FEMS Microbiology Immunology 76 (1991) 99-108 © 1991 Federation of European Microbiological Societies 0920-8534/91/$03.50 Published by Elsevier A D O N I S 0920~5349100065H

99

FEMSIM 00153

A peptide library expressed in yeast reveals new major epitopes from human immunodeficiency virus type I Pascal M a d a u l e , J e a n E d o u a r d Gairin, Serge B 6 n i c h o u a n d J e a n Rossier Laboratoire de Physiologie Nerveuse, Centre National de la Recherche Scientifique, Gif-sur- Yvette, France

Received 18 November 1990 Accepted 28 November 1990

Key words: HIV-1; Peptide library; Expression/secretion vector in yeast; Delineation of epitopes; Mapping of monoclonal antibodies 1. SUMMARY In order to characterize novel human immunodeficiency virus type 1 (HIV-1) continuous epitopes, we designed a simple method, based on recombinant DNA, providing a complete set of peptides derived from HIV-1. A library (4 x 10 4 clones) was first constructed in a new expression/ secretion vector, using as inserts small fragments of HIV-I D N A (50-150 bp) generated by random DNAse I cleavage. This peptide library, expressed in the yeast Saccharomycescerevisiae, was screened with sera of HIV-1 infected individuals and human and murine anti-HIV-1 monoclonal antibodies. Plasmids from immunoreactive colonies were recovered and the sequences of the HIV-1 derived inserts were determined. By using human sera, we have detected classical HIV-1 epitopes and identified two novel major epitopes, which may be used to improve diagnostic tests, localized in the p24 core protein and in the endonuclease. In addition, four minor epitopes were also defined by screen-

Correspondence to: J.E. Gairin. Present address: Department of Neuropharmacology, Research Institute of Scripps Clinic, La Jolla, CA 92037, U.S.A.

ing the library with monoclonal antibodies: in the protease, in the p17 core protein, in gp120 and near the C-terminal of gp41. This method is general and can be used for any protein from which a cloned cDNA is available.

2. I N T R O D U C T I O N The natural immune response against human immunodeficiency virus type 1 HIV-1 infection is often intense but inefficient in stopping disease progression. This is probably due to the immunodominance of non-neutralizing epitopes from HIV-1. Thus, characterization of all the epitopes, including the minor ones, becomes a major issue in designing a passive [1] or active immunization against AIDS. Current methods for epitope mapping relie mostly on chemically synthesized peptides and computer models [2,3]. However such approaches may be unsatisfactory due to inappropriate choice of sequences synthesized or limitations of chemistry. Thus, we developed an alternative approach, based on recombinant DNA, which is readily accessible to most research laboratories. In this new method, a continuous

100 epitope is produced as peptides of various length, 15 to 50 amino acid (a.a.) residues, thereby increasing the chance that one of them adopts a conformation recognized by the antibodies. The expression system was based in yeast which offers several advantages over Escherichia coli: it has no toxin for mammals and carries out many post-translational modifications, even with foreign proteins [4]. Previous works have demonstrated that MFa-1 gene (coding for the c~-mating factor, one of the two peptide hormones involved in yeast sexual recognition) could be used to express proteins in yeast [4-6]. The a-factor biosynthesis (see ref. 7 for review) proceeds through a large precursor where the N-terminal region is presumably involved in targeting the protein on a secretion pathway while the C-terminal region contains four hormonal domains separated from each other by specific Lys-Arg cleavage sites (Fig. 1A). When foreign D N A is placed downstream of the first encoded Lys-Arg in MFa-1, the resulting fusion protein is often processed such that the recombinant peptide is essentially devoid of MFa-1 encoded residues. N- or O-glycosylation may occur in a mannose rich fashion [4]. Finally the peptide is often released into the media. These properties seemed promising for producing peptides of immunological interest. An a-factor based vector was first modified in order to obtain proper termination for both transcription and translation regardless to the insert. This plasmid was called pSE-X, standing for secretion-X. A library was then constructed in this vector using, as inserts, short fragments obtained at random by partial DNAse I digest of HIV-1 proviral DNA. Screening this library with human sera, major epitopes from HIV-1 were characterized: a well known epitope in gp41 and also two unexpected epitopes, in the p24 core protein and in the p34 endonuclease, which may be used for improving diagnostic tests. Using monoclonal antibodies, additional novel epitopes were defined in the envelope glycoproteins gp41 and gpl20, in the protease and in the p17 core protein. Interestingly, one monoclonal antibody was successfully mapped albeit an extensive analysis with synthetic peptides had failed, demonstrating the validity of our approach.

