Molecular Immunology, Vol. 29, No. 10, pp. 1175-l 183, 1992 Printed in Great Britain.
0161-5890/92 $5.00 + 0.00 0 1992 Pergamon Press Ltd
SUBTYPING OF HUMAN IMMUNODEFICIENCY VIRUS ISOLATES WITH A PANEL OF MONOCLONAL ANTIBODIES: IDENTIFICATION OF CONSERVED AND DIVERGENT EPITOPES ON p17 AND ~25 CORE PROTEINS* V&RONIQUEROBERT-HEBMANN,~ ST~~PHANEEMILIANI,~ MARIANA RESNICOFF,~ FRBDBRIC JEANS and CHRISTIAN DEVAUX~§ tCRBM du CNRS, Centre de Tri des Mokcules Brd Henri IV, 34060 Montpellier,
Anti-HIV, Institut de Biologie-Facultb de Mkdecine, France; and $Immunotech S.A., Campus Universitaire de Luminy, 13288 Marseille. France
(First received 29 February 1992; accepted in revised form 8 April 1992) Abstract-We have investigated the feasibility and significance of subtyping of human immunodeficiency virus (HIV) isolates with monoclonal antibodies (mAb) raised against the core proteins of HIV. A panel of 37 mAb tested for reactivity with HIV1 oligopeptides was used to analyse the antigenic relatedness among 14 HIV isolates which included 12 isolates of HIV1 from different geographical origins and 2 isolates of HIV2. Three out of these 37 mAb reacted with conserved epitopes expressed by all 14 HIV isolates tested. These reagents which included 2 mAb reacting with the 285-310 amino acid sequence of p25 and 1 mAb reacting with an epitope of p25 not mapped by the peptides’ approach, also reacted with a non-human primate lentivirus. Five mAb reacting either with the 1l-25 or 121-132 amino acid sequences of p17 or the 302-320 amino acid sequence of ~25 reacted with strain-specific epitopes. The other 29 mAb reacted with polymorphic epitopes and thereby define subfamily and subtype-specific markers.
INTRODUCTION The Human Immunodeficiency Virus (HIV) is the primary aetiological agent of Acquired Immunodeficiency Syndrome (AIDS) and associated diseases (Barr&Sinoussi et al., 1983; Gallo et al., 1984). HIV and related viruses belong to the subfamily of lentiviridae. These viruses share a similar genetic organization (Delassus and Wain-Hobson, 1988; Cullen, 1991); the viral genes encode core structural proteins (gag), enzymes @ol), envelope proteins (env) and several regulatory proteins. Survey of the worldwide spreading of HIV has demonstrated a major distinction in the partition of AIDS-causing viruses (Piot et al., 1988). HIV1 isolates have been predominantly recovered from individuals in the United States, Europe and Central Africa whereas HIV2 isolates have been found in West African individuals. A high degree of polymorphism has been demonstrated between HIV1 and HIV2 isolates (Clavel et al., 1986; Guyader et al., 1987). Genetic variability has been also described within the group of HIV1 isolates (Hahn et al., 1985; Willey et al., 1986). A number of studies have indicated that HIV isolates differ in their biological properties and can therefore be classified according to their tropism, their capacity
*This work was supported by institutional grants from CNRS, INSERM and ANRS. $To whom correspondence should be addressed.
and to form syncytia (Cheng-Mayer et al., 1988; Stevenson et al., 1990; Von Briesen et al., 1990; York-Higgins et al., 1990). The high degree of genetical polymorphism found among HIV isolates accounts for these different biological properties (Fisher et al., 1988; Masuda et al., 1990; Rey et al., 1991; Fredriksson et al., 1991). The study of genetic relatedness among human and non-human primate lentiviruses could help to elucidate the history and evolution of this group of viruses. With respect to their structural gene variability, this could be carried out by investigating the spectrum of HIV isolates existing in different geographic regions. Among the structural genes of the lentiviruses, the variability is mainly distributed within the env region whereas the gag and pol genes are more conserved among the different isolates. Therefore the structural proteins p17 and p25 which are the main components of the gag gene products represent appropriate antigens for serologic analysis of isolates relatedness. In the past few years, several laboratories including our own (Ferns et al., 1987; Niedrig et al., 1989, 1991; Tersmette et al., 1989; Hinkula et al., 1990; Shang et al., 1991; Robert et al., 1991) have attempted to generate mAb against HIV core antigens in order to use them as typing tools. We show here, how a panel of such mAb is able to differentiate among 12 HIV1 isolates from American, European and African origins, two HIV2 isolates and one Simian Immunodeficiency Virus (SIV) isolate. to replicate
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V~RONIQUE ROBERT-HEBMANN et al. MATERIALS
AND METHODS
Monoclonal antibodies. mAb RL4.11.14, RL4.72.1 (Tatsumi et al., 1990a), RL16.24.5 (Robert et al., 1991) and M01.34.1, M09.42.2, M09.50.2 (Robert-Hebmann et al., 1992) have been obtained in our laboratory. mAb C.V.K. has been obtained by J. Chassagne (Chassagne et al., 1986) and provided by F. Barre-Sinoussi (Institut Pasteur, Paris, France). mAb K7.17, L14.17, K3.24 and L6.24 (Tatsumi et al., 1990a) have been provided by M. Tatsumi (NIH, Tokyo, Japan). mAb M26 (Di Marzo Veronese et al., 1988) has been provided by F. Di Marzo Veronese under transfer agreement with NIH/ADAMHA (NIH/ADAMHA, Bethesda, MD, U.S.A.). mAb 8D2, 8H7 and 3H7 (Niedrig et al., 1988, 1989) have been provided by H. Gelderblom (Robert Koch Institut, Berlin, Germany). mAb CLB-14, CLB-16, CLB-21 and CLB-47 (Tersmette et al., 1989) have been provided by J. Huisman (Netherlands Red Cross Blood Tranfusion Service, Amsterdam, The Netherlands). mAb 14D4E11, 3DlOG9, 15F8C7, llDllF2, lG5C8,9A4C4, llClOB10, 23A5G5 and 9B5C12 (Janvier et al., 1990) have been provided by B. Mandrand (UM 103 CNRS-Biomerieux, France) under transfer agreement with “Agence Nationale de Recherches sur le SIDA” (ANRS, Paris, France). mAb 21 l/92,406/01, 714/01, 1109jOl and 21 l/84 (Robert-Hebmann et al., 1992) have been provided by J. Larroque (Biosoft, Clonatec, Paris, France). mAb 3 l-l 1, 15-21, 47-2 and 169-2 (Robert-Hebmann et al., 1992) have been provided by F. Traincard (Hybridolab, Paris, France). The main characteristics of these mAb are summarized in Table 1. Viruses. HIVl-GER (Robert et al., 1991) HIVl-ROI and HIVl-SPA (MR unpublished) have been isolated in our laboratory. The other viruses HIVl-BRU (Barre-Sinoussi et al., 1983), HIV2-ROD (Clavel et al., 1986) and HIVl-ELI (Alizon et al., 1986) were provided by L. Montagnier (Institut Pasteur, Paris, France); HIVlNDK (Ellrodt et al., 1984) and HIVl-PAS (Robert et al., 1991) were provided by J. C. Chermann (INSERM U322, Marseille, France); HIVl-GON (A. Georges, unpublished), HIVl-OMB (Robert et al., 1991), originally isolated by A. Georges (Institut Pasteur, Bangui, RCA), HIV2-1169 (F. Barre-Sinoussi, unpublished) and SIV-SM (Marx et al., 1991) were all provided by F. Barre-Sinoussi (Institut Pasteur, Paris, France); HIVl-MN (Gallo et al., 1984) HIVl-RF (Popovic et al., 1984) and HIVl-SF2 (Levy et al., 1984) were provided by H. Holmes (MRC AIDS Reagent, Project, Herts, U.K.). These viruses were propagated in human T cells (CEM) under conditions previously described (Tatsumi et al., 1990b). Concentrated virus preparations were obtained from the cell-free supernatant by 100 OOOg ultracentrifugation for 2 h. The viral pellet was resuspended hundred-fold concentrated in NTE buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA pH 7.4). Viral particles (1 mg/ml) were inactivated by 56°C incubation for 30 min and treated with 0.1% Triton X 100. Recombinant proteins. The polyhedrin fusion-HIVlgag (~17, p25 and most of p9) protein (fus-gag)
recovered from baculovirus infected cells was provided by P. Boulanger (Institut de Biologie, Montpellier, France). Enzyme-linked immunosorbent assay (ELISA). ELISA plates (Nunc, Paisley, U.K.) were coated overnight with 10 pgg/ml of inactivated virus in 100 mM sodium carbonate buffer pH 9.6. After coating, plates were washed and saturated with phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA). 100 ~1 of mAb at an appropriate dilution (defined by lack of reactivity with uninfected CEM cells), were incubated 1 h at room temperature, after which the plates were washed three times with PBS-0.05% Tween 20. Bound immunoglobulins (Ig) were detected by adding 100 ~1 of goatanti-mouse IgG H + L peroxidase conjugate (lo3 fold diluted) (Immunotech S.A., Marseille, France) for 1 h, followed by washing and subsequent incubation with 0-phenylenediamine (OPD) (Sigma) as substrate. ELISA using recombinant proteins was performed as previously described (Robert-Hebmann et al., 1992). Amino acid sequence comparison. p17 and p25 amino acid sequences of 17 HIV1 (namely: BRU (LAI); HXB2; MN; JH3; CDC41; OYI; SF2; HAN; RF; ELI; 22; NDK; MAL; BHlO; BH5; PV22; WMJ2), 7 HIV2 (namely: ROD; NIHZ; ST; ISY; BEN; GHl; D205) and 3 SIV (namely: MM251; MM142; SMMH4) from Genbank (Bolt Beranek and Newman, Cambridge, MA, USA) and Los Alamos HIV sequence Database (Group T-10, Los Alamos, NM, U.S.A.) were compared. The sequence of HIVl-BRU was used as a standard and the number of different amino acids or deletion-insertion was calculated for each position.
