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The Journal of Immunology, 1999, 162: 6155-6161.
Copyright © 1999 by The American Association of Immunologists

Selection of HIV-Specific Immunogenic Epitopes by Screening Random Peptide Libraries with HIV-1-Positive Sera

Giuseppe Scala1,2,*,{ddagger}, Xueni Chen1,*, Weimin Liu*, Jean Noel Telles*, Oren J. Cohen*, Mauro Vaccarezza*, Tatsu Igarashi{dagger} and Anthony S. Fauci*

* Laboratory of Immunoregulation, and {dagger} Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892; and {ddagger} Dipartimento di Medicina Sperimentale e Clinica, Universita‘ degli Studi di Catanzaro, Catanzaro, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Efforts to develop a protective HIV-1 vaccine have been hindered by difficulties in identifying epitopes capable of inducing broad neutralizing Ab responses. In fact, the high mutation rate occurring in HIV-1 envelope proteins and the complex structure of gp120 as an oligomer associated with gp41 result in a high degree of antigenic polymorphism. To overcome these obstacles, we screened random peptide libraries using sera from HIV-infected subjects to identify antigenic and immunogenic mimics of HIV-1 epitopes. After extensive counterscreening with HIV-negative sera, we isolated peptides specifically recognized by Abs from HIV-1-infected individuals. These peptides behaved as antigenic mimics of linear or conformational HIV-1 epitopes generated in vivo in infected subjects. Consistent with these findings, sera of simian HIV-infected monkeys also recognized the HIV-specific epitopes. The selected peptides were immunogenic in mice, where they elicited HIV-specific Abs that effectively neutralized HIV-1 isolates. These results demonstrate that pools of HIV-1 mimotopes can be selected from combinatorial peptide libraries by taking advantage of the HIV-specific Ab repertoire induced by the natural infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A number of studies in animal models demonstrate a protective role of Abs against HIV-1 challenge (1, 2). In this regard, a strong correlation was observed between protection against infection and levels of neutralizing Abs in nonhuman primates infected with HIV-1 or simian HIV (SHIV)3 (3, 4, 5). Adoptive immunotherapy with HIV-specific Igs (6) and passive immunization with anti-HIV envelope mAbs in chimpanzees and in PBL-SCID mice (7, 8) have also resulted in protection against HIV challenge. Synergistic neutralization of HIV-1 by anti-HIV Igs combined with envelope-specific mAbs has also been reported (9). These results suggest that an effective vaccine should elicit an HIV-specific Ab response (10, 11). However, these studies have failed to identify epitopes capable of inducing an effective neutralizing Ab response. In fact, the identity of the immunogenic epitopes has been determined in only a few mAbs and has remained elusive in the case of the human polyclonal immune response to multiple B cell epitopes, which are primarily conformational in nature and cannot be identified from primary sequences (12). In the case of HIV-1, the high mutation rate occurring in HIV-1 envelope proteins and the complex structure of gp120 as an oligomer associated with gp41 (13, 14) result in the generation of numerous epitopes. Moreover, some epitope specificities may change during the course of disease as a result of viral evolution, Ab affinity maturation, and viral escape (15). In developing a protective vaccine, it would be advantageous to identify those epitopes that are specifically recognized by Abs generated by HIV-1-infected subjects. In fact, these epitopes might include a substantial proportion of the epitope repertoire generated among a large panel of HIV-infected subjects harboring different HIV-1 quasispecies over several years of infection. Moreover, these epitopes would be immunogenic, since they have been selected for their binding to serum Abs; in fact, they would function as antigenic mimics of HIV-1 and would induce Abs reacting with HIV-1 when utilized as immunogens. This possibility was tested by screening random peptide libraries (RPL) using HIV-positive sera as ligands. We identified numerous epitopes that behaved as antigenic and immunogenic mimics of HIV-1 or SHIV epitopes generated in the natural course of infection. The selected epitopes induced a neutralizing Ab response when used as immunogens in mice. These findings indicate that the antigenic polymorphism of HIV can be matched by a collection of epitopes selected for their affinity to human HIV-1 Abs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Screening of phage-displayed RPL

