B-cell continuous epitopes of the SIVmac-251 envelope protein in experimentally infected macaques

0 INSTITUT PASTEURELSEVIER Paris 1995

Res. Virol. 1995, 146, 19-32

B-cell continuous epitopes of the SIVmac-251 envelope protein in experimentally infected macaques V. Tanchou (l), P. Sgro-Serpente (l), H. Durand cl), A.M. Aubertin D. Dormont c3), A. Venet c4) and R. Benarous (l) (*)

(*),

‘I’ Signalisation, Inflammation et Transj-ormation cellulaire, INSERM-U332, Cochin de Genetique Moltkulaire, Universite’ Paris V, 22, rue M&k&t, 7.5014 Paris “’ INSERM-U74, Universite’ Louis Pasteur, Fact&t! de Medecine, 3, rue Koeberte, 67000 Strasbourg (France), f3) Laboratoire de Neuropathologie expt+imentale et Neurovirologie, Centre de Recherches du Service de Sante’ des Armkes, Commissariat a I’Energie Atomique, Direction des Sciences du Vivant, Depatiement de Toxicologic experimentale, B.P. 6, 92265 Fontenay-aux-Roses (France), and (41 Iaboratoire d’bnmunologie et d’oncologie des Maladies r&ovirales, INSERM-UI52, Institut Cochin de Genetique mol.kulaire, 27, rue du Faubourg Saint-Jacques, 75014 Paris

Institut

SUMMARY The humoral immune response of 34 macaques experimentally infected with SIVmat-251 was studied using a combination of an epitope library and synthetic peptides. The course of the immune response was checked for up to 9 months postinfection with a panel of clones expressing SIV fragments. A systematic study was performed with synthetic peptides covering the whole transmembrane (TM) and external (SU) envelope proteins. Seven major immunodominant epitopes were characterized. Four are localized in the SU protein: one in the VI region (Ill-1301, one in the Cys loop of the V3 region (311330) and two in the C-terminal end (501520 and 511530). Three are localized in the TM protein: one in the extracellular domain (601-6191, one in the anchor domain (731-750) and one in the intracytoplasmic domain (861681). Among these epitopes, only one, 601619, was found to be reactive with all sera and can be defined as the principal immunodominant epitope. Key-words: SIV, B lymphocyte, Epitope, Immunodominance; response, Macaque, Sequencing, Peptides, Envelope proteins.

INTRODUCTION The simian immunodeficiency virus (SIV) is a member of the lentivirus subfamily related to the human immunodeficiency viruses (HIV1 and

Submitted

March

(*) Corresponding

17, 1994, author.

accepted

July

27,

1994.

HIV, Humoral

HIV2) responsible for AIDS in humans (Blattner et al., 1988; Barre-Sinoussi et d., 1983; Alizon et al., 1984). The extensive similarity between SIV and HIV at the level of genetic organization (40% to 60% with HIV1 and 60% to 80% with

20

V. TANCHUU

ET AL.

HIV2) (Chakrabarti et al., 1987; Franchini et al, 1987), and the fact that clinical manifestations of simian AIDS are similar to those of human AIDS (Desrosiers, 1990; Letvin and King, 1990) suggest that SIV is well suited as a model system for the study of AIDS progression and vaccine development (Gardner and Luciw, 1989). Antigenic variation related to genomic variability may be one mechanism by which these lentivimses avoid elimination by the host immune system (Montelaro et al., 1984; Clements et d., 1980; Ellis et aZ., 1987). From this point of view, hypervariability of the V3 loop of HIVl, the major site of neutralizing antibodies (Goudsmit et al., 1988; Kinney Thomas et al., 1988; Matsushita et al., 1988), suggests that HIV may be able to escape neutralization by mutations in this region. In SIV, the most extensive variation is observed in the VI, V2 and V4-like variable domains of the external envelope protein (Johnson et al., 1991; Overbaugh Ed al., 1991; Baier et uZ., 1991; Burns and Desrosiers, 1991).

several domains of SIV envelope proteins. In particular, we have used a combination of an expression epitope library and a collection of 87 overlapping peptides covering the whole SIV envelope protein. When this study was nearly completed, similar works were reported (McBride et al., 1993; Miller et al., 1992). Comparison of their results with ours underlines the importance of several continuous epitopes of the SIV envelope as major targets for the host immune system of infected animals.