3. MATERIALS A N D M E T H O D S 3.1. Monoclonal antibodies and human sera All the human sera used were strongly reactive to most of the HIV proteins in a Western blot analysis. The murine monoclonal antibodies 25.7, 41.1, 110.1, 110.4 and 110.5 are a kind gift of Genetic System [8], and the other monoclonal antibodies against p17 (named CVK) [9], gpl20 (named gpI123 and gpI112) and the pol gene product were kindly provided respectively by Jean-Claude Gluckman and David Klatzmann (HSpital Piti6-Salpdtri6re, Paris), Jean-Loup Romet-Lemone (Centre National de Transfusion Sanguine, Les Ulis, France) and Karin Moelling (Max Planck Institut ffir Molekfilare Genetik, Berlin). The human monoclonal antibody was given by Claude Desgranges [10]. Human HIV-1 positive sera were obtained from a blood facility at the Institut Pasteur (Paris). 3.2. Construction of the expression vector pSE-X and its control pGAG Detailed construction of pSE-X will be published somewhere else. Briefly, this plasmid based in pUC18 (Fig. 1) has five pieces of foreign DNA: first, a 600-bp fragment of the c~-factor gene (MFa-1) containing the promoter and the coding sequence up to the first Lys-Arg cleavage site; second, a 35-bp fragment of pEX2 providing a stop codon for each open reading frame [11] of MFa-1; third, a 400-bp fragment of TRP5 carrying signals for gene termination [6]; fourth, the URA3 gene as a selectable marker in yeast; fifth, the 2-/~m D N A as a yeast origin of replication. The polycloning region has been removed. A positive control, called p G A G , was obtained by cloning into the HindIII site of pSE-X, a 587-bp PstI-ApaI fragment of HIV-1 DNA [12], encoding parts of p24 and p13. 3.3. Construction of the expression library The library was constructed using as inserts random short fragments of pBT-1 (a pUC18 plasmid containing a complete integrated provirus of HIV-1, BRU isolate [12]). Fragmentation was performed using DNAse I as described by Anderson [13] except that the conditions were adjusted

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to obtain smaller fragments, ranging from 50 to 150 bp, and linkers were not used. Since D N A s e I makes about two nicks for one double strand cut [13], care was observed not to melt the D N A until the nicks were repaired. Inserts were ligated with pSE-X prepared for blunt end cloning at the HindlII site. E. coli (SCS1, Stratagene) transformed with the ligation mix gave rise to 4 x 10 4 independent clones. Plasmid D N A was analysed from 12 random colonies: 11 had inserts (average

3.4. Immunological screening of the library Transformed yeast cells were spread on Petri dishes containing a selective media [6] for URA3 plasmids. After 30 h Of culture at 30 ° C the yeast colonies, usually 104, were transfered onto nitrocellulose filters, lysed in situ, and analysed like a Western blot (defatted milk procedure) with human sera or monoclonal antibodies as described previously [15]. However most human sera needed to be immunoadsorbed as follow: baker's yeast (Fould-Springer) was dispersed in water (1 ml of water per g of yeast) and kept at 1 0 0 ° C for 20 min. The p H was adjusted to 7.3 with N a O H and 0.05 volumes of 1.5 M N a C I / 0 . 1 M sodium phosphate buffer, p H 7.3, was added. Sera were diluted in the yeast lysate at 1:300 and incubated overnight at 4 o C with gentle shaking. The mixture was spun and the supernatant used directly for incubation with the nitrocellulose filters. Peroxidase-coupied goat anti-human or anti-murine I g G (Pasteur Diagnostic) were used as second antibodies and staining was performed with diaminobenzidine in presence of nickel [16] until the negative control clones became slightly visible. Plasmids present in each positive yeast clone were rescued in E. coli [14] and prepared; PstI fragments, bearing the inserts (Fig. 1B), were subcloned into M13 m p l 9 and sequenced by the dideoxy chain termination procedure [17] using either a classical M13 primer or the oligonucleotide T G C C A G C A T T G C T G C T priming within MFa-1.