Human sera were collected from HIV-1-positive or HIV-1-negative control subjects. Criteria for definition of long-term nonprogression or of AIDS were as previously described (16). Two peptide libraries composed of random nonamers displayed on the N terminus of pVIII major coat protein of filamentous phages, either unconstrained (pVIII9aa) (17) or flanked by two cysteines (pVIII9cys-aa-cys) (18), were screened as described (19). In the immunoaffinity selection, serum IgG was linked to magnetic microbeads (tosyl-activated Dynabeads M450; Dynal, Lake Success, NY) previously coated with an anti-human (Fc-specific) polyclonal Ab (goat anti-human IgG Fc-specific; Sigma, St. Louis, MO) at 200 µg/ml of beads suspension. A total of 2 x 1011 transducing units (TU) of phage particles were applied to IgG-coated beads and incubated for 16 h at 4°C. After extensive washing, bound phages were eluted with 0.1 M HCl/glycine buffer (pH 2.2) and neutralized.

Immunoscreening was performed as follows: TG-1 cells were infected with eluted phages at a multiplicity of infection (MOI) of 1 x 10-3 and plated at a density of 1 x 104 TU/plate. The following day, bacterial colonies were collected, amplified, and superinfected with M13K07 at an MOI of 50. A total of 2 x 103 colonies were replated on a lawn of TG-1 cells in the presence of 35 µg/ml of isopropyl-1-thio-ß-D-galactoside (IPTG). Plates were layered with nitrocellulose filters for 16 h at 37°C. Filters were incubated with serum at a 1:50 dilution in immunoscreening buffer (5% nonfat dry milk, 0.1% Nonidet P-40, 3 x 1011 wild-type phages, 5 x 109 M13K07 UV-killed phages/ml, 10 µl of TG1 bacterial extract) for 16 h, at 4°C. Positive colonies were detected by an anti-human (Fc-specific) alkaline phosphatase-conjugated Ab (Sigma).

For ELISA, microtiter plates were coated with anti-M13 Ab (Pharmacia, Piscataway, NJ) at 10 µg/ml overnight at 4°C in coating buffer. A total of 50 µl of cleared phage supernatant with an equal volume of blocking buffer were incubated for 1 h at 37°C. Plates were washed extensively and supplemented with human serum at 1:100 dilution, followed by an overnight incubation at 4°C. After washing, wells were coated with an anti-human (Fc-specific) alkaline phosphatase-conjugated Ab. Plates were washed and developed. Results were expressed as the difference between OD405 nm and OD620 nm by an ELISA reader.

Affinity purification of phagotope-specific human Abs

Dishes with a 60-mm diameter were coated with 5 x 1011 CsCl-purified phages overnight at 4°C. After washing and blocking, human serum (1:100 dilution) was added and incubated for 16 h at 4°C. After extensive washing, bound Abs were eluted with glycine-HCl buffer (pH 2.2). Ab concentration was determined by an in house ELISA with a low detection level of 1–2 ng/ml.

Immunization of mice

Phages were CsCl-purified and used at a concentration of 6 x 1012 particles/ml in 0.9% NaCl with an equal volume of CFA or IFA. Four- to five-wk-old female BALB/c and C57B1 mice were immunized by i.p. injection of 200 µl of Ag emulsion at weeks 0, 3, 6, 9, 12 and bled on day 0 and days 7–10 after each additional injection. Serum IgG were purified from mouse sera with T-Gel Adsorbent (Pierce, Rockford, IL).

Virus neutralization

Neutralization of HIVIIIB and NL4-3 molecular clones was measured in a MT-2 assay (20). Briefly, cell-free virus (500 tissue culture-infective dose50/ml) and serial dilutions of mouse IgG were incubated in triplicate at 37°C for 1 h before the addition of MT-2 cells (5 x 104/well). At 6–8 days postinfection, neutralization was quantified by staining viable cells with neutral red, followed by colorimetric determination of uptake at 540 nm. In the case of AD8, neutralization assay was performed on PHA-activated PBMC as described (21).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Affinity selection of HIV-1 mimotopes