Several immunoreactive regions have been described in the external (SU) as well as in the transmembrane (TM) envelope protein of SIV (Miller et al., 1992 ; Kent et al., 1991; Kodama et al., 1991), which is relatively more conserved in its sequence.

Peptides

Some of these biologically important epitopes, and in particular those which might elicit neutralizing antibodies, have been identified using monoclonal antibodies raised in mice against purified proteins or disrupted SIV (Benichou et aZ., 1992; Kent etal., 1991; Kodama et aZ., 1991; McBride et al., 1993).

MATERIALS

AND METHODS

Animals and viruses Rhesus macaques (Macaca mulatta) described in this study were infected with SIVmac-25 1 (gift of R. Desrosiers), as described elsewhere (Venet et al., 1992; Legrand et al., 1992).

Small overlapping peptides were provided by the French agency against AIDS (ANRS) (catalog numbers: SP90439 to SP490647 and SP91082 to SP91089). Construction of an SIV epitope library

In this paper, we describe a systematic study of the humoral immune response of 34 macaques experimentally infected with SIVmac-25 1 against

This library was constructed as described (Stanley and Luzio, 1984; Luzio et al., 1989) in the bacterial expression vector pTEX : SIVmac-25 1 proviral DNA was randomly fragmented by partial digestion with DNase I (Boehringer, 104-159). Large fragments of around 400 bp were then isolated from agarosegel slices and polished with Escherichia coli DNA polymerase I (Biolabs-209s). BamHl adaptor ligation was performed on both fragments and vector. The ligation mixture was electrotransferred to the recipient bacterial cell MC 1061. In this system, SIV DNA fragments are cloned at the 3’ end of the LacZ gene and the epitopes are expressedas a fusion protein with P-galactosidase, under the control of the PR promoter from bacteriophage h and the temperature sensitive h repressor (~1857).

IT

SIV

=

Th4

=

In order to define these targets, one possible approach is to study the natural antiviral immune response through analysis of the immunoreactivity of sera from infected animals at the molecular level of different viral antigens.

HIV

= =

fusion protein. human immunodeficiency

virus.

simian immunodeficiency transmembrane (envelope

virus. protein).

SIVmac-251

ENVELOPE

B-CELL

21

EPITOPES

Screening of the library

ELISA

The epitope library (about 50,000 colonies) is spread onto 24x24 cm L-broth agar plates (NUNC2/40835) and grown overnight (16 to 20 h) at 30°C then transferred to nitrocellulose filters (Schleicher and Schuell-401191) and incubated for 2 h at 42°C to express the fusion proteins, while the master plates are replaced in the incubator at 30°C for 8 h for colonies to regrow. The colony blot procedure was performed by electrotransfer as described above (Stanley, 1988). Screening was done according to published procedures (Stanley and Luzio, 1984) using sera from four different infected monkeys (macaques no. 471, 483, 495 and 501) 9 months after infection, at 1:200 to 1:500 dilutions. Secondary antibodies used were either peroxidase-conjugated (CAPPEL, 3210-0081) goat anti-monkey IgG or phophatase-alkaline-conjugated (Biosys, BI25 15) goat anti-human IgG.

Wells of Dynatech plates were coated overnight at 4°C with 50 pl of solutions containing either FPs (1 pg/ml) or peptides (5 pg/ml) diluted in 0.1 M NaCO pH 10.0 and then treated with PBS containing 1 a/o non-fat milk and 0.1% Tween-20 for 2 h at room temperature. Incubations with sera or eluted antibodies were performed for 2 h at room temperature. Affinity-purified biotinylated anti-human IgG antibodies (Vector, BA-3000) were used as secondary antibodies. End point titres were determined as the serum dilution giving a reactivity 3-fold above the average of the control values and were expressed as logic, of reciprocal dilutions.