RESULTS
Reactivity of the mAb with HIVl-BRU, recombinant molecules and oligopeptides
HZVl-BRU
A selection of 37 mAb specific for the HIV-core antigens which originated from 13 independent fusion experiments was used for subtyping of HIV isolates. These mAb were produced against HIV1 (BRU, IIIB, NDK or KB strain, respectively) or HIV2 (ROD strain). Their fine specificity has been previously analysed by reacting them with 35 HIVl-BRU overlapping synthetic linear peptides (Robert-Hebmann et al., 1992). Of these 37 mAb, 20 have been precisely mapped by the peptides’ approach. Failure in mapping 17 mAb binding site was a consequence of the lack of HIVl-BRU binding (for two of them), or was related to the conformational requirement of their target-epitopes (for the others). Table 1 shows the reactivity of these mAb with HIVlBRU, HIVl-BRU gag recombinant molecules (fus-gag), and summarizes the result of our previously published mapping study. Analysis of p 17 and p25 antigenic polymorphism
In order to quantitate the p17 and p25 antigens variability among HIV isolates, viral amino acid sequences were compared. A computer program was used to construct diagrams of p17 and p25 proteins variability. The
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Subtyping of HIV isolates with mAb Table 1. mAb used in this study ELISA reactivity1 Hybridoma designation* Anti-p17 mAb L14.17 3H7 31-11 15-21 21 l/92 RL16.24.5 K7.17 C.V.K. M01.34.1 Anti-p25 mAb CLB-2 1 15F8C7 47-2 14D4Ell 8H7 714/01 1109/01 8D2 lG5C8 RL4.72.1 M26 406/O1 CLB-14 L6.24 169-2 23A5G5 3DlOG9 M09.42.2 M09.50.2 9A4C4 llDllF2 1IClOBlO K3.24 9BSC12 CLB-16 CLB-47 R14.11.14 21 l/84
Immunogent
HIVI-BRU
fus-gag
BRU peptides
HIVl-BRU HIVl-IIIB HIVl-BRU HIVl-BRU HIVl-IIIB HIVl-NDK HIVl-KB HIVl-BRU HIVZROD
+ + + + + -
+ + + + + -
+ + -
+ + -
1l-25 (P2) Ill-123 (P12) 121-132 (P13) 121-132 (P13) ND ND ND ND ND
HIV1 ~25 HIVl-IIIB HIVl-BRU ~25 HIVl-IIIB HIVl-IIIB HIVl-IIIB HIVl-IIIB HIVI-IIIB ~25 HIVl-IIIB HIVl-NDK HIV1 -1IIB HIVl-IIIB HIV1 HIVl-BRU HIVZROD ~25 HIVl-IIIB ~25 HIVl-IIIB HIV2-ROD HIVZROD ~25 HIV l -IIIB ~25 HIVl-IIIB ~25 HIVl-IIIB HIVI-KB HIVl-IIIB/HIVZ HIV1 HIV1 HIV1 -NDK HIVl-IIIB
+ + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + +
ND ND 201-218 (P21) 201-218 (P21) 201-218 (P21) 201-218 (P21) 201-218 (P21) 201-218 (P21) 201-218 (P21) 219-233 (P22) ND 233-253 (p23) ND ND ND 285-304 (P28) 285-304 (P28) 285-3 10 (P28-P29) 285-310 (P28-P29) 302-320 (P30) 302-320 (P30) 302-320 (P30) ND ND ND ND ND ND
-I+ +
-I+ + -f + + + + + + + -I-t + + + + + + + + +
*Hybridoma were constructed by fusion of either NSl-Ag4.1 (3H7, M26, 8D2, 8H7), SP2/O.Ag14 (L14.17, 311/01, K7.17, CLB-21, 15FSC7, 14D4El1, 714/01, 1109/01, lG5C8, 406/01, CLB-14, L6.24, 23A5G5, 3DlOG9, 9A4C4, llDllF2, llClOBl0, K3.24,9B5C12, CLB-16, CLB-47,211/84) or X63-Ag8.653 (31-11, 15-21, RL16.24.5, C.V.K., M01.34.1, 47-2, RL4.72.1, 169-2, M09.42.2, M09.50.2, RL4.11.14) mouse myeloma cells with spleen cells from BALB/c immunized mice. The mAb express either IgGl, IgG2a, IgG2b, or IgG3 isotype (Robert-Hebmann et al., 1992). tMice were immunized with either inactivated HIV1 or HIV2 viruses or purified viral proteins. HIVZROD = HIV type 2 ROD strain; ~25 HIVl-IIIB = purified ~25 protein from HIV type 1 strain HTLV-IIIB. $mAb were tested for binding to HIVl-BRU, polyhedrin-fusion-HIVl-gag (fus-gag) and 35 overlapping synthetic linear HIVl-BRU peptides (Pl to P35) totally covering the p17 (Pl to P13) and ~25 (P14 to P35) proteins. Their reactivity with HIVl-BRU and peptides has been previously published (Robert-Hebmann et af., 1992). The reactivity of a given mAb for a given antigen was considered positive ( +) when absorbance was over 3 times the background. jAmin acid numbers of the first and last residues of target peptide are given according to the HIV sequence Database Group T-10 (Los Alamos National Laboratory, NM, U.S.A.) and peptide code is shown between brackets. The peptide code is given according to that previously reported (Robert-Hebmann et al., 1992).
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VBR~NIQUEROBERT-H~B~~NNet al.
