AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 7, Number 3, 1991 Mary Ann Liebert, Inc., Publishers

Production of a «^/-Specific Monoclonal Antibody by the Use of a Synthetic Peptide RITA DE SANTIS,1 ANNA MARIA ANASTASI,1 STANISLAO MARCOLINI,1 GUIDO VALESINI,2 MARIO PEZZELLA,3 NICOLETTA VONESCH,3 ELENA STURCHIO,3 and ANTONIO MELE1

ABSTRACT Monoclonal antibodies have been generated against a synthetic peptide of the nef protein of human immunodeficiency virus type 1 (HIV-1) in order to further characterize the biochemical and functional nature of this protein and its role in the control of HIV-1 transcriptional regulation. Earlier studies indicated nef to be a negative regulatory factor for viral transcription, whereas more recent studies report evidence against this original hypothesis. Nef is a protein of 206 amino acids of approximately 27 kD in most HIV-1 isolates; however, in some other isolates a truncated form of 124 amino acids has been described. A peptide sequence of six amino acids, corresponding to a region of the nef protein exhibiting high-sequence homology to thymosin a, protein, has been synthesized by Merrifield solid-phase methodology. This peptide is coded by a sequence located upstream to the stop codon described in some HIV-1 isolates and then is maintained in both complete and truncated forms of the nef protein. F14.il is a «e/peptide-specific monoclonal antibody (IgG2a/k) exhibiting the ability to recognize natural nef protein in either radioimmunoassay, radioimmunoprecipitation assay, or immunocytochemical analysis. Since F14.il is able to identify nefprotein in the cytoplasm of lymphocytes from HIV-infected seronegative subjects it may prove useful in monitoring the expression of nef during the silent HIV-1 infection.

INTRODUCTION

The

basal transcription of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) is regulated by the interaction of several cellular and viral proteins.1 Replication of the virus is dependent on the functional expression of some small virus-coded regulatory proteins, such as tat and rev (formerly known as the artltrs) proteins. Tat gene is essential for high levels of viral gene expression and replication since in HIV-1 mutants lacking a functional tat, transcription is initiatied but viral RNA molecules do not elongate beyond the HIV-1 LTR. Rev is also required for HIV replication since mutations in rev abolish viral infectivity. The posttranscriptional action of rev selectively increases the levels of gag and env RNAs by activat-

ing the sequence-specific nuclear export of incompletely spliced HIV-1 RNA species. Nef gene (648 bp) begins in a different reading frame at the

end of the env gene sequence and extends into the 3' LTR where it terminates in the U3 region.2 In some HIV-1 isolates a premature termination codon truncates the gene product at 124 amino acids, whereas most other isolates are predicted to express a protein of 206 amino acids of approximately 27 kD.3 The p27 protein is N-terminal myristilated4 and shares structural homology with a highly charged region within the intracy-

toplasmatic phosphorylation

site of the catalytic subunit of cAMP-dependent protein kinase (cAMP-PK) and other members of the protein kinase family.5'6 It has been shown that frameshift or premature termination

'Department of Biotechnology, Menarini Ricerche Sud, Via Tito Speri, 10 00040 Pomezia, Rome, Italy, institute Clínica Medica I, University of Rome "La Sapienza", Rome, Italy. 'Institute of Infectious Diseases, University of Rome "La Sapienza", Rome, Italy. 315

domain of human interleukin-2

receptor (IL-2R) and the adenine triphosphate (ATP) -binding

316

DE SANTIS ET AL.