To select for B cell epitopes specifically recognized by serum Abs of HIV-1-infected subjects, RPLs displayed on phages were screened with HIV-1-positive sera. Given the fact that sera from long-term nonprogressor (LTNP) subjects show higher titers of neutralizing Abs than sera from AIDS patients (22), initial screening was performed with LTNP sera. To maximize the detection of HIV-specific peptides, phagotopes were first selected by immunoaffinity purification with the IgG of one HIV-1-positive individual and then subjected to immunoscreening by using a second HIV-1-positive serum. Positive colonies were tested by ELISA for reactivity with sera from multiple HIV-infected individuals and counterscreened with an equivalent number of sera from HIV-negative subjects. In selection 1, LTNP sera 6090 and 3976 were utilized for immunoaffinity and immunoscreening steps, respectively. Similarly, selection 2 was performed by using LTNP sera 3872 and 8075. Selection 1, performed on a cysteine-constrained pVIII9cys-aa-cys library (18), resulted in the identification of five HIV-specific clones; selection 2, conducted on an unconstrained pVIII9aa library (17), led to isolation of five additional phagotopes (Fig. 1Goa). All the selected clones were found to react with 22 LTNP sera and 25 AIDS sera with a recognition frequency (f) ranging from 23 to 64%; all clones tested negative by ELISA with 50 HIV-negative sera. It was highly unlikely that the frequency distribution of each phagotope between HIV-positive and -negative sera could have occurred by chance (p < 0.001, as determined by using the Fisher exact test; p values were adjusted for multiple testing using the Bonferroni method). Accordingly, the clones were considered HIV-1-specific phagotopes. Each serum manifested a distinct pattern of reactivity with the pool of phagotopes (Fig. 1Goa). Some sera, such as 8873 and 1276, recognized most phagotopes indicating a broad Ab specificity, whereas sera 2214, 5223, and 8075 reacted with only one phagotope. Clone p217 was restricted in its reactivity to a subset of LTNP sera (f, 23); however, it was completely unreactive with a pool of AIDS sera (Fig. 1Goa). Analysis of the reactivities of sera for each phagotope showed that Ab titers to p163, p217, and p335 were significantly higher in sera from LTNPs than from AIDS patients (p < 0.05; Fig. 1Gob). These results suggest that Ab responses to these epitopes might afford a degree of protection against disease progression.



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  		FIGURE 1. ELISA reactivities of HIV-specific phagotopes. a, LTNP and AIDS subjects were selected as reported (16). HIV-specific phagotopes were identified from RPL pVIII9aa-cys (selection 1), or pVIII9aa (selection 2) by serial steps of screening and phage colony purification, as described in Materials and Methods. Results are expressed as fold increase of the average values of the phagotopes over values of the wild-type phages. Values were considered positive when at least 4-fold higher than the background signal of wild-type phage. Average values of at least four independent assays are shown as: {square} < 4;4 < {square} < 10; 10.1 < < 20; and {blacksquare} > 20. The far right column indicates the recognition frequency of positive sera by each phagotope (f). The percentages of significant phage reactivity in the HIV-positive and control groups were compared using the Fisher exact test; p values (p < 0.001) were adjusted for multiple testing using the Bonferroni method. b, Comparison of ELISA reactivities of phagotopes with sera of LTNP (hatched bars) vs AIDS subjects (black bars). Data are expressed as mean ± SEM of fold increase for each phagotope. Statistical analysis was performed according to the one-sided Student’s t test; *, p < 0.05.

 
Characterization of HIV-1 mimotopes

The amino acid sequences of the phage-displayed peptides are shown in Fig. 2Go. A BLAST analysis revealed that the p195 epitope shares sequence homology with the gp120 V1 region (residues 112–120) of HIV1-U16374, a primary isolate from an acute seroconverter (23); the p217 sequence matched with the gp120 C2 region (residues 198–205) of HIV1-U116077, a primary isolate from an AIDS patient (24) (Fig. 2Goa). Residues within these regions have been predicted to be immunologically accessible by selected mAbs and by x-ray crystal structure (13, 14). Moreover, the p197 epitope mapped to a region of gp41 (residues 602–605) of the HIVANT70 primary isolate (25). This region is conserved among primary isolates of HIV subtypes A through G and defines a disulfide-bonded structure recognized by a human mAb (Fig. 2Go, a and b; and 26). Thus, the peptides expressed on p195, p217, and p197 are antigenic mimics of epitopes expressed in primary HIV isolates from subjects at different stages of disease. No sequence homology with HIV proteins was found in the remaining clones, suggesting that they may represent immunological mimics of conformational HIV-1 epitopes (Fig. 2Goc).



