be considered in designing experimental vaccines.
INTRODUCTION viruses (SIVs) belong to a primate lentiviruses, first isolated from rhesus monkeys (SIVmac) and subsequently from several other monkey species. ' ·2 This group of lentiviruses shares many biological, pathogenic, genetic, and antigenic properties with the human immunodeficiency viruses (HIV-1 and HIV-2), the etiological agents of AIDS. In particular, SIVmac is very closely
Thefamily
simian immunodeficiency
of nonhuman
related to HIV-2.3·4 Because of these similarities between SIVs and HIVs, the SIV macaque model is particularly attractive for the development and testing of vaccine strategies against human AIDS, and initial vaccine trials in macaques, using an inacti¬ vated whole SIVmac vaccine, suggest that a protective immunity may be obtained against SIVmac.5·6
METHODS In the case of HIV-1, several targets for neutralizing antibod¬ ies have been identified,7-12 among which the principal deter¬
minant is contained within a loop located in the third variable domain (V3 loop) of the external glycoprotein gpl20.13,14 The V3 loop however induces isolate-specific neutralizing antibod¬ ies, probably due to its sequence diversity among HIV-1 iso¬ lates.13 In the case of SIV, a strain-specific neutralizing deter¬ minant present in the transmembrane glycoprotein of SIVmac has been recently described,15 but very little is known about neutral¬ izing antibodies directed against the external glycoprotein of SIVs gpl30. In this report, we analyze two MAbs, designated MATG2033 and MATG2014, raised against SIVmac251 gpl40 and displaying a cell-free virus neutralizing activity in vitro. The antigenic domain recognized by these two MAbs has been mapped in the external envelope glycoprotein using a SIVmac peptide library expressed in Saccharomyces cerevisiae. This neutralizing determinant does not correspond to the presumed V3 loop-like domain of SIVmac.I6 MAbs were raised in mice against SIVmac, isolate 251, as follows. A recombinant vaccinia virus expressing the modified env gene was constructed. As for the HIV-1 env protein,17 the cleavage site between gpl30 and gp32 (a.a. 546 and 547) was
'Unité de Recombinaison et Expression Génétique and 2Hybridolab, Institut Pasteur, 28 rue du Dr Roux, 75024 Paris Cedex 15 France. 'Commissariat à l'Energie Atomique, Département de toxicologie Experimentale, CRSSA, 92265 Fontenay aux Roses Cedex, France. Transgène S.A., 67032 Strasbourg, Cedex, France. 1165
1166
BENICHOU ET AL. Table 1. Characterization of SIV„
MAbs
Ig subtype
No.
2003 2008 2014 2016 2022 2024 2029 2033 2037 b
Gl G2b G2b G2a Gl Gl Gl Gl Gl
'
Antibody b
Antibody
titer
3
6.6 2 4 3 5
X x
104 104
X
105 104 105
gpl40
concentration
10s
6.7 3.4 10.3 6.9 19.5 12 5.1
4 104
2.9
x
x x
MAbs
104
Neutralizing
'
titer
'
1024
64
Subtypes were determined using a mouse monoclonal antibody isotyping kit from Amersham. The titer is defined as the reciprocal of the ascite dilution giving 50% of the maximum
absorbance value. c Expressed in mg/ml of ascite fluid. d Neutralization activity for SIVmac251 is expressed dilution where at least 1 out of 4 wells had no detectable
mutated and the hydrophobic transmembrane domain (a.a. 166 189 of gp32) was deleted. The live recombinant virus served as immunogen. Six-week-old female BALB/c mice were immu¬ nized intraperitoneally with 5.107 plaque-forming units (pfu) of virus in complete Freund's adjuvant, followed by two boosts in incomplete Freund's adjuvant at 1 week intervals. A third boost in phosphate-buffered saline (PBS) after 1 week was performed intravenously. Three days later, the spleen cells were fused to P3x63-Ag8U! (ATCC CRL 1897) cells and selected in HAT medium as usual.18 A total of 33 hybridomas reactive against purified SIVmac gpl40 produced by recombinant vaccinia virus in an enzyme-linked immunosorbent assay (ELISA) were ob¬ tained. The hybridoma corresponding to 9 of these MAbs was subcloned and injected to mice to generate ascite fluids. The ELISA titers, IgG contents, and immunoglobulin subclasses of the MAbs were determined (Table 1 ). A radioimmunoprecipita¬ tion assay (RIPA) was performed as previously described17 with all the MAbs on supernatants and extracts