Sequencing

Panel of clones expressing tein fragments

Plasmid DNAs from purified clones were prepared according to Birnboim and Doly (1979) and sequenced by the dideoxynucleotide chain termination method with the “Sequenase Kit-Version 2” (U.S.B., Cleveland, Ohio), using the following oligonucleotide primers located both sides from the BamHl cloning site: 5’ sequencing primer (OL88): 5’-GGGGATTGGTGGCGACGACTCCTGG-3’ and 3’ sequencing primer (OLVT): 5’-CTAGAG CCGGATCGATCCGGTC-3’. Clones were localized in the SIV sequence according to the nucleotide and protein sequences of SIVmac-251 published by Chakrabarti (Chakrabarti et al., 1987). Preparation proteins

and solubilization

of the fusion

Fusion proteins o;Ps) were prepared as previously described (Stanley, 1983). Briefly, recombinant bacteria were grown at 30°C to a density of 7x lo7 cells/ml (A =0.2) and then incubated for 2 h at 42°C. The ce 6% s were harvested by centrifugation at 2,000 g for 15 min. washed in 100 mM NaCl, 50 mM Tris-HCl pH 8.0, lysed in 15% sucrose, 50 mM TrisHCl pH 8.0 and 2 mg!ml of lysosyme, for 15 min on ice, and treated with 100 mM MgCl, and 200 p&ml of DNase I (Sigma, D-0876) on ice for 15 min. Inclusion bodies containing insoluble fusion proteins were resuspended with a 1% Triton-Xl00 0.5 % deoxycholate, 100 mM NaCl and 10 mM Tris-HCl pH 7.4 detergent solution for 5 min on ice, washed 3 times in 1.75 M guanidinium-HCl, 1 M NaCl and 1% TritonXl00 and solubilized after sonication in 8 M urea, 50 mM NaCl and 1 mM EDTA.

assays

RESULTS SJY envelope

pro-

After two rounds of screening, clones were selected to be sequenced and analysed further. Among these clones, 32 expressed large fragments covering the whole TM protein. Several of these clones were overlapping with the C-terminal end of the SU protein. This panel of clones was used (i) to titrate by serum dilution the antibodies against different regions of the envelope protein in order to define immunodominant regions that are the targets of the immune system of the infected animals, (ii) to analyse the course of the immune response of infected animals for various times after infection and (iii) to compare the imtnunoreactivity of sera from a panel of 34 different infected animals together with 14 preimmune sera used as controls.

Detection of immunodominant TM protein

regions

in the

The panel of clones defined above was used to analyse immunoreactivity of sera from 34 infected macaques against different regions of the TM protein. Sera from these infected macaques were used at several dilutions from 1: 100 to l:lO,OOO. The results obtained with two of them are shown in figure 1.

22

V. TANCHOU

ET AL..

in group I, as clones displaying a weak immunoreactivity, indicated with one +. 2) Several clones still reacted with sera at 1:2,000 dilution (panels b) and sometimes at 15,000 (panels c). They are listed in table I, in group II, as clones displaying an intermediate reactivity indicated with two +. 3) Finally, 4 clones (clones 9, 11, 22 and 43) conserved strong immunoreactivity with sera diluted 1:10,000 (panels d). These strong reactive clones define group III, indicated with three + in table I. For group I and group II clones, the pattern of reactivity was not identical from one serum to another. In contrast, for the strongest reactive clones of group III, a similar pattern of immunoreactivity was obtained for all sera from different infected macaques. This indicates that these regions of strong immunoreactivity encode major immunodominant epitopes recognized as preferential targets by the host immune system of all infected animals. Course of immunoreactivity period after infection Fig. 1. Immunoreactivity of the panel of clones against sera from infected macaques. Serum from macaque no. 483 on the left and from macaque no. 495 on the right, 9 months aftex infection, were used at the following dilutions: 1:lOO (a), 1:2,000 (b), 1:5,000 (c) and l:lO,OOO (d). The respective amino acid localizations of the different clones mentioned in the text was as follows: clone 1=428-581, clone 4=478-614, clone 7=497-615, clone 9=513-631. clone 11=520-667, clone22=546-682, clone 37=493-594, clone 39 =496594, clone 43=548-698, clone 46=493-595.