4. R E S U L T S 4.1. Identification of major epitopes using human sera Sera from ten HIV-1 infected individuals were obtained from the Institut Pasteur. Each serum was positive for all the viral structural proteins in a Western blot analysis (not shown). In a pre-

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Fig. 2. lmmunodetection of recombinant HIV-1 peptides in total yeast lysates. Well charaterised yeast clones were spread as long parallel patches on a Petri dish and analysed as described for library immunoscreen, except that the nitrocellulose filter was cut in strips containing proteins transferred from each clone, allowing comparison of various antibodies. Lanes A to J, h u m a n sera from 10 seropositive patients; lane m, h u m a n monoclonal antibody (see text); lanes a, b, c, d, and e: mouse monoclonal antibodies, respectively 41.1, 25.7, 110.4, 110.5, and 110.1. Clone numbers 1 to 13 and control pSE-X and pGag are indicated on the top.

liminary experiment, serum G detected strongly the yeast positive control (producing the 23 kDa fragment of gag) and was therefore initially used to screen the library. Seven colonies were detected (clones 1 to 7, Fig. 2) and analysed: plasmid D N A present in each positive clone was recovered and the sequence of each insert was determined; the several overlapping sequences obtained delineated two epitopes (Table 1), one in p24 (a.a. 318-346) and the other in gp41 (a.a. 591-615). This gp41 epitope is the major viral epitope already mapped

[18]. Two mouse monoclonal antibodies, 25.7 and 41.1, both raised against the full-size HIV-1 proteins, have been previously mapped to these positions (L. Montagnier, personal communication). These monoclonal antibodies detected our seven recombinant peptides, either in total yeast lysates (Fig. 2, clones 1-7) or in yeast culture supernatant analysed by ELISA (enzyme linked immunosorbent assay), a more sensitive assay [24]. These seven clones were also analysed with the other human sera from HIV-1 infected individuals (Fig. 2). The gp41 epitope (clones 1 4) was strongly detected with a large majority of sera, confirming that it represents a major epitope. A careful examination of Fig. 2 suggests the presence of two epitopes in the set of p24 clones: clones 5 and 6 were equally detected by the monoclonal 25.7 (Fig. 2, lane b), demonstrating that the corresponding epitope is produced similarly by both clones. However, one human serum (B) strongly detected clone 5 but not at all clone 6. In fact, clone 5 has a longer insert than clone 6, encoding 17 extra amino acids (a.a. 287-303, Table 1) where a novel epitope detected by serum B may lie. Alternatively, the longer peptide produced by clone 5 may fold to form a conformational epitope. Serum F, which gave almost no reaction with clones 1 7 (Fig. 2), was used to screen the library in a search for other epitopes and detected two colonies, one strongly (clone 8) and the other moderately (clone 9). These clones 8 and 9 appeared to contain an epitope from the p32 endonuclease (a.a. 749-773), a pol-encoded protein. The shorter peptide sequence encoded by clone 9 could explain its weaker reactivity. This epitope was also unambiguously detected, although weakly, by three other sera (A, B, and C) in total yeast lysates (Fig. 2). This suggests that it may represent a major HIV-1 epitope which could be used for diagnostic purpose. Interestingly, this peptide contains two cysteinyl residues, a feature in common with the major epitope of gp41 [18]. A mouse monoclonal antibody raised against the pol gene product failed to detect clones 8 and 9. The library was then screened with this antibody and one yeast clone was detected (clone 19). Sequence analysis of its insert delineated a 75 a.a. long

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Table 1

104 peptide from the pol gene product (a.a. 102-176) corresponding to the last 66 residues domain of the protease and the first 9 residues of the p66/51 protein [19]. 4.2. Epitope mapping of anti- HIV-1 monoclonal antibodies A human monoclonal antibody, derived from lymphocytes of an early volunteer for vaccination trial, was first used to screen the library [10]. This antibody, directed against gp41, failed to react with synthetic peptides supposed to represent antigenic domains of gp41 (C. Desgranges, personal communication). However, screening of the library with this antibody detected only one yeast clone (clone 10) which produces the last 38 residues of gp41 (Table 1). Interestingly, this clone uses the HIV-1 env stop codon, instead of one of the three vector derived stop codons. Since the synthetic peptide composed of the last 15 residues of the gp41 did not react with this human antibody, this epitope is probably not located at the C-terminal itself. Alternatively, the epitope may be unusually large or require a post-translational modification that yeast is able to perform. In a total yeast lysate, this epitope is not recognized by any of the 10 HIV-positive human sera (Fig. 2). Then, this sequence is, at best, weakly immunogenic in the response against natural HIV-1 infections. Others have described an epitope within the 129 last residues of gp41 [20]. The library was also screened with five mouse monoclonal antibodies raised against full-size gp120. Monoclonals 110.4 and 110.5, both mapped to position 303-323, neutralize the infectivity of some HIV-1 strains [8]. Monoclonal 110.1 had been mapped to position 489-511, a well-known epitope of gp120 [8,20]. The monoclonals gp1123 and gpI112 were not previously mapped. Two clones were detected with 110.1 (clones 11 and 13, lane e, Fig. 2), one clone was detected with 110.4 and 110.5 (clone 12, lanes c and d, Fig. 2), and three clones with gpI123 and gpI112 (clones 14, 15 and 16). Sequence analysis of the inserts obtained from the positive clones (Table 1) confirmed the previous mapping of antibodies 110.1, 110.4 and 110.5. The three overlapping sequences of inserts from clones 14, 15 and 16 (Table 1) delineated a