from BHK 21 cells infected with the recombinant vaccinia virus expressing the native SIVmac25I envelope glycoproteins (VV-TG-SIV-4176) (Fig. 1). All the MAbs, except MATG2003, immunoprecipi¬ tated SIVmac envelope glycoproteins from the supernatant and/or from the cell pellet. Only MATG2003 failed to signifi¬ cantly precipitate SIVmac251 glycoproteins, but reacted in ELIS A with recombinant gp 140 at the titer of 3 x 104 (Table 1 ). These anti-SIVmac25, gpl40 MAbs were then tested for their ability to neutralize SIVmac251 using an assay described previ¬ ously.19 Briefly, twofold serial dilutions of ascites were incu¬ bated with 10 TCID50 (50% tissue culture infectious dose) of virus in a total volume of 50 µ . After 1 h of incubation at 37°C, 50 µ of culture medium containing 3.104 HUT-78 cells were added to each well. After a second hour of incubation at 37°C, the plate was washed twice with PBS, and cells were resus¬ pended in 200 µ of culture media containing 2 µg/ml of polybrene. This assay was performed in quadruplicate. The presence or absence of syncytia was detected at days five and seven by double microscopic examination. The neutralizing titer was determined as the dilution where at least one out of four
the reciprocal synctia.
as
of the
highest
ascite
1(2)345678
to
9
10
9
10
200—^ 94—M 68
~B
43—m
29
V s
1(2)345678
Radioimmunoprecipitation assays of SIVmac25] enve¬ lope glycoproteins with 9 MAbs. Both supernatants (S) and pellets (P) from BHK 21 cells infected overnight with the wild-type (—) or the recombinant vaccinia virus (+) expressing the native SIVmac glycoproteins were tested with 9 MAbs in ascite form. Lane 1: SIVmac25,-infected macaque serum; lane 2 FIG. 1.
MATG2014; lane 3: MATG2003; lane 4: MATG2033; lane 5 MATG2037; lane 6: MATG2022; lane 7: MATG2016; lane 8 MATG2029; lane 9: MATG2008; lane 10: MATG2024.
NEUTRALIZING DOMAIN IN THE
SIVmac gpl30
1167
2014 140
160
180
200
amino acid
-PVS__WW_TSSCUQNNCTCLEQEQMISCKFrMTCL^^
position
2033
IS
reactivity
with MAbs
2033
2014
139-182
c
176-216
+
161-210
+
155-202
+
5S
165-200
+
55
161-208
+
PI
153-170
P2
161-180
P3
171-190
P4
181-200
P5
175-188
2S
196-208
P6 _
FIG. 2. Epitope mapping of MATG2033 and MATG2014 MAbs. (A) Clones 1S to 6S were selected by screening a random library of SIVmac251 peptides expressed in yeast (B). The synthetic peptides PI to P6 were tested in ELISA (200 ng/well). MATG2033 and MATG2014 MAbs were used at a 1:1000 dilution, the second antibody was coupled to peroxidase and reactions were revealed in the presence of O-phenylenediamine and H202. The 139 to 216 amino acid sequence of the SIVmac251 gpl 30 is shown at the top, as well as the deduced epitope positions of MATG2033 and 2014. wells had no detectable syncytia. A single MAb, MATG2014, had a strong neutralizing activity against SIVmac251, displaying a titer of 1:1024 corresponding to an antibody concentration of 10 µg of IgG/ml. Another MAb, MATG2033, had a marginal effect, displaying a neutralizing titer of 1:64 corresponding to an IgG concentration of 65 µg/ml, while the other seven MAbs failed to perturb the infectivity of cell-free viruses at the highest concentration tested, 1:32 (Table 1). The affinity of the two neutralizing MAbs for the native and denatured SIVmac25I gpl40 was determined by measuring the dissociation constants (Kd) according to the method of Friguet et al.20 M ATG2014 displayed ^ of 1 x 10"9Mand5 x 10~'° M, respectively, for the native and denatured gpl40, while MATG2033 had a lower affinity (Kd 2 x 10~8 M and 1 x IO-8 M) for the same respective proteins (Table 2). This result suggests that the epitopes recognized by the MAbs are well exposed on the native protein, as expected from the neutralizing activity. It should be also noted that the weaker neutralizing activity of MATG2033 may be explained by its lower affinity for the SIVmac envelope glycoprotein. In addition, since denaturation of the antigen has no effect on the MAb affinities, the epitopes recognized by the MAbs most likely are not conformational. Given the neutralizing activity of MATG2033 and 2014, delineation of the epitopes recognized by these MAbs was performed according to a method already described.21 Briefly, a peptide expression library (6.104 clones) was constructed in S. =