From these results, three main classes of clones could be distinguished according to the immunoreactivity displayed at different sera dilutions. 1) Clones which reacted weakly at a 1: 100 dilution (panels a) were no longer detectable at a 1:2,000 or 1:5,000 dilution (panels b and c). This was the case of clones 1 to 4, 7, 37, 39 and 46 (right and left panels). These clones detectable only at 1:lOO serum dilution are listed in table I

over a 4-month

One example of such a study is shown in figure 2: serum from the experimentally infected macaque no. 495, collected various times before (panel B) and after (panel C to G) inoculation, was used at 1: 1,000 dilution against the panel of clones. Serum from a seronegative macaque was used as control in panel A. Two to four weeks after infection (panels C and D), the first positive clones clearly detectable were located in the TM protein (clones 9, 11, 22 and 43). They all belonged to the immunoreactive group III. Two months after the infection (panel E), these clones reacted very strongly and several others became positive. After 3 and 4 months (panels F and G), reactivity against various clones in Env proteins became clearly detectable. After 4 months, the first positive clones still displayed a strong reactivity, and almost all other clones were positive. Similar results were found with the other infected macaques tested.

SIVmac-251

ENVELOPE

c1eavsge

s,,e

%J

Tll

B-CELL

23

EPITOPES

ED

........... /.......... ...........

AD;:.-.-::::.-:.I .........

,D

111

FP.5 FP.6 I r.* FP.44 FP.41 Fr.10 FP.11 FP.,I Fr.Is FP.18 FP.19 cr.38 FP.IO FP.21 FP.24 FP.25 FP.26 Cr.18

II

FP., FP.2 FP.3 FP.4

I

FP.37 Fr-46 Fr.39 Fr.7 FP.40

IMMUNOREACTIVE GROUP

FUSION PROTEINS

Table

I. Diagram showing the relative position of the different overlapping clones expressing fragments of the Th4 protein and C-terminal part of gp120 SU protein. The different clones are listed according to their immunoreactivity. The cleavage site between SU gp120 and TM gp41 proteins is indicated by the arrow. The different domains of the TM protein are indicated: ED (extracellular domain), AD (anchor domain) and ID (intracytoplasmic domain).

Identification immunodominant fusion proteins

and quantitation analysis epitopes by ELISA

of on

the envelope protein were indeed due to differences in antibody concentration in the sera of infected animals.

In order to confirm that the differences in immunoreactivity shown in figures 1 and 2 were not due to differences in the rate of expression of P-galactosidase FPs produced by the different clones, Western blots were performed using similar amounts of the different purified FPs. These purified FPs, loaded at similar protein concentration, still displayed the differences in immunoreactivity previously detected with streaked colonies (data not shown). So, the differences observed in immunoreactivity between regions of

Quantitation of these differences was performed by ELISA assays with various purified FPs from the 3 immunoreactive groups described above. Results obtained with serum no. 483, 9 months postinfection, are shown in figure 3 and confirm the classification into three immunoreactive groups. Respective titres of antibodies which reacted against the various purified FPs were calculated by endpoint analysis and are indicated. With other sera from the panel of infected macaques, a similar pattern of reactivity distin-

24

V. TANCHOU

Fig. 2. Immunoreactivity

ET AL.

of the panel of clones.