Anlao
ACID WtlBER
Fig. 1. Superimposition of peptide location on the diagrams of variability of the p17 and ~25 proteins constructed by comparison of amino acid sequences of different HIV and SIV isolates. The HIVI-BRU sequence was used as a standard. A difference in amino acids at a given position to the sequence of prototype is depicted by a vertical bar equal to 1 unit in the ordinate scale. Sequence alignments were made according to the highest percentage of homology to the prototype at each position; therefore a deletion or addition that could result in a frame shift is illustrated by a vertical bar. (A) Sequence variations among HIV1 isolates (n = 17; see Materials and Methods for details); (B) Sequence variations among HIV1 (n = 17), HIV:! (n = 7) and SIV (n = 3).
variability was evaluated with respect to the sequence of HIVl-BRU. A diagram of variability of HIV1 p17 and ~25 sequences was constructed using known sequences of 16 HIV1 isolates, and another diagram of primates lentivirus p17 and ~25 sequences variability was constructed using the previous 16 HIV1 sequences together with that of 7 HIV2 and 3 SIV. Figure 1 superimposes the location of HIVl-BRU peptides shown to be target for mAb (peptides P2, P12, P13, P21, P22, P23, P28, P29 and P30) on the diagrams of variability of HIV1 (LA) and primate lentiviruses (1 B). This schematic representation allows to predict whether or not the target epitope for a given mAb is expected to be shared among virus isolates. For example, epitopes of P29 would be expected to be quite conserved among HIV1 and HIV2 isolates whereas epitopes expressed by P13 might be of two types, either highly polymorphic or conserved among HIV1 isolates. This was indeed verified by serotyping of HIV isolates. Serotyping of HIV isolates
The results of the serotyping of HIV isolates with mAb by ELISA are summarized in Fig. 2. This study revealed appreciable heterogeneity in epitope expression. With the exception of 3 mAb (M09.42.2, M09.50.2 and 9B5C12) which bound epitopes shared by HIVl, HIV2 and SIV isolates, all other mAb revealed an antigenic polymorphism within the core molecules of the primate lentivirus isolates tested. Among these mAb, 2 (23A5G5 and 3DlOG9), bound an epitope(s) located in the P28 peptide and shared by the HIV1 and HIV2 isolates tested with the exception of HIVl-GER and HIVl-SPA.
A few mAb, such as L14.17 (reacting with a sequence in P2), 9A4C4, llDllF2 and llClOBl0 (reacting with a sequence in P30), or 15-21 (reacting with a sequence in P13) reacted with polymo~hic antigens of HIV. The restricted pattern of reactivity of mAb 15-21 is compatible with the fact that about half of the PI3 sequence diverges among the isolates (see Fig. 1). Interestingly, another mAb reacting with P13 (mAb 31-11, known to bind an epitope of P13 distinct from that of 15-21; Ro~rt-Hebmann et al., 1992), showed a broad reactivity pattern among isolates. This is consistent with the fact that another part of P13 is conserved among HIV 1. Among the mAb that bound epitopes having conformational requirements, some, like RL16.24.5, L6.24, K3.24 and CLB-16 also exhibited a relatively restricted pattern of cross-reactivity. Over two-thirds of the virus isolates had altered expression of the majority of epitopes recognized on HIVl-BRU using this panel of mAb. For example, all seven mAb to P21 (amino acids 201-21s) reacted with HIVi-BRU, HIVl-RF and HIVl-NDK, their patterns of reactivity differed on HIVI-ROI (suggesting the existence of at least two distinct epitopes in P21), and they did not react with the other viruses tested. The majority of mAb tested clearly define subtype specific epitopes. Interestingly for typing purposes, some of these mAb showed totally distinct patterns of crossreactivity. For example, K7.17 reacted with HIV1 -BRU, HIVl-RF and HIVl-NDK but not with the other HIV1 isolates whereas M01.34.1 showed the opposite pattern of reactivity.
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Subtyping of HIV isolates with mAb
VIRUSES I
Fig. 2. Summary of serotyping of virus isolates. Reactivity of 37 anti-HIV mAb with 15 HIV and SIV isolates. The name of isolates tested and their geographical origin (Eur. = Europe; Ame. = America; Afr. = Africa) are indicated. The viruses were propagated in CEM cells and ELISA coated plates were prepared as described. mAb binding to virus isolates was measured and expressed with respect to the absorbance at 492 nm n = ELISA-positive reaction (absorbance > 1.5); N = weak reaction (0.5 < absorbance c 1.5); 0 = ELISA-negative reaction (absorbance < 0.5). The first vertical column (-) shows the lack of mAb reactivity with uninfected CEM cell supernatant treated under the same experimental conditions as viral samples. Each value has been calculated from the average of quadruplicate assays.
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VBRONIQUE ROBERT-HEBMANN et al.
11-25 HIV1 HIV1 HIV1 HIV1 HIV1 HIV1 HIV2
BRU pq, SF2 RF ELI EIDK ROD
(P2) GELDRWEKIRLRFGG . ..____~____.__ ____K__________ _K__K________R_ _K__K__________ _K__*__R_______ yJ(y_EL_R__~____
201-218 HIV1 BRU HIV1 MI1 HIV1 SF2 HIV1 RF HIV1 ELI HIV1 NDK HIV2 ROD
121-132 HIV1 HIV1 HIV1 HIV1 HIV1 HIV1 HIV2
(P21)
KE?l-11m EXAEWDRVHP _____________L-_ _____________L__ _____________L__ _____~_________ R_~_________VQ__
BRU MI1 SF2 RF ELI
DT...GHSSQVSQPN __G*Jp~~________ G-... _~~________ --..:IIG------_*~~_______
NLIK
-.....:-------
ROD
TSRFTAJ--EKCX--
285-320 HIV1 HIV1 HIV1 HIV1 HIV1 HIV1 HIV2
(P13)
BRU M.tI SF2 RF ELI NDK ROD
(P28,P29,P30)
IRQGPKEPFRDYVDRFYKTLPAEXJASQEXXNWMT~ ~_________________________RT___ D-------__________~_______~________D__..___~ ______~_________D________ ________________D_______~ _~___~___Q~_______~_____~p*______Q_
Fig. 3. Amino acid sequence alignments of HIVl-BRU gag regions 11-25 (P2), 121-132 (P13), 201-218 (P21) and 285-320 (P28, P29, P30) and regions of similarity from five different HIV1 isolates and HIV2-ROD. A dash represents an amino acid identity with HIVl-BRU. A point is used to prevent insertion-deletion frame shift.
Sequence comparison among the major antigenic regions of the HIV core molecules As shown in Fig. 3, sequence comparison confirms that the shared P28-P29 epitope(s) defined by M09.42.2 and M09.50.2 is (are) highly conserved among the HIV1 and HIV2 isolates tested in this study. Moreover, the sequence YVDRFY of P28 and P29 is also conserved among a number of other HIV1 (HXB2R, JH3, JRCSF, OYI, NYSCG, NL43, CDC4, HAN, and Z226), HIV2 (NIHZ, ISY, ST, BEN, D194, GHl, and D205), and SIV (CPZ, MM251, MM142, MM239, MNE, SMMH4, and SMMPBJ) isolates, whereas a permutation of Y for F was reported for HIVl-MAL and HIVl-U455 (Group T-10, Los Alamos, U.S.A.). The pattern of reactivity observed using mAb that bound P2, P13 or P28 is also consistent with the sequence variability found in these regions among the different isolates. For example, the private reactivity of mAb 15-21 is compatible with the presence in P13 of the DTGHS sequence which is only found in the HIVl-BRU isolate, whereas another anti-HIVl-BRU mAb 31-11 that also reacts with P13 is more likely interacting with amino acids of the SSQVSQNY sequence. Although the published 201-218 amino acid sequence of HIVl-SF2 is identical to that of HIVl-BRU, and the sequences of HIVl-MN, HIVl-ELI and HIVl-RF are identical one to the other within this region of ~25, we found that mAb specific for P21 only bound a limited number of HIV isolates (including BRU, RF and NDK but not SF2, ELI and MN). Another set of unexpected data concerns the lack of reactivity of mAb to P12, P22, and P23 with HIVl-ELI, HIVl-SF2 and HIVl-MN. This result might be related to in vitro variation of strains, laboratory contamination, or more likely differences in epitope accessibility.