cerevisiae with an expression/secretion vector pSE-X, using as inserts small fragments (100-200 bps) of SIVmac25I proviral DNA generated by random DNAsel cleavage. The vector contains an expression cassette based on the -mating factor gene allowing the expression of a fusion protein.22 The C-termi¬ nal portion is encoded by the insert and the N-terminal portion is a fragment of the prepro-a factor molecule. The recombinant peptide usually takes the yeast secretory pathway, may be glycosylated, and is then released in the yeast culture medium. The SIVmac peptide library (about 3 x 104 yeast clones produc¬ ing SIVmac25i peptides) was immunologically screened using MATG2033 and 2014 MAbs at a 1:500 dilution. Several yeast immunoreactive clones were detected, their plasmid recovered and DNA sequencing of the inserts permitted deduction of the primary structure of the SIVmac peptides produced by the positive yeast clones. As shown in Figure 2A, the yeast recom¬ binant peptides produced by clones 3S-6S are recognized by both MAbs, while MATG2033 and MATG2014 reacted only with peptides produced by clone 1S (a. a. 139-182) and clone 2S (a.a. 176-216), respectively. The two MAbs appeared to recog¬ nize distinct overlapping epitopes from the same domain of the S1V_ gpl30. To confirm and delineate more precisely this
epitope mapping, overlapping synthetic peptides corresponding to the domain were analyzed by ELISA (Fig. 2B). Altogether, the data in Figure 2 indicate that the antigenic sites recognized by MATG2033 and 2014 are situated, respectively, within residues 171-180 and 176-188 of the SIVmac25] gpl30.
BENICHOU ET AL.
1168 1.1.
Table 2. Dissociation Constants (Kd of MATG2014 and
1.0.
SIV„
gpl40
MAbs
0.7.
MATG2014 MATG2033
0.6
1 2
x x
HIV-2B
gpl40
gpl40D*
10"9M 5 10-8M 1
x x
10~'°M 10~8M
>
6.5 1.8
x x
10"8M 10"~7M
agpl40D denaturated gpl40 obtained by incubation of concentrated protein, 10 min at 100°C, in 0.06 M Tris-HCl buffer (pH 6.8) containing 5% ß-mercaptoethanol and 2.5 % sodium dodecylsulfate. =
°
05
ü
Ö 0.4.
obtained from uninfected control macaques, and no reactivity was observed. Thus, this neutralizing domain usually induces a specific antibody response during the course of an experimental infection by SIVmac251. four
0.3
0.2
sera
0.1
0.0
RESULTS DDD D
GDDD
P2
P3
P5
161-180
171-190
175-188
D
D
D
FIG. 3.
Reactivity of SIVmac-infected macaque sera against SIVmac251 neutralizing domain. Filled square: sera from 6 experimentally SIVmac251-infected macaques. Open square: sera from 4 uninfected control macaques. See Fig. 2B for ELISA method. Sera were used at a 1:100 dilution. Each point repre¬ sents the mean of duplicate values. The amino acid position of the synthetic peptides P2, P3, and P5 are indicated. To evaluate the antigenicity of the neutralizing domain in the natural course of infection, serum samples from 6 experimen¬ tally SIVma
Neutralizing Domain in the External Envelope Glycoprotein of Simian Immunodeficiency Virus
Identification of a
BENICHOU,1 ROGER LEGRAND,3 NAOKO NAKAGAWA,4 THÉRÈSE FAURE,4 GUILLAUME VOGT,3 DOMINIQUE DORMONT,3 PIERRE TIOLLAIS,1 TRAINCARD,2 FRANÇOIS MARIE-PAULE KIENY ,4 and PASCAL MADAULE1 SERGE
ABSTRACT Two murine monoclonal antibodies (MAbs), designated MATG2014 and MATG2033, were generated. They are reactive with the external envelope glycoprotein gpl30 of the simian immunodeficiency virus of macaque monkey (SIVmac2Si), and display a cell-free virus neutralizing activity in vitro. In addition, MATG2014 cross-reacts with HIV-2Rod gpl40. Epitope mapping of these MAbs was performed by screening an SIVmac peptide library expressed in yeast and confirmed using synthetic peptides. MATG2014 and MATG2033 recognize two overlapping epitopes localized in an 18 residue domain between amino acid 171 and 188 of the SIV-ac2s1 gpl30. Sera from experimentally SIV-infected macaques are immunoreactive with this neutralizing domain. Sequence comparison with related SIV and HIV-2 viral strains indicates a low variability of this region, consistent with the cross-reactivity of MATG2014 with HIV-2Rod gpl40. This domain should then be considered in designing experimental vaccines.