The panel of clones was tested against sera from infected macaque no. 495 various times before infection (B) and 2 weeks (C), 1 month (D), 2 months (E), 3 months (F) and 4 months (G) after infection. In panel A serum from another seronegative macaque was used as control. All sera were used at l:l,CQO dilution.

guishing the three groups stantly observed (data not the same clones (number 9, had the strongest reactivity. differences in ELISA titres clone were observed from (data not shown).

of clones was conshown). In particular, 11,22 and 43) always However, significant against each specific one serum to another

In table I, all overlapping clones covering the TM protein are ordered according to both their position in the protein sequence and their immunoreactivity. From comparison between the primary structures of group I weak clones (+) with those of the stronger reactive ones from group II (++) and III (tt+), striking features could be seen: on the one hand, without exception, all strongly reactive clones possessed sequence fragments of variable length beyond amino acid Phe 615 of gp140 (corresponding to position 88 of the TM protein). On the other hand, all weakly reactive clones from group I (+) lacked any sequence beyond this position. It can thus be concluded that around and beyond this position (Phe 615 in gp140), there

may exist one or several immunodominant topes.

epi-

In this region of the TM protein, a first immunodominant epitope, corresponding to the smallest fragment beyond Phe 615 found in one of the strongest reactive clones (clone 9, +++), can be defined between positions Phe 6 15 and Thr 63 1. It is noteworthy that no clones which contained the sequence corresponding to this epitope (clones from group II and group III) displayed identical immunoreactivity. This underlines the influence of other domains of the protein on the immunoreactivity of a particular epitope.

Immunoreactivity of synthetic peptides ing the whole transmembrane protein

cover-

In order to precisely map the immunoreactive epitopes detected with fragments of the TM pro tein expressed as fusion proteins and to look for additional epitopes possibly present in the C-terminal region of this protein, ELISA assays were

SIVmac-251

ENVELOPE

B-CELL

25

EPITOPES

Antibodies titers

a0.6 -

SERUM

FP-11

58

FP-22:

5.5

FP-43

5.2

FP-9

5.4

FP-la:

48

---

FP-6

43

--*--

FP-7

--*-..

FP-4

_._. +.-.

bgal

:

3.6 34

(47)

DILUTIONS Antlbodics titers

b0.6

E c z w

-

601619:

5

-

731.750.

38

-

711.730:

3.9

-

861-361:

3.6

-

662.680:

3.8

0.4

8

0.2

SERW

Fig. 3. Titration

by ELISA

651.670:

2.6

771.790.

23

761.780:

2

781400:

2

MLUTtONS

of the immunoreactivity

against various FPs and synthetic peptides.

The serum used was from infected macaque no. 483, 9 months postinfection. a) FF’s corresponding to the various curves, as well as non-recombinant P-galactosidase (47) are indicated on the right; b) curves corresponding to the various peptides are indicated on the right. Titres of antibodies reacting with these Fps and peptides were calculated by endpoint analysis and are indicated.

V. TANCHOU

26

performed against a panel of 35 overlapping peptides of 20 residues in length covering the whole TM protein, using sera from the panel of infected macaques. As an example, figure 4 shows results obtained with sera from two infected macaques (representative of the panel of infected animals) and one preimmune serum (representative of the panel of 14 preimmune sera). The peptide 601619, indicated in a solid bar, which contained the principal immunodominant epitope, was the most reactive (fig. 4A and 4B). This peptide was not reactive with non-immune serum (fig. 4C). Besides this peptide, a few others indicated in dotted bars were found frequently but inconsistently reactive. This was, in particular, the case

ET AL.

for peptides 731-750 and 861-881, which were among the most reactive. Titration of serum from infected macaque no. 483,9 months postinfection, against various peptides is shown in figure 3b. l&es against these peptides are indicated on the right. Similar titres were obtained with sera from other infected macaques (data not shown).

Determination of the external envelope protein continuous epitopes using synthetic peptides Fifty-two synthetic overlapping peptides of 20 residues in length, covering the whole external envelope protein, were tested in

A

ID

AD

ED

Fig. 4. Immunoreactivity

in ELISAs

of a panel of 35 peptides covering the whole TM protein.