DISCUSSION The aim of this study was the analysis of the antigenic relatedness among several HIV isolates by anti-core mAb serotyping and comparison of cross-reactivity patterns to viral sequences alignment, when those sequences were made available. We showed here that mAb can be used as reference standards for analysing primate lentivirus strains since subfamily, subtype and strain-specific reagents were identified. In this study, a complete correlation between the sequence alignment and the cross-reactivity pattern was found using the mAb reacting with the subfamily-specific epitopes expressed by the P28-P29 peptides or the strainspecific epitopes expressed by the P2, P13, and P30 peptides. However, a few discrepancies were observed with the mAb reacting with the subtype-specific epitopes expressed by the P12, P21, P22 and P23 peptides that showed an abnormal serotype with HIVl-ELI, HIVlMN and HIVl-SF2. In vitro variation of strains after long-term culture or undetected laboratory contamination, as previously reported by others (Tersmette et al., 1989; Wain-Hobson et al., 1991), or more likely differences in epitope accessibility, might account for such results. Another parameter to take into account concerns the method used for virus serotyping. As previously reported (Ferns et al., 1987; Niedrig et al., 1988, 1991), depending on the test system used, a few mAb showed various degrees of cross-reaction reflecting the conservation and availability of their respective epitopes. In the ELISA, RL4.72.1 reacted with HIVl-BRU, HIVl-NDK, HIVlPAS and HIV2-ROD, and was negative for HIVl-ELI, HIVl-OMB, HIVl-GER, whereas it bound all those strains in a previous study using an immunofluorescence (IF) analysis (Robert et al., 1991). Since we could not exclude changes in serotype as a consequence of main-
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Subtyping of HIV isolates with mAb tenance of the viruses in culture, RL4.72.1 was tested again and simultaneously on several isolates by both methods to determine whether or not the viruses tested in ELISA were variants. The result of this study was consistent with our previous data, suggesting that the difference resided only in the method used (data not shown). Given their broad reactivity, mAb to the epitope shared by the P28 and P29 peptides can be used for the detection of members of the lentiviridae subfamily. Niedrig et al. (1989) have described a mAb which reacts with an epitope located within this region of ~25 and expression of which is shared by HIVl-IIIB, HIV2-ROD and SIV-MAC. Here we confirm and extend this observation by showing the mAb M09.42.2 and M09.50.2 reacted strongly with all human lentiviruses and a simian lentivirus tested to date in ELISA. This result is in agreement with the sequence alignment analysis showing that the sequence YVDRFY is shared among HIVl, HIV2 and SIV. Interestingly, this sequence is not found in Visna virus (Sargan et al., 1991). Therefore, this epitope might be a marker for primate lentiviruses. The P28-reacting mAb described here, bound all HIV1 and HIV2 isolates tested with the exception of HIVlGER and HIVl-SPA, but failed to react with SIV-SM despite sequence similarities with HIV2-ROD in this region. Since HIVl-GER and HIVl-SPA apparently showed an original serotype, the epitope recognized turns out to be predominantly expressed by human lentiviruses and might therefore be considered as a subfamily-specific marker. The majority of mAb tested in this study reacted with subtype-specific epitopes. Frequently, these subtypespecific epitopes were expressed by HIV isolates from different geographical origins. For example, the P21reacting mAb bound some European and African isolates of HIV1 (e.g. HIVl-BRU and HIVl-NDK), but failed to react with other European and African isolates of HIV1 (e.g. HIVl-PAS and HIVI-GON). Similar observations have been reported by Ferns et al. (1987) who have found that the anti-p25 mAb EBlA9 produced against a European isolate called HIVl-CLBl reacts with two American HIV isolates (HIVl-IIIB and HIVl-ARV2/ SF2), with an African isolate (HIVl-Z129) but fails to bind another American isolate (HIVI-MN); whereas the anti-p25 mAb CD12B4 also produced against HIVlCLBl binds HIVl-IIIB and HIVl-MN and fails to bind HIVl-ARV2 and HIVl-Z129. HIVl-GER and HIVl-SPA, two viruses recently isolated in our laboratory from patients with AIDS, showed an interesting pattern of reactivity: they expressed the epitopes recognized by the anti-P28-29 mAb and the 9B5C12 epitope (epitopes shared by all HIV and SIV tested to date) and the M01.34.1 epitope (which bound a few HIV1 and one HIV2 isolates), but did not express several epitopes commonly shared by human lentiviruses, like the RL4.11.14 target-epitope and those expressed by the P13 and P28 peptides. The molecular cloning of HIVl-GER is under progress to investigate the reason for such a polymorphism in this European isolate. MLMM 29,10--8.
A careful selection of mAb of known specificity and choice of a testing method are required for significant serotyping of HIV strains. With respect to this rule, HIV serotyping by mAb could also be used for the identification of variant HIV isolates, to the study of virus transmission, and for monitoring recombinations between HIV genomes in in vitro models. AcknowIedgements-VR
and SE were fellows of the “Ministere de la Recherche et de I’Enseignement Superieur” (MRES). MR was supported by a postdoctoral training grant from the “Agence National de Recherches sur le SIDA” (ANRS). We are grateful to the ANRS, MRC, NIH, Institut Pasteur, Biosoft, Hybridolab, Drs F. Barre-Sinoussi, P. Boulanger, J. C. Chermann, F. Di Marzo Veronese, H. Gelderblom, A. Georges, H. Holmes, J. Huisman, J. Larroque, B. Mandrand, L. Montagnier, M. Tatsumi and F. Traincard for providing mAb and virus isolates. We also thank Drs P. Corbeau and S. Hong for critical reading of the manuscript.
REFERENCES Alizon M., Wain-Hobson S., Montagnier L. and Sonigo P. (1986) Genetic variability of the AIDS virus: nucleotide sequence analysis of two isolates from African patients. Cell 46, 63-74. Barre-Sinoussi F., Chermann J. C., Rey F., Nugeyre M. T., Chamaret S., Gruest J., Dauguet C., Axler-Blin C., VezinetBrun F., Rouzioux C., Rozenbaum W. and Montagnier L. (1983) Isolation of a T-lymphotropic retrovirus from a patient at risk for Acquired Immune Deficiency Syndrome (AIDS). Science 220, 868-871. Chassagne J., Verrelle P., Dionet C., Clavel F., Barre-Sinoussi F., Chermann J. C., Montagnier L., Gluckman J. C. and Klatzmann D. (1986) A monoclonal antibody against LAV gag precursor: use for viral protein analysis and antigenic expression in infected cells. J. Zmmun. 136, 1442-1445. Cheng-Mayer C., Seto D., Tateno M. and Levy J. A. (1988) Biologic features of HIV-l that correlate with virulence in the host. Science 240, 80-82. Clavel F., Guitard D., Brun-Vezinet F., Chamaret S., Rey M. A., Santos-Ferreira M. O., Laurent A. G., Dauguet C., Katlama C., Rouzioux C., Klatzmann D., Champalimaud J. L. and Montagnier L. (1986) Isolation of a new human retrovirus from West Africa patients with AIDS. Science 223, 343-346. Cullen B. R. (1991) Human Immunodeficiency Virus as a prototypic complex retrovirus. J. Viral. 65, 1053-1056. Delassus B. R. and Wain-Hobson S. (1988) Molecular genetics of the AIDS retrovirus. Bull. Inst. Pasteur 86, 243-261. Di Marzo Veronese F., Copeland T. D., Oroszlan S,, Gallo R. C. and Sarngadharan M. G. (1988) Biochemical and immunological analysis of Human Immunodeficiency Virus gag gene products p17 and ~24. J. Virol. 62, 795-801. Ellrodt A., Barre-Sinoussi F., Le Bras P., Nugeyre M. T., Brun-Vezinet F., Rouzioux C., Segond P., Caquet R., Montagnier L. and Chermann J. C. (1984) Isolation of human T-lymphotropic retrovirus (LAV) from Zairan married couple, one with AIDS, one with prodomes. Lancet i, 1383-1385. Ferns R. B., Tedder R. S. and Weiss R. A. (1987) Characterization of monoclonal antibodies against the Human Immunodeficiency Virus (HIV) gag products and their use