INTRODUCTION viruses (SIVs) belong to a primate lentiviruses, first isolated from rhesus monkeys (SIVmac) and subsequently from several other monkey species. ' ·2 This group of lentiviruses shares many biological, pathogenic, genetic, and antigenic properties with the human immunodeficiency viruses (HIV-1 and HIV-2), the etiological agents of AIDS. In particular, SIVmac is very closely
Thefamily
simian immunodeficiency
of nonhuman
related to HIV-2.3·4 Because of these similarities between SIVs and HIVs, the SIV macaque model is particularly attractive for the development and testing of vaccine strategies against human AIDS, and initial vaccine trials in macaques, using an inacti¬ vated whole SIVmac vaccine, suggest that a protective immunity may be obtained against SIVmac.5·6
METHODS In the case of HIV-1, several targets for neutralizing antibod¬ ies have been identified,7-12 among which the principal deter¬
minant is contained within a loop located in the third variable domain (V3 loop) of the external glycoprotein gpl20.13,14 The V3 loop however induces isolate-specific neutralizing antibod¬ ies, probably due to its sequence diversity among HIV-1 iso¬ lates.13 In the case of SIV, a strain-specific neutralizing deter¬ minant present in the transmembrane glycoprotein of SIVmac has been recently described,15 but very little is known about neutral¬ izing antibodies directed against the external glycoprotein of SIVs gpl30. In this report, we analyze two MAbs, designated MATG2033 and MATG2014, raised against SIVmac251 gpl40 and displaying a cell-free virus neutralizing activity in vitro. The antigenic domain recognized by these two MAbs has been mapped in the external envelope glycoprotein using a SIVmac peptide library expressed in Saccharomyces cerevisiae. This neutralizing determinant does not correspond to the presumed V3 loop-like domain of SIVmac.I6 MAbs were raised in mice against SIVmac, isolate 251, as follows. A recombinant vaccinia virus expressing the modified env gene was constructed. As for the HIV-1 env protein,17 the cleavage site between gpl30 and gp32 (a.a. 546 and 547) was
'Unité de Recombinaison et Expression Génétique and 2Hybridolab, Institut Pasteur, 28 rue du Dr Roux, 75024 Paris Cedex 15 France. 'Commissariat à l'Energie Atomique, Département de toxicologie Experimentale, CRSSA, 92265 Fontenay aux Roses Cedex, France. Transgène S.A., 67032 Strasbourg, Cedex, France. 1165
1166
BENICHOU ET AL. Table 1. Characterization of SIV„
MAbs
Ig subtype
No.