The following sera were used at 1: 100 dilution: serum from infected macaques no. 483 (A) and no. 471 (B) and from a non-immune macaque (C). Immunoreactivity of peptide 601-619, corresponding to the principal immunodominant epitope, is indicated by a solid bar. Immunoreactivity of the other epitopes is indicated by dotted bars. Open bars represent negative or non-specific reactivities. Localization of the peptides in the different domains of the TM protein is indicated by the arrows: ED=extracellular domain, AD=anchor domain and ID=intracytoplasmic domain.

SIVrnac-251 ENVELOPE

ELISA assays against sera from 34 infected macaques, 9 months after infection. Results obtained with two of these sera as well as with a preimmune serum used as a control, are

B-CELL, EPlTOPES

shown in figure 5. About 13 peptides were found positive at variable levels of reactivity. Among these peptides, the most highly and frequently reactive were those represented in dot-

PEPTIDES

B

PEPTfDES

C

PEPTIOES

Fig. 5. Immunoreactivity

27

in ELISAs of a panel of 52 synthetic peptides covering the whole SU protein. The following sera were used at 1:lOO dilution: serum from a non-i~une macaque (A) and from infected macaques no. 483 (B) and no. 471 (C). Peptides corresponding to the major immunodominant epitope or to secondary epitopes described in the text and found to be frequently positive are represented in dotted bars. Negative peptides or peptides rarely found to be positive (less than 10% of the sera tested) are represented in open bars (ie. peptide 261-280).

28

V. TANCHOU

ET AL.

ted bars: peptides 111-130, 311-330, 501-520 and 511-530. In summary, after studying the reactivity of sera from 34 infected macaques together with

14 preimmune sera, against 87 synthetic peptides covering the whole SU and TM proteins, 7 pep tides (see table II) were found more frequently and were more highly reactive. Figure 6 shows

l

l l

l

l

l

l

:

l l

a 0

l

:

l

0

l

8

l

0

l

!

:

l

:

:

1

0

l

0 l

I ----___

l l

l

0

l

l

l m

0

0 : fl

l

l :

t

i

I I

J

su

I TM

PEPTIDES

Fig. 6. Immunoreactivity

of the 7 major immunodominant

epitopes of the envelope protein.

Immunoreactivity of sera from 34 infected macaques (closed circles on the left) together with sera from 14 non-immune macaques (open circles on the right) are tested in ELBA at 1:lOO dilution. Respective localization of those peptides in the SU and TM proteins is indicated below. Peptide 101-120 was used as a reference for a weakly reactive peptide, and peptide 492-509 corresponds to a control peptide of an irrelevant sequence (human p561ck purchased from Chiron Mimotope). Positive reactions were defined as any A,, value higher than three times the average of the control sera values shown in open circles.

SIVmac-251

ENVELOPE

the reactivity obtained with these 7 peptides against the panel of the 34 immune sera (closed circles) and against the panel of the 14 preimmune sera (open circles). The numerous peptides found to be negative or weakly positive are not shown here. These results clearly demonstrate that only one peptide in the TM protein, 601-619, was found to be constantly reactive in 100 % of infected macaques and generally displayed the highest reactivity. By comparison, peptides 731-750 and 861-881 were found to be positive in, respectively, 50% and 85 % of sera from infected animals (table II). In the SU protein, 4 major reactive peptides were found to be frequently reactive (between 70% to 94 % of the sera) (table II). However, as shown in figure 6, this reactivity was highly variable in intensity. The results obtained with the peptide 101-120, found to be positive for 55 % of the immune sera, were representative of the reactivity obtained with the 9 weakly reactive peptides not shown in figure 6.