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Peng C., Ho B. K., Chang T. W. and Chang N. T. (1989) Role of Human Immunodeficiency Virus type l-specific protease in core protein maturation and viral infectivity, J. Virol. 63, 2550-2556. Piot P., Plummer F. A., Mhalu F. S., Lamboray J. L., Chin J. and Mann J. M. (1988) AIDS: An international prospective. Science 239, 573-579. Popovic M., Sarngadharan M. G., Read E. and Gallo R. C. (1984) Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224, 497-500. Rey F., Donker G., Spire B., Devaux C. and Chermann J. C. (1991) Selection of an HIV-l variant following anti-CD4 treatment of infected cells. AIDS Forschung 5, 243-246. Robert V., Resnicoff M., Chermann J. C. and Devaux C. (199 1) Characterization of monoclonal antibodies identifying type and strain-specific epitopes of Human Immunodeficiency Virus type 1. Mol. cell. Biochem. 102, 115-123. Robert-Hebmann V., Emiliani S., Jean F., Resnicoff M., Traincard F. and Devaux C. (1992) Clonal analysis of murine B-cell response to the Human Immunodeficiency Virus type 1 (HIVl)-gag pl7 and p25 antigens. Molec. Immun. 29, 729-738. Sargan D. R., Bennet I. D., Cousens C., Roy D. J., Blacklaws B. A., Dalziel R. G., Watt N. J. and McConnel I. (1991) Nucleotide sequence of EVl, a British isolate of Maedi-Visna virus. J. gen. Viral. 72, 1893-1903. Shang F., Huang H., Revesz K., Chen H. C., Herz R. and Pinter A. (1991) Characterization of monoclonal antibodies against the Human Immunodeficiency Virus matrix protein, ~17”““: identification of epitopes exposed at the surfaces of infected cells. J. Virol. 65, 4798-4804. Stevenson M., Haggerty S., Lamonica C., Mann A. M., Meier C. and Wasiak A. (1990) Cloning and characterization of Human Immunodeficiency Virus type 1 variants diminished in the ability to induce syncitium-independent cytolysis. J. Virol. 64, 3792-3803. Tatsumi M., Devaux C., Kourilsky F. and Chermann J. C. (1990a) Characterization of monoclonal antibodies directed against distinct conserved epitopes of Human Immunodeficiency Virus type 1 core proteins. Molec. cell. Biochem. 96, 127-136. Tatsumi M., Jean F., Robert V., Chermann J. C. and Devaux C. (19906) Use of monoclonal antibodies for the detection and quantitation of HIV1 core protein ~25: comparative evaluation of in aitro HIV1 infection by immunofluorescence, antigen capture ELISA and reverse transcriptase assays. Res. Virol. 141, 649-661. Tersmette M., Winkel I. N., Groenink M., Gruters R. A., Spence R. P., Saman E., Van Der Groen G., Miedema F. and Huisman J. G. (1989) Detection and subtyping of HIV-l isolates with a panel of characterized monoclonal antibodies to HIV ~249”“. Virology 171, 149-155. Von Briesen H., Andreasen R. and Rubsamen-Waigmann H. (1990) Systematic classification of HIV biological subtypes on lymphocytes and monocytes/macrophages. Virology 178, 597-602. Wain-Hobson S., Vartanian J. P., Henri H., Chenciner N., Cheynier R., Delassus S., Martins L. P., Sala M., Nugeyre M. T., Guetard D., Klatzmann D., Gluckman J. C., Rozenbaum W., Barre-Sinoussi F. and Montagnier L. (1991) LAV revisited: Origins of the early HIV-l isolates from Institut Pasteur. Science 252, 961-964.
Subtyping of HIV isolates with mAb Willey R. L., Rutledge R. A., Dias S., Folks T., Theodore T., Buckler C. E. and Martin M. A. (1986) Identification of conserved and divergent domains within the envelope gene of the Acquired Immunodeficiency Syndrome retrovirus. Proc. natn. Acad. Sci. U.S.A. 83, 5038-5042.
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York-Higgins D., Cheng-Mayer C., Bauer D., Levy J. A. and Dina D. (1990) Human Immunodeficiency Virus type 1 cellular host range, replication, and cytopathicity are linked to the envelope region of the viral genome. J. Virol. 64, 4016-4020.
0161-5890/92 $5.00 + 0.00 0 1992 Pergamon Press Ltd
SUBTYPING OF HUMAN IMMUNODEFICIENCY VIRUS ISOLATES WITH A PANEL OF MONOCLONAL ANTIBODIES: IDENTIFICATION OF CONSERVED AND DIVERGENT EPITOPES ON p17 AND ~25 CORE PROTEINS* V&RONIQUEROBERT-HEBMANN,~ ST~~PHANEEMILIANI,~ MARIANA RESNICOFF,~ FRBDBRIC JEANS and CHRISTIAN DEVAUX~§ tCRBM du CNRS, Centre de Tri des Mokcules Brd Henri IV, 34060 Montpellier,
Anti-HIV, Institut de Biologie-Facultb de Mkdecine, France; and $Immunotech S.A., Campus Universitaire de Luminy, 13288 Marseille. France
(First received 29 February 1992; accepted in revised form 8 April 1992) Abstract-We have investigated the feasibility and significance of subtyping of human immunodeficiency virus (HIV) isolates with monoclonal antibodies (mAb) raised against the core proteins of HIV. A panel of 37 mAb tested for reactivity with HIV1 oligopeptides was used to analyse the antigenic relatedness among 14 HIV isolates which included 12 isolates of HIV1 from different geographical origins and 2 isolates of HIV2. Three out of these 37 mAb reacted with conserved epitopes expressed by all 14 HIV isolates tested. These reagents which included 2 mAb reacting with the 285-310 amino acid sequence of p25 and 1 mAb reacting with an epitope of p25 not mapped by the peptides’ approach, also reacted with a non-human primate lentivirus. Five mAb reacting either with the 1l-25 or 121-132 amino acid sequences of p17 or the 302-320 amino acid sequence of ~25 reacted with strain-specific epitopes. The other 29 mAb reacted with polymorphic epitopes and thereby define subfamily and subtype-specific markers.
INTRODUCTION The Human Immunodeficiency Virus (HIV) is the primary aetiological agent of Acquired Immunodeficiency Syndrome (AIDS) and associated diseases (Barr&Sinoussi et al., 1983; Gallo et al., 1984). HIV and related viruses belong to the subfamily of lentiviridae. These viruses share a similar genetic organization (Delassus and Wain-Hobson, 1988; Cullen, 1991); the viral genes encode core structural proteins (gag), enzymes @ol), envelope proteins (env) and several regulatory proteins. Survey of the worldwide spreading of HIV has demonstrated a major distinction in the partition of AIDS-causing viruses (Piot et al., 1988). HIV1 isolates have been predominantly recovered from individuals in the United States, Europe and Central Africa whereas HIV2 isolates have been found in West African individuals. A high degree of polymorphism has been demonstrated between HIV1 and HIV2 isolates (Clavel et al., 1986; Guyader et al., 1987). Genetic variability has been also described within the group of HIV1 isolates (Hahn et al., 1985; Willey et al., 1986). A number of studies have indicated that HIV isolates differ in their biological properties and can therefore be classified according to their tropism, their capacity
*This work was supported by institutional grants from CNRS, INSERM and ANRS. $To whom correspondence should be addressed.