22-residue domain from the gp120 (a.a. 91-112). Interestingly, the peptides produced by clones 15 and 16 contain an antigenic site of the gpl20 (HEDIISLWDQSLK) recognized by T-lymphocytes from human volunteers who have been immunized with a recombinant vaccinia virus expressing the gene of the HIV envelope glycoprotein gp160 of the HTLVIIIB, BH10 isolate [21]. All the human sera failed to detect those epitopes in total yeast lysates (Fig. 2); since the recombinant peptides are highly produced (1 /LI of yeast culture supernatant is sufficient in ELISA for detection with monoclonal antibodies), the titer of the corresponding antibodies must be low in the sera from HIV-1 infected individuals. Finally, a mouse monoclonal antiboby, raised against the whole virus and directed against the p17 core protein, was used to screen the library. This antibody was important to analyse because it presents a cross-reactivity with some cellular component, which could reflect some structural homology between HIV-1 and its host [9]. A PEPSCAN analysis had been previously performed (testing the immunoreactivity of each possible hexapeptide chemically synthesized according to the p17 sequence) and no peptide was found to react with this antibody (J.-C. Gluckman and D. Klatzmann, personal communication). Nevertheless, two yeast clones were strongly detected in the library. Sequence analysis of the corresponding inserts (clones 17 and 18, Table 1) defined a 43-residue domain from p17 (a.a. 75-117). This epitope should not be considered as a major one since no strong immunoreactivity was observed with HIV positive human sera (not shown).

5. DISCUSSION We describe in this article a novel approach to define antigenic determinants or epitopes of proteins. Using HIV-positive human sera, we could identify unexpected highly antigenic epitopes. The system is also very efficient to map monoclonal antibodies since all the antibodies tested so far do react with specific clones. We will discuss now particular properties of this approach. No preliminary data is required for defining the

105

epitopes. Mapping the antigenic sites is classically performed by analysing the immunoreactivity of numerous peptides chemically synthesized. Computer models predicting the antigenic sites of a protein have been used initially, in order to reduce the number of peptides to synthesize. Since these models proved to be inefficient [18], the recent trend is to synthesize all possible peptides of a given length (for example residues 1-6, 2-7, 3-8, and so on), a technique known as PEPSCAN. This approach requires the sequence of the protein, a goal usually obtained by DNA sequencing. DNA sequences are easy to interpret with most cellular cDNA, but when analysing viral DNA, determination of which open reading frame is actually directing protein synthesis may be almost impossible. Indeed, it took years to demonstrate that some open reading frames were used by HIV-1 [221; still today the debate is open about the significance of an open reading frame present on the minus strand of HIV-1. In our system, all possible peptides should be synthesized regardless of the reading frame or the strand polarity. Then, defining the reading frame is useless for our approach. Actually, sequence data are not required to construct a library as described. The only critical reagent needed is a stock of DNA rich in the coding sequence for the protein of interest. Larger peptides have a better antigenic structure. We constructed the library such that the majority of the yeast clones will produce 20-30residue long peptides. A random analysis of the clones confirmed that this size was predominant in the library. Most of the immunoreactive clones however, encode 40-50 residue long peptides, suggesting that only those longer molecules were readily detected in our experiments. We recently observed that a monoclonal antibody against gp41 had a three orders of magnitude drop for its affinity when compared between full-size gpl60 and a synthetic 25 a.a.-long peptide. This drop was less than one order of magnitude when the 52-residue long peptide produced by clone 3 was used instead [24]. Two explanations are possible: first, longer peptides may adopt a conformation resembling more to a native protein; second, if the main antigenic site for an antibody is only 5 or 10 residues long, extra amino acids might also be