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  		FIGURE 2. Amino acid sequences of the HIV-specific phagotopes. The amino acid sequences of peptides displayed on the HIV-specific phagotopes are shown as single letter codes. a, Homology between the amino acid sequences of p195, p217, and p197 and discrete regions of HIV gp160. Gray boxes indicate identity; similarity among amino acid residues is indicated as gray. b, Consensus homology of p197 with a gp41 domain conserved between HIV-1 subtypes A trough G. c, Amino acid sequences of epitopes with no obvious sequence homology with HIV protein domains.

 
The Ab reactivities shown in Fig. 1Goa indicate that the phage-displayed peptides behave as antigenic mimics of viral determinants generated in the course of HIV infection. Therefore, it should be possible to immunoaffinity-purify Abs specific for each phagotope from sera of HIV-infected individuals by using single phagotopes as ligands. To this end, p195, p197, p217, p287, and p335 were utilized to purify the phagotope-specific Abs from LTNP serum 6090. The phagotope-specific Abs, purified from serum to IgG concentrations of 5–10 ng/ml, recognized HIV-1 proteins by ELISA (Fig. 3Goa). This reactivity was specifically displaced by the related phagotopes, but not by wild-type or unrelated phages (Fig. 3Gob, and data not shown). Moreover, peptides corresponding to the epitopes displayed on phages p195, p197, and p335 effectively displaced the binding of Abs to HIV-1, indicating that these peptides acquire in solution a conformation similar to the one expressed by both the phage-displayed peptides and the HIV epitopes (Fig. 3Goc). Only a partial competition was observed in the case of peptide 217 and 287, indicating that expression of these peptides on the surface of the phages is essential to acquire a conformation mimicking HIV epitopes (Fig. 3Goc), as previously suggested (27). When the phagotope-specific Abs were tested in immunoblotting, a distinctive reactivity was found. Ab-195 and Ab-217 recognized gp160 and gp120, consistent with the mapping of these epitopes at envelope regions. In addition, Ab-287 and Ab-335 also detected HIV-gp160 and gp120, indicating that they recognized envelope-specific epitopes (Fig. 3God). No bands were detected by p197-specific Abs, indicating an intrinsic inability of these Abs to bind to the cognate epitope under denaturing conditions (Fig. 3God).



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  		FIGURE 3. Phagotope-specific Abs bind to HIV-1. a, ELISA reactivities of immunoaffinity-purified Abs with HIV-1. Abs were immunoaffinity-purified from LTNP serum 6090 using single phagotopes as ligands and tested for ELISA reactivity against HIV-1 virions by using a standard ELISA kit (Organon, West Orange, NJ). Purified Abs were tested at 5–10 ng/ml; HIV-negative (CS) and serum 6090 (HIV-1S) were tested at 1:100 dilution. Data are expressed as mean ± SEM of four independent determinations. b, The binding of phagotope-specific Abs to HIV-1 is specifically displaced by the related phagotopes. ELISA reactivities of single immunopurified Abs to HIV-1 were tested in the presence of the indicated concentrations of p195 ({blacksquare}), p197 ({diamondsuit}), p217 (•), p287 ({blacktriangleup}), and p335 ({blacktriangledown}). The binding of each Ab to HIV was also tested in presence of increasing concentrations of wild-type phages (open symbols). c, Displacement of HIV-1 binding by peptides corresponding to the phage-displayed epitopes shown in Fig. 2Go, a and c. ELISA reactivities of single immunoaffinity-purified Abs with the related phagotopes were tested in the presence of increasing concentrations of peptides 195 ({blacksquare}), 197 ({blacktriangleup}), 217 ({diamondsuit}), 287 ({square}), and 335 (•). d, HIV-1 immunoblotting with phagotope-specific human Abs. Immunoaffinity-purified Abs were tested at 60 ng/ml for binding to HIV-1 proteins in Western blot analysis by a diagnostic kit (Aquila Biopharmaceuticals, Framingham, MA); 6090 LTNP serum was tested at 1:1000 dilution.