and to form syncytia (Cheng-Mayer et al., 1988; Stevenson et al., 1990; Von Briesen et al., 1990; York-Higgins et al., 1990). The high degree of genetical polymorphism found among HIV isolates accounts for these different biological properties (Fisher et al., 1988; Masuda et al., 1990; Rey et al., 1991; Fredriksson et al., 1991). The study of genetic relatedness among human and non-human primate lentiviruses could help to elucidate the history and evolution of this group of viruses. With respect to their structural gene variability, this could be carried out by investigating the spectrum of HIV isolates existing in different geographic regions. Among the structural genes of the lentiviruses, the variability is mainly distributed within the env region whereas the gag and pol genes are more conserved among the different isolates. Therefore the structural proteins p17 and p25 which are the main components of the gag gene products represent appropriate antigens for serologic analysis of isolates relatedness. In the past few years, several laboratories including our own (Ferns et al., 1987; Niedrig et al., 1989, 1991; Tersmette et al., 1989; Hinkula et al., 1990; Shang et al., 1991; Robert et al., 1991) have attempted to generate mAb against HIV core antigens in order to use them as typing tools. We show here, how a panel of such mAb is able to differentiate among 12 HIV1 isolates from American, European and African origins, two HIV2 isolates and one Simian Immunodeficiency Virus (SIV) isolate. to replicate
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V~RONIQUE ROBERT-HEBMANN et al. MATERIALS
AND METHODS
Monoclonal antibodies. mAb RL4.11.14, RL4.72.1 (Tatsumi et al., 1990a), RL16.24.5 (Robert et al., 1991) and M01.34.1, M09.42.2, M09.50.2 (Robert-Hebmann et al., 1992) have been obtained in our laboratory. mAb C.V.K. has been obtained by J. Chassagne (Chassagne et al., 1986) and provided by F. Barre-Sinoussi (Institut Pasteur, Paris, France). mAb K7.17, L14.17, K3.24 and L6.24 (Tatsumi et al., 1990a) have been provided by M. Tatsumi (NIH, Tokyo, Japan). mAb M26 (Di Marzo Veronese et al., 1988) has been provided by F. Di Marzo Veronese under transfer agreement with NIH/ADAMHA (NIH/ADAMHA, Bethesda, MD, U.S.A.). mAb 8D2, 8H7 and 3H7 (Niedrig et al., 1988, 1989) have been provided by H. Gelderblom (Robert Koch Institut, Berlin, Germany). mAb CLB-14, CLB-16, CLB-21 and CLB-47 (Tersmette et al., 1989) have been provided by J. Huisman (Netherlands Red Cross Blood Tranfusion Service, Amsterdam, The Netherlands). mAb 14D4E11, 3DlOG9, 15F8C7, llDllF2, lG5C8,9A4C4, llClOB10, 23A5G5 and 9B5C12 (Janvier et al., 1990) have been provided by B. Mandrand (UM 103 CNRS-Biomerieux, France) under transfer agreement with “Agence Nationale de Recherches sur le SIDA” (ANRS, Paris, France). mAb 21 l/92,406/01, 714/01, 1109jOl and 21 l/84 (Robert-Hebmann et al., 1992) have been provided by J. Larroque (Biosoft, Clonatec, Paris, France). mAb 3 l-l 1, 15-21, 47-2 and 169-2 (Robert-Hebmann et al., 1992) have been provided by F. Traincard (Hybridolab, Paris, France). The main characteristics of these mAb are summarized in Table 1. Viruses. HIVl-GER (Robert et al., 1991) HIVl-ROI and HIVl-SPA (MR unpublished) have been isolated in our laboratory. The other viruses HIVl-BRU (Barre-Sinoussi et al., 1983), HIV2-ROD (Clavel et al., 1986) and HIVl-ELI (Alizon et al., 1986) were provided by L. Montagnier (Institut Pasteur, Paris, France); HIVlNDK (Ellrodt et al., 1984) and HIVl-PAS (Robert et al., 1991) were provided by J. C. Chermann (INSERM U322, Marseille, France); HIVl-GON (A. Georges, unpublished), HIVl-OMB (Robert et al., 1991), originally isolated by A. Georges (Institut Pasteur, Bangui, RCA), HIV2-1169 (F. Barre-Sinoussi, unpublished) and SIV-SM (Marx et al., 1991) were all provided by F. Barre-Sinoussi (Institut Pasteur, Paris, France); HIVl-MN (Gallo et al., 1984) HIVl-RF (Popovic et al., 1984) and HIVl-SF2 (Levy et al., 1984) were provided by H. Holmes (MRC AIDS Reagent, Project, Herts, U.K.). These viruses were propagated in human T cells (CEM) under conditions previously described (Tatsumi et al., 1990b). Concentrated virus preparations were obtained from the cell-free supernatant by 100 OOOg ultracentrifugation for 2 h. The viral pellet was resuspended hundred-fold concentrated in NTE buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA pH 7.4). Viral particles (1 mg/ml) were inactivated by 56°C incubation for 30 min and treated with 0.1% Triton X 100. Recombinant proteins. The polyhedrin fusion-HIVlgag (~17, p25 and most of p9) protein (fus-gag)
recovered from baculovirus infected cells was provided by P. Boulanger (Institut de Biologie, Montpellier, France). Enzyme-linked immunosorbent assay (ELISA). ELISA plates (Nunc, Paisley, U.K.) were coated overnight with 10 pgg/ml of inactivated virus in 100 mM sodium carbonate buffer pH 9.6. After coating, plates were washed and saturated with phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA). 100 ~1 of mAb at an appropriate dilution (defined by lack of reactivity with uninfected CEM cells), were incubated 1 h at room temperature, after which the plates were washed three times with PBS-0.05% Tween 20. Bound immunoglobulins (Ig) were detected by adding 100 ~1 of goatanti-mouse IgG H + L peroxidase conjugate (lo3 fold diluted) (Immunotech S.A., Marseille, France) for 1 h, followed by washing and subsequent incubation with 0-phenylenediamine (OPD) (Sigma) as substrate. ELISA using recombinant proteins was performed as previously described (Robert-Hebmann et al., 1992). Amino acid sequence comparison. p17 and p25 amino acid sequences of 17 HIV1 (namely: BRU (LAI); HXB2; MN; JH3; CDC41; OYI; SF2; HAN; RF; ELI; 22; NDK; MAL; BHlO; BH5; PV22; WMJ2), 7 HIV2 (namely: ROD; NIHZ; ST; ISY; BEN; GHl; D205) and 3 SIV (namely: MM251; MM142; SMMH4) from Genbank (Bolt Beranek and Newman, Cambridge, MA, USA) and Los Alamos HIV sequence Database (Group T-10, Los Alamos, NM, U.S.A.) were compared. The sequence of HIVl-BRU was used as a standard and the number of different amino acids or deletion-insertion was calculated for each position.
RESULTS
Reactivity of the mAb with HIVl-BRU, recombinant molecules and oligopeptides
HZVl-BRU
A selection of 37 mAb specific for the HIV-core antigens which originated from 13 independent fusion experiments was used for subtyping of HIV isolates. These mAb were produced against HIV1 (BRU, IIIB, NDK or KB strain, respectively) or HIV2 (ROD strain). Their fine specificity has been previously analysed by reacting them with 35 HIVl-BRU overlapping synthetic linear peptides (Robert-Hebmann et al., 1992). Of these 37 mAb, 20 have been precisely mapped by the peptides’ approach. Failure in mapping 17 mAb binding site was a consequence of the lack of HIVl-BRU binding (for two of them), or was related to the conformational requirement of their target-epitopes (for the others). Table 1 shows the reactivity of these mAb with HIVlBRU, HIVl-BRU gag recombinant molecules (fus-gag), and summarizes the result of our previously published mapping study. Analysis of p 17 and p25 antigenic polymorphism
In order to quantitate the p17 and p25 antigens variability among HIV isolates, viral amino acid sequences were compared. A computer program was used to construct diagrams of p17 and p25 proteins variability. The
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Subtyping of HIV isolates with mAb Table 1. mAb used in this study ELISA reactivity1 Hybridoma designation* Anti-p17 mAb L14.17 3H7 31-11 15-21 21 l/92 RL16.24.5 K7.17 C.V.K. M01.34.1 Anti-p25 mAb CLB-2 1 15F8C7 47-2 14D4Ell 8H7 714/01 1109/01 8D2 lG5C8 RL4.72.1 M26 406/O1 CLB-14 L6.24 169-2 23A5G5 3DlOG9 M09.42.2 M09.50.2 9A4C4 llDllF2 1IClOBlO K3.24 9BSC12 CLB-16 CLB-47 R14.11.14 21 l/84
Immunogent
HIVI-BRU
fus-gag
BRU peptides
HIVl-BRU HIVl-IIIB HIVl-BRU HIVl-BRU HIVl-IIIB HIVl-NDK HIVl-KB HIVl-BRU HIVZROD
+ + + + + -
+ + + + + -
+ + -
+ + -
1l-25 (P2) Ill-123 (P12) 121-132 (P13) 121-132 (P13) ND ND ND ND ND
HIV1 ~25 HIVl-IIIB HIVl-BRU ~25 HIVl-IIIB HIVl-IIIB HIVl-IIIB HIVl-IIIB HIVI-IIIB ~25 HIVl-IIIB HIVl-NDK HIV1 -1IIB HIVl-IIIB HIV1 HIVl-BRU HIVZROD ~25 HIVl-IIIB ~25 HIVl-IIIB HIV2-ROD HIVZROD ~25 HIV l -IIIB ~25 HIVl-IIIB ~25 HIVl-IIIB HIVI-KB HIVl-IIIB/HIVZ HIV1 HIV1 HIV1 -NDK HIVl-IIIB
+ + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + +
ND ND 201-218 (P21) 201-218 (P21) 201-218 (P21) 201-218 (P21) 201-218 (P21) 201-218 (P21) 201-218 (P21) 219-233 (P22) ND 233-253 (p23) ND ND ND 285-304 (P28) 285-304 (P28) 285-3 10 (P28-P29) 285-310 (P28-P29) 302-320 (P30) 302-320 (P30) 302-320 (P30) ND ND ND ND ND ND
-I+ +
-I+ + -f + + + + + + + -I-t + + + + + + + + +
*Hybridoma were constructed by fusion of either NSl-Ag4.1 (3H7, M26, 8D2, 8H7), SP2/O.Ag14 (L14.17, 311/01, K7.17, CLB-21, 15FSC7, 14D4El1, 714/01, 1109/01, lG5C8, 406/01, CLB-14, L6.24, 23A5G5, 3DlOG9, 9A4C4, llDllF2, llClOBl0, K3.24,9B5C12, CLB-16, CLB-47,211/84) or X63-Ag8.653 (31-11, 15-21, RL16.24.5, C.V.K., M01.34.1, 47-2, RL4.72.1, 169-2, M09.42.2, M09.50.2, RL4.11.14) mouse myeloma cells with spleen cells from BALB/c immunized mice. The mAb express either IgGl, IgG2a, IgG2b, or IgG3 isotype (Robert-Hebmann et al., 1992). tMice were immunized with either inactivated HIV1 or HIV2 viruses or purified viral proteins. HIVZROD = HIV type 2 ROD strain; ~25 HIVl-IIIB = purified ~25 protein from HIV type 1 strain HTLV-IIIB. $mAb were tested for binding to HIVl-BRU, polyhedrin-fusion-HIVl-gag (fus-gag) and 35 overlapping synthetic linear HIVl-BRU peptides (Pl to P35) totally covering the p17 (Pl to P13) and ~25 (P14 to P35) proteins. Their reactivity with HIVl-BRU and peptides has been previously published (Robert-Hebmann et af., 1992). The reactivity of a given mAb for a given antigen was considered positive ( +) when absorbance was over 3 times the background. jAmin acid numbers of the first and last residues of target peptide are given according to the HIV sequence Database Group T-10 (Los Alamos National Laboratory, NM, U.S.A.) and peptide code is shown between brackets. The peptide code is given according to that previously reported (Robert-Hebmann et al., 1992).