recognized and would stabilize greatly the immune complex. This lower affinity for short peptides might explain why we could map the monoclonal antibody against p17 since the PEPSCAN which failed was performed with peptides of only six residues long. Alternatively, we cannot exclude that a post-translational modification, properly performed in yeast, is essential to the antigenicity of this p17 epitope. Then, this technique fills a gap between synthetic peptides which are smaller, and much larger ones produced by expression systems where the DNA of interest has been fragmented with restriction enzymes [22,23]. Only minor biases are observed in the library. An important question to address with libraries is their bias level. In our case, we had to check whether all potential antigenic peptides are produced in the library. This can be estimated using several monoclonal antibodies. We used six different monoclonal antibodies, which were reactive in Western blot analysis. All of them detected clones at a frequency of 10 .3 to 10 -4, demonstrating a good representation of the library. It should be pointed out however, that we used genomic HIV-1 DNA as starting material; the few peptides encoded on separate axons cannot then be synthesized. We also observed that a few inserts had a common extremity, indicating that DNAse I had some preference in the partial digest of HIV-1 DNA. Nevertheless, those are very minor problems since no antibody failed to detect its antigenic site. Finally, the recombinant peptides are not toxic for yeast since all of the recombinant clones have similar doubling time compared to a control, and a good cell viability at 4 ° C (not shown). This is quite unusual for an expression system, especially when the promoter used leads to a strong and constitutive expression. In this system, however, the recombinant gene product is quickly directed into the Golgi apparatus, thereby minimizing interactions with many cellular components and being also protected against degradation [5,7]. The only striking difference from clone to clone is their ability to secrete their recombinant peptide: about two thirds of the clones secrete large quantities of peptide (up to 1 rag/l), while the other ones secrete only a small amount (not shown).

106 T h e m a j o r i t y of the d e t e c t e d clones secrete their r e c o m b i n a n t peptide, thus p r o v i d i n g an unlimited source of antigen in a relatively clean form. Since we have o b t a i n e d several a n t i g e n i c p e p t i d e s f r o m HIV-1, a very low cost b l o o d screening test s h o u l d b e easy to design. Similarly, the p e p t i d e s p r o d u c e d in yeast could b e used for immunization. I n conclusion, we designed a novel, s i m p l e a n d inexpensive m e t h o d to d e t e c t c o n t i n u o u s epitopes. A s d e m o n s t r a t e d with the r a p i d m a p p i n g of novel e p i t o p e s in the gp120, gp41, p17 a n d p r o t e a s e o f HIV-1, this technique is very efficient for m o n o clonal antibodies. I n the case of h u m a n sera, highly i m m u n o g e n i c e p i t o p e s were also m a p p e d since we c o u l d quickly characterize n o t o n l y the classical m a j o r e p i t o p e o f gp41, b u t also u n e x p e c t e d epi-, topes in the e n d o n u c l e a s e a n d in p24 gag. Interestingly, s o m e e p i t o p e s are strongly d e t e c t e d b y a small subset of sera, for e x a m p l e clones 6 a n d 7 are m o s t l y d e t e c t e d b y s e r u m G. D e l i n e a t i o n of such o c c a s i o n a l l y strong epitopes, together with a clinical follow up of the few c o r r e s p o n d i n g p a tients, could help to design p r o g n o s t i c tests a n d p o s s i b l y vacccines. Similar w o r k m a y b e u n d e r t a k e n for o t h e r pathogens, a u t o i m m u n i t y diseases o r for m a p p i n g e p i t o p e s d e f i n i n g f u n c t i o n a l d o m a i n s in a protein. D N A a n d strains d e s c r i b e d in this r e p o r t are available for research.

ACKNOWLEDGEMENTS W e a c k n o w l e d g e Pierre Sonigo a n d M a r c A l i z o n for the gift of H I V - 1 D N A , M i c h e l L a b o u e s s e , F r a n c o i s L a c r o u t e a n d A l a n M y e r s for y e a s t strains, J e a n - C l a u d e G l u c k m a n , D a v i d K l a t z m a n n , J e a n - L o u p R o m e t - L e m o n e , C l a u d e Desgranges a n d K a r i n M o e l l i n g for the gift of m o n o clonal antibodies, Luc M o n t a g n i e r a n d A n n B e a u m o n t for critical review of the m a n u s c r i p t , R a n d o l p h V. Lewis for his help a n d advice a n d Pascal Bochet for his help in h a n d l i n g with sequences. This w o r k was s u p p o r t e d b y grants f r o m A g e n c e N a t i o n a l e de Recherche sur le S I D A , Association p o u r la R e c h e r c h e sur le C a n c e r a n d F o n d a t i o n p o u r la Recherche M~dicale.

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A peptide library expressed in yeast reveals new major epitopes from human immunodeficiency virus type 1.

In order to characterize novel human immunodeficiency virus type 1 (HIV-1) continuous epitopes, we designed a simple method, based on recombinant DNA,...
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