DISCUSSION

In the present paper, we describe the analysis of the natural host immune response of macaques experimentally infected with SIVmac-25 1. This study was performed using an SIV epitope library constructed in the pTEX bacterial expression vector (Stanley, 1988; Haylemerle et al., 1986) expressing large protein fragments. This enabled us to characterize large immunoreactive regions including linear and conformational epitopes. The epitope library was used in combination with epitope-mapping techniques based on small synthetic peptides in order to precisely map these epitopes. Although some discrepencies between the two techniques can be noted, which are probably related to differences in immunoaffinity of large P-galactosidase fusion proteins and small synthetic peptides for reactive sera, similar and complementary results were obtained by means of these two approaches. For instance, it is noteworthy that clones localized in the SU protein, by contrast with those localized

B-CELL Table

II. Amino

Peptide 11 I- 130 3 1 l-330 501-520 5 1 l-530 601-619 73 l-750 861-881

29

EPITOPES

acid sequence of the 7 major immunodominant epitopes. Sequence

MRCNKSETDRWGLTKSSTTI MKCRRPGNKTVLPVTIMSGL LVEITPIGLAPTDVKRYTTG F’TDVKRY’ITGGTSRNKRGVF YLKDQAQLNAWFAFRQYFH Acm PSYFQQTHTQQDPALPTREG GGRWILAIPRRIRQGLELTLL

Positive sera (%) 80 70 94 85 100 Acm 50 85

“Acm” corresponds to an acetamidomethyl radical localized on the Cys residues. Peptides 11 I-130, 31 l-330, 501-520 and 511-530 are localized in the SU protein; peptides 601-619, 731-750 and 861-881 are localized in the TM protein. The percentage of sera yielding positive reactions to each peptide is indicated on the right and calculated as described in “Materials and Methods”.

in the TM protein, were not selected by our immunoscreening procedure. This is probably due to the fact that expression of large fragments (300-400 bp) of the SU protein is toxic for bacteria. The course of the immune response up to 9 months after the infection was studied with a panel of clones covering the whole TM protein and the C-terminal end of the SU protein. Results showed that (i) the pattern of reactivity is different from one infected animal to another; (ii) in each animal, changes in immunoreactivity are detectable during the course of infection; however, no obvious correlation between the pattern of immunoreactivity and the clinical status of the animals could be shown ; and (iii) antibodies directed against the principal immunodominant epitope in the TM protein are present in the serum of all the infected animals ; they appear very early and persist throughout the infection. This work is a systematic study of the reactivity of 87 overlapping peptides covering the whole SIV envelope protein performed with a large number of infected (34) and preimmune (14) macaques. It enabled us to draw reliable conclusions about the continuous epitopes which are the main targets of the immune response of infected

30

V. TANCHOU

hosts. Seven peptides, 4 in the SU protein and 3 in the TM protein, were the most reactive ones. They should correspond to the major continuous immunoreactive epitopes of the SIV envelope protein. Among these 7 major epitopes, only one was reactive against all 34 sera tested, the other being positive in most (between 50 to 94%) but not all cases. Tbis epitope, 601-619, corresponds to the well known principal immunodominant epitope of the TM protein described in SIV (Shafferman et aZ., 1989), as well as previously in HIV (Gnann et al., 1989; Goudsmit et al., 1990; Xu et al., 1991). It is noteworthy that this peptide was also found to be reactive with 93% of infected sera tested by Miller et al. in their study (Miller et aZ., 1992). The interest of this region resides in the relatively high level of sequence conservation found in the family of the lentiviruses (Myers et al., 1991). It is also known that the two highly conserved Cys residues are critical for the recognition of the envelope precursor proteases, and are essential for precursor env cleavage (Dedera et al, 1992; Syu et al., 1991). Besides this principal immunodominant epitope, 2 major epitopes (731-750 and 861-881) were found in the TM protein, localized in the C-terminal portion of the protein, respectively in the anchor domain and the intracytoplasmic tail, By contrast with Miller et al. (1992) who found corresponding epitopes reactive only in 13 and 7%, respectively, our results were as high as 50 and 85% of the sera t.Bkd.