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VBR~NIQUEROBERT-H~B~~NNet al.
Anlao
ACID WtlBER
Fig. 1. Superimposition of peptide location on the diagrams of variability of the p17 and ~25 proteins constructed by comparison of amino acid sequences of different HIV and SIV isolates. The HIVI-BRU sequence was used as a standard. A difference in amino acids at a given position to the sequence of prototype is depicted by a vertical bar equal to 1 unit in the ordinate scale. Sequence alignments were made according to the highest percentage of homology to the prototype at each position; therefore a deletion or addition that could result in a frame shift is illustrated by a vertical bar. (A) Sequence variations among HIV1 isolates (n = 17; see Materials and Methods for details); (B) Sequence variations among HIV1 (n = 17), HIV:! (n = 7) and SIV (n = 3).
variability was evaluated with respect to the sequence of HIVl-BRU. A diagram of variability of HIV1 p17 and ~25 sequences was constructed using known sequences of 16 HIV1 isolates, and another diagram of primates lentivirus p17 and ~25 sequences variability was constructed using the previous 16 HIV1 sequences together with that of 7 HIV2 and 3 SIV. Figure 1 superimposes the location of HIVl-BRU peptides shown to be target for mAb (peptides P2, P12, P13, P21, P22, P23, P28, P29 and P30) on the diagrams of variability of HIV1 (LA) and primate lentiviruses (1 B). This schematic representation allows to predict whether or not the target epitope for a given mAb is expected to be shared among virus isolates. For example, epitopes of P29 would be expected to be quite conserved among HIV1 and HIV2 isolates whereas epitopes expressed by P13 might be of two types, either highly polymorphic or conserved among HIV1 isolates. This was indeed verified by serotyping of HIV isolates. Serotyping of HIV isolates
The results of the serotyping of HIV isolates with mAb by ELISA are summarized in Fig. 2. This study revealed appreciable heterogeneity in epitope expression. With the exception of 3 mAb (M09.42.2, M09.50.2 and 9B5C12) which bound epitopes shared by HIVl, HIV2 and SIV isolates, all other mAb revealed an antigenic polymorphism within the core molecules of the primate lentivirus isolates tested. Among these mAb, 2 (23A5G5 and 3DlOG9), bound an epitope(s) located in the P28 peptide and shared by the HIV1 and HIV2 isolates tested with the exception of HIVl-GER and HIVl-SPA.
A few mAb, such as L14.17 (reacting with a sequence in P2), 9A4C4, llDllF2 and llClOBl0 (reacting with a sequence in P30), or 15-21 (reacting with a sequence in P13) reacted with polymo~hic antigens of HIV. The restricted pattern of reactivity of mAb 15-21 is compatible with the fact that about half of the PI3 sequence diverges among the isolates (see Fig. 1). Interestingly, another mAb reacting with P13 (mAb 31-11, known to bind an epitope of P13 distinct from that of 15-21; Ro~rt-Hebmann et al., 1992), showed a broad reactivity pattern among isolates. This is consistent with the fact that another part of P13 is conserved among HIV 1. Among the mAb that bound epitopes having conformational requirements, some, like RL16.24.5, L6.24, K3.24 and CLB-16 also exhibited a relatively restricted pattern of cross-reactivity. Over two-thirds of the virus isolates had altered expression of the majority of epitopes recognized on HIVl-BRU using this panel of mAb. For example, all seven mAb to P21 (amino acids 201-21s) reacted with HIVi-BRU, HIVl-RF and HIVl-NDK, their patterns of reactivity differed on HIVI-ROI (suggesting the existence of at least two distinct epitopes in P21), and they did not react with the other viruses tested. The majority of mAb tested clearly define subtype specific epitopes. Interestingly for typing purposes, some of these mAb showed totally distinct patterns of crossreactivity. For example, K7.17 reacted with HIV1 -BRU, HIVl-RF and HIVl-NDK but not with the other HIV1 isolates whereas M01.34.1 showed the opposite pattern of reactivity.
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Subtyping of HIV isolates with mAb
VIRUSES I
Fig. 2. Summary of serotyping of virus isolates. Reactivity of 37 anti-HIV mAb with 15 HIV and SIV isolates. The name of isolates tested and their geographical origin (Eur. = Europe; Ame. = America; Afr. = Africa) are indicated. The viruses were propagated in CEM cells and ELISA coated plates were prepared as described. mAb binding to virus isolates was measured and expressed with respect to the absorbance at 492 nm n = ELISA-positive reaction (absorbance > 1.5); N = weak reaction (0.5 < absorbance c 1.5); 0 = ELISA-negative reaction (absorbance < 0.5). The first vertical column (-) shows the lack of mAb reactivity with uninfected CEM cell supernatant treated under the same experimental conditions as viral samples. Each value has been calculated from the average of quadruplicate assays.
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VBRONIQUE ROBERT-HEBMANN et al.
11-25 HIV1 HIV1 HIV1 HIV1 HIV1 HIV1 HIV2
BRU pq, SF2 RF ELI EIDK ROD
(P2) GELDRWEKIRLRFGG . ..____~____.__ ____K__________ _K__K________R_ _K__K__________ _K__*__R_______ yJ(y_EL_R__~____
201-218 HIV1 BRU HIV1 MI1 HIV1 SF2 HIV1 RF HIV1 ELI HIV1 NDK HIV2 ROD
121-132 HIV1 HIV1 HIV1 HIV1 HIV1 HIV1 HIV2
(P21)
KE?l-11m EXAEWDRVHP _____________L-_ _____________L__ _____________L__ _____~_________ R_~_________VQ__
BRU MI1 SF2 RF ELI
DT...GHSSQVSQPN __G*Jp~~________ G-... _~~________ --..:IIG------_*~~_______
NLIK
-.....:-------
ROD
TSRFTAJ--EKCX--
285-320 HIV1 HIV1 HIV1 HIV1 HIV1 HIV1 HIV2
(P13)
BRU M.tI SF2 RF ELI NDK ROD
(P28,P29,P30)
IRQGPKEPFRDYVDRFYKTLPAEXJASQEXXNWMT~ ~_________________________RT___ D-------__________~_______~________D__..___~ ______~_________D________ ________________D_______~ _~___~___Q~_______~_____~p*______Q_
Fig. 3. Amino acid sequence alignments of HIVl-BRU gag regions 11-25 (P2), 121-132 (P13), 201-218 (P21) and 285-320 (P28, P29, P30) and regions of similarity from five different HIV1 isolates and HIV2-ROD. A dash represents an amino acid identity with HIVl-BRU. A point is used to prevent insertion-deletion frame shift.