 
HIV-1 mimotopes react specifically with sera of SHIV-infected monkeys

SHIV recombinant viruses expressing HIV env on the backbone of SIV isolates are a useful model of HIV-1 infection in primates (28). SHIV-infected monkeys raise high titers of neutralizing Abs that correlate with long-lasting protection from subsequent challenge with pathogenic SHIV (29) or SIV-mac239 (30). Since the HIV-specific phagotopes are immunogenic mimics of HIV-1 env proteins ( Figs. 1–3GoGoGo), they should be recognized by Abs of SHIV-infected animals. To test this hypothesis, sera of nine SHIV-infected monkeys and of four uninfected control animals were tested for ELISA reactivity with the pool of HIV mimotopes. As in the case of HIV-1-infected subjects (Fig. 1Goa), sera of SHIV-infected monkeys recognized the HIV-1 phagotopes with variable frequencies (Fig. 4Go). As previously noted, phagotopes p32, p54, and p689 did not match any HIV sequences in the database (Fig. 2Goc). The fact that certain SHIV sera recognized these phagotopes suggests that they are conformational mimics of discrete regions of gp160, Nef, Tat, or Vpu, since these are the only HIV-specific sequences within SHIV. Phagotope p163 and p483, which were consistently recognized by LTNP and AIDS sera (Fig. 1Goa), did not react with SHIV sera, suggesting that they are antigenic mimics of HIV-1 epitopes encoded for by gag or pol genes. Sera from uninfected monkeys tested negative in ELISA (Fig. 4Go).



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  		FIGURE 4. ELISA reactivities of monkey sera with HIV-specific phagotopes. Sera of naive monkeys (SHIV Negative) and SHIV-infected animals were tested for binding to HIV-1 phagotopes. Results are expressed as fold increase of OD405 nm values of tested phagotope over the OD405 nm values of wild-type phage. Cutoff values were set as detailed in the legend to Fig. 1Goa. All the preinfection sera of SHIV-positive animals tested negative by ELISA (data not shown). A125, 42C, E50, and AK98 are Rhesus macaques; 4138, 4150, and 79 are cynomologous macaques; these animals were infected with SHIVMD1 (38). 17860 and 17846 are pigtail macaques infected with SHIVMD14YE (38).

 
HIV-1 mimotopes induce neutralizing Abs in mice

HIV-1-binding Abs may exert neutralizing activity in vitro if directed to accessible epitopes of infectious virions. As antigenic mimics of HIV-1 epitopes, HIV-1 phagotopes have a conformation that fits in the Ag-binding site of the related serum Abs, and would be expected to elicit Abs in vivo with specificities similar to the original serum IgG utilized to select them. To test this possibility, HIV-1 phagotopes p195, p197, p217, p287, and p335 were used to immunize BALB/c or C57B/6 mice. All mice developed comparable titers of Abs against wild-type phages and a strong Ab response to the original phagotopes used as immunogens (data not shown). Purified IgG from mice immunized with HIV-1 phagotopes exerted a significant inhibition of infection by HIV-1IIIB and NL4-3 isolates over a wide range of IgG concentrations in an in vitro acute infection system, with 50% protection observed at IgG concentrations of 0.8–3 µg/ml in neutralization assays with HIVIIIB or NL4-3, respectively (Fig. 5Go, a and b). Consistent levels of viral neutralization were also obtained in the case of the AD8 primary isolate, with the exception of p335-specific Abs, which exerted partial protection only at the highest concentrations (Fig. 5Goc).