Four major epitopes have been detected in the external envelope of SIV. The peptide 311-330 is localized in the Cys loop of the V3 region, known to be conserved in SIV (Johnson et al., 1991; Overbaugh et al, 1991), by contrast with the hypervariability of the homologous domain of HIV1 . The second major epitope found in the SU protein is localized in the C-terminal end of the protein, just before the cleavage site with the TM protein. In SIV, this epitope has been described (McBride et al., 1993; Miller et al., 1992; Shafferman et al., 1991) on the basis of sequence similarity with the corresponding region of HIVl, previously reported to be highly immunogenic (Bolognesi, 1990; Palker et al., 1987). In this

ET AL.

region, we found 2 overlapping reactive peptides (501-520 and 511-530). They could define the same epitope, but it should be emphasized that some macaques are differentially reactive against these 2 peptides. These results suggest that, in this region, more than one epitope could be defined. Finally, another major epitope was found in region 111-130, localized in the Vl region and was described by Overbaugh and colleagues (Overbaugh et al., 1991, 1992) as quite variable during progression of simian AIDS. Thus, comparison of our results with those recently reported by Miller et aI. (1992) and McBride et al. (1993) shows striking agreement in the reactivity displayed by most of the major epitopes we identified, exept for 2 epitopes of the TM protein located, in the anchor domain (731-750) and in the cytoplasmic tail (861-881). From these results obtained with a large number of infected animals, we conclude that the seven epitopes described represent the main continuous epitope targets of the host immune system in the SIV envelope. Outside these epitopes, as shown by our systematic study on 87 peptides covering the whole envelope protein, as well as by the results reported by Miller et al. and McBride et al. on a more limited collection of peptides, there is no continuous epitope displaying significant immunoreactivity. Examination of the sequences of these major epitopes shows that most of them correspond to regions of the envelope protein relatively conserved among various strains of SIV (Myers et al., 1991). So it seems that the immune response of infected animals against continuous epitopes is directed mainly to relatively conserved regions of the envelope protein. If these antibodies are not neutralizing antibodies, this could be one way for the virus, in addition to other means such as genomic variability, to escape the host immune system. Experiments should be performed to check whether or not antibodies from infected macaques, affinitypurified on these epitopes, display significant neutralizing activity. These immunodominant

epitopes could be of

SIVmac-251

ENVELOPE

great interest in irnrm.mosurveiIIance of infected macaques during the course of infection or in vaccine trials.

Acknowledgements We thank ANRS for the gift of gp41 peptides, M. Bodeus for fruitful discussions and P. Sonigo for critical reading of the manuscript. This work was supported in part by grants from ANRS.

Caractirisation des bpitopes B continus de la protiine d’enveloppe du virus SIVmac-251 chez des macaques exp6rimentalement infect&

Nous avons CtudiC la reponse humorale de 34 macaques expkimentalement infect& par le virus SIVmac-251 en utilisant en association une banque d’epitopes et des peptides synthetiques. L’Ctnde de l’evolution de la reponse immune a et& men&e sur une periode de 9 mois aprbs l’infection, grlce a differents clones exprimant divers fragments du g&tome viral. Parallelement, une etude systematique a &C realisee avec des peptides synthetiques couvrant la totalite des proteines transmembranaire (TM) et exteme (SU) de I’enveloppe. Sept Cpitopes majeurs immunodominants ont ttt caracterises. Quatte sont 1ocalisCs darts la protkine SU : un dans la region Vl (11 l-130). un darts la boucle cysteine precCdant la region V3 (3 1 l-330) et deux dans la partie C-terminale (501-520 et 511-530). Trois Cpitopes sont local&% dans la protkine TM: un darts le domaine extracellulaire (601-619), un dans la region transmembranaire (731-750) et un dans le domaine intracytoplasmique (861-881). Parmi ces tpitopes,

un seul, le 601-619,

s’est

rCvtlC

rdactif

avec tous les drums testes et peut Ctre defini comme le principal epitope immunodominant. M&s-cl&: SIV, Lymphocyte B. Epitope, Immunodominance ; VIH, RCponse humorale, Macaque, Sequencage, Peptides, Proteines d’enveloppe.

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