2003 2008 2014 2016 2022 2024 2029 2033 2037 b
Gl G2b G2b G2a Gl Gl Gl Gl Gl
'
Antibody b
Antibody
titer
3
6.6 2 4 3 5
X x
104 104
X
105 104 105
gpl40
concentration
10s
6.7 3.4 10.3 6.9 19.5 12 5.1
4 104
2.9
x
x x
MAbs
104
Neutralizing
'
titer
'
1024
64
Subtypes were determined using a mouse monoclonal antibody isotyping kit from Amersham. The titer is defined as the reciprocal of the ascite dilution giving 50% of the maximum
absorbance value. c Expressed in mg/ml of ascite fluid. d Neutralization activity for SIVmac251 is expressed dilution where at least 1 out of 4 wells had no detectable
mutated and the hydrophobic transmembrane domain (a.a. 166 189 of gp32) was deleted. The live recombinant virus served as immunogen. Six-week-old female BALB/c mice were immu¬ nized intraperitoneally with 5.107 plaque-forming units (pfu) of virus in complete Freund's adjuvant, followed by two boosts in incomplete Freund's adjuvant at 1 week intervals. A third boost in phosphate-buffered saline (PBS) after 1 week was performed intravenously. Three days later, the spleen cells were fused to P3x63-Ag8U! (ATCC CRL 1897) cells and selected in HAT medium as usual.18 A total of 33 hybridomas reactive against purified SIVmac gpl40 produced by recombinant vaccinia virus in an enzyme-linked immunosorbent assay (ELISA) were ob¬ tained. The hybridoma corresponding to 9 of these MAbs was subcloned and injected to mice to generate ascite fluids. The ELISA titers, IgG contents, and immunoglobulin subclasses of the MAbs were determined (Table 1 ). A radioimmunoprecipita¬ tion assay (RIPA) was performed as previously described17 with all the MAbs on supernatants and extracts from BHK 21 cells infected with the recombinant vaccinia virus expressing the native SIVmac25I envelope glycoproteins (VV-TG-SIV-4176) (Fig. 1). All the MAbs, except MATG2003, immunoprecipi¬ tated SIVmac envelope glycoproteins from the supernatant and/or from the cell pellet. Only MATG2003 failed to signifi¬ cantly precipitate SIVmac251 glycoproteins, but reacted in ELIS A with recombinant gp 140 at the titer of 3 x 104 (Table 1 ). These anti-SIVmac25, gpl40 MAbs were then tested for their ability to neutralize SIVmac251 using an assay described previ¬ ously.19 Briefly, twofold serial dilutions of ascites were incu¬ bated with 10 TCID50 (50% tissue culture infectious dose) of virus in a total volume of 50 µ . After 1 h of incubation at 37°C, 50 µ of culture medium containing 3.104 HUT-78 cells were added to each well. After a second hour of incubation at 37°C, the plate was washed twice with PBS, and cells were resus¬ pended in 200 µ of culture media containing 2 µg/ml of polybrene. This assay was performed in quadruplicate. The presence or absence of syncytia was detected at days five and seven by double microscopic examination. The neutralizing titer was determined as the dilution where at least one out of four
the reciprocal synctia.
as
of the
highest
ascite
1(2)345678
to
9
10
9
10
200—^ 94—M 68
~B
43—m
29
V s
1(2)345678
Radioimmunoprecipitation assays of SIVmac25] enve¬ lope glycoproteins with 9 MAbs. Both supernatants (S) and pellets (P) from BHK 21 cells infected overnight with the wild-type (—) or the recombinant vaccinia virus (+) expressing the native SIVmac glycoproteins were tested with 9 MAbs in ascite form. Lane 1: SIVmac25,-infected macaque serum; lane 2 FIG. 1.
MATG2014; lane 3: MATG2003; lane 4: MATG2033; lane 5 MATG2037; lane 6: MATG2022; lane 7: MATG2016; lane 8 MATG2029; lane 9: MATG2008; lane 10: MATG2024.
NEUTRALIZING DOMAIN IN THE
SIVmac gpl30
1167
2014 140
160
180
200
amino acid
-PVS__WW_TSSCUQNNCTCLEQEQMISCKFrMTCL^^
position
2033
IS
reactivity
with MAbs
2033
2014
139-182
c
176-216
+
161-210
+
155-202
+
5S
165-200
+
55
161-208
+
PI
153-170
P2
161-180
P3
171-190
P4
181-200
P5
175-188
2S
196-208
P6 _