Sequence comparison among the major antigenic regions of the HIV core molecules As shown in Fig. 3, sequence comparison confirms that the shared P28-P29 epitope(s) defined by M09.42.2 and M09.50.2 is (are) highly conserved among the HIV1 and HIV2 isolates tested in this study. Moreover, the sequence YVDRFY of P28 and P29 is also conserved among a number of other HIV1 (HXB2R, JH3, JRCSF, OYI, NYSCG, NL43, CDC4, HAN, and Z226), HIV2 (NIHZ, ISY, ST, BEN, D194, GHl, and D205), and SIV (CPZ, MM251, MM142, MM239, MNE, SMMH4, and SMMPBJ) isolates, whereas a permutation of Y for F was reported for HIVl-MAL and HIVl-U455 (Group T-10, Los Alamos, U.S.A.). The pattern of reactivity observed using mAb that bound P2, P13 or P28 is also consistent with the sequence variability found in these regions among the different isolates. For example, the private reactivity of mAb 15-21 is compatible with the presence in P13 of the DTGHS sequence which is only found in the HIVl-BRU isolate, whereas another anti-HIVl-BRU mAb 31-11 that also reacts with P13 is more likely interacting with amino acids of the SSQVSQNY sequence. Although the published 201-218 amino acid sequence of HIVl-SF2 is identical to that of HIVl-BRU, and the sequences of HIVl-MN, HIVl-ELI and HIVl-RF are identical one to the other within this region of ~25, we found that mAb specific for P21 only bound a limited number of HIV isolates (including BRU, RF and NDK but not SF2, ELI and MN). Another set of unexpected data concerns the lack of reactivity of mAb to P12, P22, and P23 with HIVl-ELI, HIVl-SF2 and HIVl-MN. This result might be related to in vitro variation of strains, laboratory contamination, or more likely differences in epitope accessibility.
DISCUSSION The aim of this study was the analysis of the antigenic relatedness among several HIV isolates by anti-core mAb serotyping and comparison of cross-reactivity patterns to viral sequences alignment, when those sequences were made available. We showed here that mAb can be used as reference standards for analysing primate lentivirus strains since subfamily, subtype and strain-specific reagents were identified. In this study, a complete correlation between the sequence alignment and the cross-reactivity pattern was found using the mAb reacting with the subfamily-specific epitopes expressed by the P28-P29 peptides or the strainspecific epitopes expressed by the P2, P13, and P30 peptides. However, a few discrepancies were observed with the mAb reacting with the subtype-specific epitopes expressed by the P12, P21, P22 and P23 peptides that showed an abnormal serotype with HIVl-ELI, HIVlMN and HIVl-SF2. In vitro variation of strains after long-term culture or undetected laboratory contamination, as previously reported by others (Tersmette et al., 1989; Wain-Hobson et al., 1991), or more likely differences in epitope accessibility, might account for such results. Another parameter to take into account concerns the method used for virus serotyping. As previously reported (Ferns et al., 1987; Niedrig et al., 1988, 1991), depending on the test system used, a few mAb showed various degrees of cross-reaction reflecting the conservation and availability of their respective epitopes. In the ELISA, RL4.72.1 reacted with HIVl-BRU, HIVl-NDK, HIVlPAS and HIV2-ROD, and was negative for HIVl-ELI, HIVl-OMB, HIVl-GER, whereas it bound all those strains in a previous study using an immunofluorescence (IF) analysis (Robert et al., 1991). Since we could not exclude changes in serotype as a consequence of main-
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Subtyping of HIV isolates with mAb tenance of the viruses in culture, RL4.72.1 was tested again and simultaneously on several isolates by both methods to determine whether or not the viruses tested in ELISA were variants. The result of this study was consistent with our previous data, suggesting that the difference resided only in the method used (data not shown). Given their broad reactivity, mAb to the epitope shared by the P28 and P29 peptides can be used for the detection of members of the lentiviridae subfamily. Niedrig et al. (1989) have described a mAb which reacts with an epitope located within this region of ~25 and expression of which is shared by HIVl-IIIB, HIV2-ROD and SIV-MAC. Here we confirm and extend this observation by showing the mAb M09.42.2 and M09.50.2 reacted strongly with all human lentiviruses and a simian lentivirus tested to date in ELISA. This result is in agreement with the sequence alignment analysis showing that the sequence YVDRFY is shared among HIVl, HIV2 and SIV. Interestingly, this sequence is not found in Visna virus (Sargan et al., 1991). Therefore, this epitope might be a marker for primate lentiviruses. The P28-reacting mAb described here, bound all HIV1 and HIV2 isolates tested with the exception of HIVlGER and HIVl-SPA, but failed to react with SIV-SM despite sequence similarities with HIV2-ROD in this region. Since HIVl-GER and HIVl-SPA apparently showed an original serotype, the epitope recognized turns out to be predominantly expressed by human lentiviruses and might therefore be considered as a subfamily-specific marker. The majority of mAb tested in this study reacted with subtype-specific epitopes. Frequently, these subtypespecific epitopes were expressed by HIV isolates from different geographical origins. For example, the P21reacting mAb bound some European and African isolates of HIV1 (e.g. HIVl-BRU and HIVl-NDK), but failed to react with other European and African isolates of HIV1 (e.g. HIVl-PAS and HIVI-GON). Similar observations have been reported by Ferns et al. (1987) who have found that the anti-p25 mAb EBlA9 produced against a European isolate called HIVl-CLBl reacts with two American HIV isolates (HIVl-IIIB and HIVl-ARV2/ SF2), with an African isolate (HIVl-Z129) but fails to bind another American isolate (HIVI-MN); whereas the anti-p25 mAb CD12B4 also produced against HIVlCLBl binds HIVl-IIIB and HIVl-MN and fails to bind HIVl-ARV2 and HIVl-Z129. HIVl-GER and HIVl-SPA, two viruses recently isolated in our laboratory from patients with AIDS, showed an interesting pattern of reactivity: they expressed the epitopes recognized by the anti-P28-29 mAb and the 9B5C12 epitope (epitopes shared by all HIV and SIV tested to date) and the M01.34.1 epitope (which bound a few HIV1 and one HIV2 isolates), but did not express several epitopes commonly shared by human lentiviruses, like the RL4.11.14 target-epitope and those expressed by the P13 and P28 peptides. The molecular cloning of HIVl-GER is under progress to investigate the reason for such a polymorphism in this European isolate. MLMM 29,10--8.
A careful selection of mAb of known specificity and choice of a testing method are required for significant serotyping of HIV strains. With respect to this rule, HIV serotyping by mAb could also be used for the identification of variant HIV isolates, to the study of virus transmission, and for monitoring recombinations between HIV genomes in in vitro models. AcknowIedgements-VR
and SE were fellows of the “Ministere de la Recherche et de I’Enseignement Superieur” (MRES). MR was supported by a postdoctoral training grant from the “Agence National de Recherches sur le SIDA” (ANRS). We are grateful to the ANRS, MRC, NIH, Institut Pasteur, Biosoft, Hybridolab, Drs F. Barre-Sinoussi, P. Boulanger, J. C. Chermann, F. Di Marzo Veronese, H. Gelderblom, A. Georges, H. Holmes, J. Huisman, J. Larroque, B. Mandrand, L. Montagnier, M. Tatsumi and F. Traincard for providing mAb and virus isolates. We also thank Drs P. Corbeau and S. Hong for critical reading of the manuscript.
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