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  		FIGURE 5. Assays of the neutralization of HIV-1 by phagotope-specific Abs. C57B16 mice were immunized with either wild-type phages ({square}) or with p195 ({blacksquare}), p197 ({circ}), p217 (•), p287 ({triangleup}), or p335 ({blacktriangleup}). IgG were purified from immunized mice and tested for inhibition of HIVIIIB (a) or NL4-3 (b) infection in the MT2 assay (20). Neutralization of AD8 infection was performed on PHA-activated PBMC (21) (c). Results are expressed as percentages of protection and are representative of three independent experiments. IgG from BALB/c immunized mice gave comparable results (not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The quest for a prophylactic HIV vaccine has been frustrated in part by the high variability of HIV envelope sequences and by its complex oligomeric structure. Furthermore, a vaccine formulation should take into account the subtype prevalence in different geographic regions as well as the insurgence of new HIV isolates (31). Combinatorial peptide libraries express a large collection of peptide sequences (108 or more) that mimic both linear and conformational epitopes of folded protein domains, and even carbohydrate structures (32) that contribute to the generation of viral escape variants (33). Such libraries might offer a unique opportunity to overcome the complexity of the HIV epitope repertoire by allowing the selection of a pool of HIV-specific mimotopes using HIV-specific Abs generated in representative cohorts of HIV-infected subjects. To this end, we screened phage-displayed RPLs and selected numerous epitopes that behaved as antigenic and immunogenic mimics of HIV epitopes generated in the natural course of infection in humans as well as in SHIV-infected monkeys. In further support of the HIV-specificity of the selected phagotopes, sera from six HIV-infected chimpanzees also recognized HIV phagotopes (G.S. et al., unpublished observations) with a pattern similar to the reactivities of human sera. Of interest, LTNP sera manifested higher Ab titers to certain epitopes compared with AIDS sera, suggesting that the immune response to these epitopes might afford some degree of protection against disease progression.

Although the above phagotopes were selected by using Abs from HIV-infected subjects likely carrying different isolates, the neutralization data shown in Fig. 5Go indicate that the specific Abs induced in mice bind to HIVIIIB, NL4-3, and AD8 virus isolates. This is consistent with the data shown in Fig. 3Go, where Abs that had been immunoaffinity purified from patients’ sera by using single epitopes as ligands reacted with HIVIIIB proteins in ELISA and in Western blot analysis (Fig. 3Go, a and d). This indicates that the epitopes displayed on the pVIII coat of the phages could mimic HIV-1 epitopes from different strains. This possibility is further supported by the data shown in Fig. 4Go, where epitopes reacted with Abs from monkeys infected with chimeric SHIVs displaying an envelope from DH12, a dual-tropic strain of HIV-1 (34). Moreover, as shown in Fig. 1Goa, epitopes selected for reactivity with a given serum were recognized by Abs from numerous subjects likely infected with different HIV-1 quasispecies. The above evidence underscores the capacity of a phage displayed peptide to mimic multiple HIV epitopes present in vivo on glycosylated gp120 without the constraint of a high sequence homology. Consistent with this possibility, phage mimotopes of hypervariable region 1 of hepatitis C virus induces Abs cross-reacting with a large number or viral variants (35). Although the selected phagotopes were recognized by most of the HIV-positive sera, it is unclear at present to what extent they represent the complexity of the epitope repertoire of HIV envelope proteins. In this regard, we are extending the pool of HIV-1 mimotopes by screening peptide libraries with additional HIV-positive sera.

The selected phagotopes fulfilled the requirement for an effective immunogen. In fact, Abs from phagotope-immunized mice neutralized HIV-1 strains in vitro, suggesting that they bind well to the virus under physiologic conditions and could possibly prevent or inhibit HIV infection when induced in phagotope-immunized primates. In support of this possibility, serum Abs of SHIV-infected monkeys showed a strong reactivity with the phage-displayed epitopes. In addition, bacteriophages are excellent immunogens that induce a specific T cell-dependent Ab response by parenteral as well as oral administration (36, 37).

Taken together, our results indicate that a collection of HIV-1 mimotopes can be retrieved from combinatorial phage libraries by taking advantage of the specific Ab repertoire induced by natural infection, and thus may be useful in the development of effective HIV-1 vaccines.


   Acknowledgments
 
We thank D. C. Montefiori for advice in viral neutralization assays; C. Allahan for statistical analysis; T.-W. Chun, J. Arthos, and B. Mathieson for critical reading of the manuscript; M. A. Martin for discussing and reviewing the manuscript; R. Cortese for providing the RPLs; P. Monaci, A. Nicosia, and C. Prezzi for helpful discussions; and P. Walsh for excellent editorial assistance.


   Footnotes
 
1 These authors contributed equally to this work. Back

2 Address correspondence and reprint requests to Dr. Giuseppe Scala, Laboratory of Immunoregulation/National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 6A08, 10 Center Drive, Bethesda, MD 20892-1576. E-mail address: Back

3 Abbreviations used in this paper: SHIV, simian HIV; RPL, random peptide libraries; LTNP, long-term nonprogressor; f, recognition frequency. Back

Received for publication December 21, 1998. Accepted for publication March 3, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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