FIG. 2. Epitope mapping of MATG2033 and MATG2014 MAbs. (A) Clones 1S to 6S were selected by screening a random library of SIVmac251 peptides expressed in yeast (B). The synthetic peptides PI to P6 were tested in ELISA (200 ng/well). MATG2033 and MATG2014 MAbs were used at a 1:1000 dilution, the second antibody was coupled to peroxidase and reactions were revealed in the presence of O-phenylenediamine and H202. The 139 to 216 amino acid sequence of the SIVmac251 gpl 30 is shown at the top, as well as the deduced epitope positions of MATG2033 and 2014. wells had no detectable syncytia. A single MAb, MATG2014, had a strong neutralizing activity against SIVmac251, displaying a titer of 1:1024 corresponding to an antibody concentration of 10 µg of IgG/ml. Another MAb, MATG2033, had a marginal effect, displaying a neutralizing titer of 1:64 corresponding to an IgG concentration of 65 µg/ml, while the other seven MAbs failed to perturb the infectivity of cell-free viruses at the highest concentration tested, 1:32 (Table 1). The affinity of the two neutralizing MAbs for the native and denatured SIVmac25I gpl40 was determined by measuring the dissociation constants (Kd) according to the method of Friguet et al.20 M ATG2014 displayed ^ of 1 x 10"9Mand5 x 10~'° M, respectively, for the native and denatured gpl40, while MATG2033 had a lower affinity (Kd 2 x 10~8 M and 1 x IO-8 M) for the same respective proteins (Table 2). This result suggests that the epitopes recognized by the MAbs are well exposed on the native protein, as expected from the neutralizing activity. It should be also noted that the weaker neutralizing activity of MATG2033 may be explained by its lower affinity for the SIVmac envelope glycoprotein. In addition, since denaturation of the antigen has no effect on the MAb affinities, the epitopes recognized by the MAbs most likely are not conformational. Given the neutralizing activity of MATG2033 and 2014, delineation of the epitopes recognized by these MAbs was performed according to a method already described.21 Briefly, a peptide expression library (6.104 clones) was constructed in S. =
cerevisiae with an expression/secretion vector pSE-X, using as inserts small fragments (100-200 bps) of SIVmac25I proviral DNA generated by random DNAsel cleavage. The vector contains an expression cassette based on the -mating factor gene allowing the expression of a fusion protein.22 The C-termi¬ nal portion is encoded by the insert and the N-terminal portion is a fragment of the prepro-a factor molecule. The recombinant peptide usually takes the yeast secretory pathway, may be glycosylated, and is then released in the yeast culture medium. The SIVmac peptide library (about 3 x 104 yeast clones produc¬ ing SIVmac25i peptides) was immunologically screened using MATG2033 and 2014 MAbs at a 1:500 dilution. Several yeast immunoreactive clones were detected, their plasmid recovered and DNA sequencing of the inserts permitted deduction of the primary structure of the SIVmac peptides produced by the positive yeast clones. As shown in Figure 2A, the yeast recom¬ binant peptides produced by clones 3S-6S are recognized by both MAbs, while MATG2033 and MATG2014 reacted only with peptides produced by clone 1S (a. a. 139-182) and clone 2S (a.a. 176-216), respectively. The two MAbs appeared to recog¬ nize distinct overlapping epitopes from the same domain of the S1V_ gpl30. To confirm and delineate more precisely this
epitope mapping, overlapping synthetic peptides corresponding to the domain were analyzed by ELISA (Fig. 2B). Altogether, the data in Figure 2 indicate that the antigenic sites recognized by MATG2033 and 2014 are situated, respectively, within residues 171-180 and 176-188 of the SIVmac25] gpl30.
BENICHOU ET AL.
1168 1.1.
Table 2. Dissociation Constants (Kd of MATG2014 and
1.0.
SIV„
gpl40
MAbs
0.7.
MATG2014 MATG2033
0.6
1 2
x x
HIV-2B
gpl40
gpl40D*
10"9M 5 10-8M 1
x x
10~'°M 10~8M
>
6.5 1.8
x x
10"8M 10"~7M
agpl40D denaturated gpl40 obtained by incubation of concentrated protein, 10 min at 100°C, in 0.06 M Tris-HCl buffer (pH 6.8) containing 5% ß-mercaptoethanol and 2.5 % sodium dodecylsulfate. =
°
05
ü
Ö 0.4.
obtained from uninfected control macaques, and no reactivity was observed. Thus, this neutralizing domain usually induces a specific antibody response during the course of an experimental infection by SIVmac251. four
0.3
0.2
sera
0.1
0.0
RESULTS DDD D
GDDD
P2
P3
P5
161-180
171-190
175-188
D
D
D
FIG. 3.
Reactivity of SIVmac-infected macaque sera against SIVmac251 neutralizing domain. Filled square: sera from 6 experimentally SIVmac251-infected macaques. Open square: sera from 4 uninfected control macaques. See Fig. 2B for ELISA method. Sera were used at a 1:100 dilution. Each point repre¬ sents the mean of duplicate values. The amino acid position of the synthetic peptides P2, P3, and P5 are indicated. To evaluate the antigenicity of the neutralizing domain in the natural course of infection, serum samples from 6 experimen¬ tally SIVma