mutations in the nef gene lead to an increase in virus replication up to 100-380% compared with wild-type virus.7 The target sequence of nef action has been identified in the HIV-1 LTR between 156 and 340 nucleotides upstream of the RNA start site as a eis acting transcriptional inhibitory element previously designated negative regulatory element (NRE).8 The ability of nef to repress HIV-1 LTR transcription in a dosedependent manner suggests a possible role of nef during the silent period of HIV-1 infection. This hypothesis is also supported by the observation that transfection of the nef gene in infected T lymphocytes downregulates the expression of CD4

receptor.6

The recombinant p27 protein expressed in bacterial cells has been shown to react with human sera from patients at different stages of acquired immunodeficiency syndrome (AIDS), indicating that the protein product of nef is immunogenic in vivo.9 Nevertheless, two independent groups recently produced evidence against the negative regulatory role of nef on HIV-1 transcription and viral growth,,0- ' ' while others reported differential effects of nef on HIV-1 replication, supporting the hypothesis that nef could be important in the establishment of HIV-1

latency.12

The aim of our investigation was to generate a «¿/-specific monoclonal antibody by the use of synthetic peptide corresponding to the sequence 83-88 Ala-Ala-Val-Asp-Leu-Ser of the HIV-1 nef protein. The selected peptide shares homology with the sequence 3-8 Ala-Ala-Val-Asp-Thr-Ser of thymosin a, protein (Fig. 1). It is interesting that the 83-88 nef peptide is conserved in several nef amino acid sequences of HIV-1 as shown in Table 1, and is maintained in all truncated forms described (HXB2 123 aa, BH10 124 aa, BH8 152 aa). We used the ne/-specific monoclonal antibody to detect the presence of nefprotein in peripheral blood mononuclear cells (PBMC) from both HIV-1-infected subjects and their seronegative regular sexual partners. Our goal is to further investigate the role of nef in the control of HIV-1 replication and AIDS progression.

MATERIALS AND METHODS

Peptide synthesis and carrier conjugation The

peptide corresponding to

the 83-88

(Ala-Ala-Val-Asp-

Leu-Ser) sequence of nef protein was synthesized by Merrifield

solid-phase methodology using 4-methylbenzhydrylamine resin produce a peptide with C-terminal amide.13 T-Boc-amino acids were used and side-chain protecting groups were benzyl for serine and threonine cyclohexyl ester for aspartic acid. Each coupling reaction was performed by preformed symmetric anhydride. At the end of the synthesis, the peptide was deprotected and cleaved from the resin by treatment with hydrogen fluoride. The crude peptide was purified by gel chromatography and then to

Ala Ala Val

83-88 Net

3-8

Thymosin

FIG. 1. mosin a,

Asp

Leu Ser

Thr

ai .

Sequence homology peptide.

between

—

nef peptide

—

and

thy-

Table 1 HXB2 BH10 BH8 PCV12 PV22 BRU RF CDC4 Z6 SF2 MAL ELI Z321

ALA-ALA-VAL-ASP-LEU-SER ______

______

______

______

______

GLY LEU

______

_____

LEU PHE LEU PHE

ILE

-----

GLY GLU GLY —

—

-

-

-

—

—

-

-

—

-

—

—

-

-

-

Source: Human retrovirus and AIDS (1988); Los Alamos National Lab, Los Alamos, NM 87545.

by reverse-phase high-performance liquid chromatography (HPLC) with a final purity greater than 98%. Nef'peptide was chemically conjugated to either bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) by A'-ethyl-Ar'-(3-dimethyl aminopropyl)-carbodiimide hydrochloride (EDCI) or glutaraldehyde, respectively. A'e/peptide-KLH and Ne/peptide-BSA conjugates have been produced for mice immunization and hybridoma screening, respectively, in order to avoid the generation of clones possibly reacting with either carriers or cross-linking reagents.

Hybridoma production Monoclonal antibodies to nef protein were obtained by immunizing 2-month-old Balb/c mice with 50 p,g of ne/peptide-KLH in Freund's complete adjuvant intraperitoneally 15 and 8 days before fusion. Three days before fusion, mice were boosted intraperitoneally with 50 p.g of «e/peptide-KLH in saline. Two days and one day before fusion, 15 p,g of «e/peptide-KLH in saline was injected in equal amounts both intraperitoneally and intravenously. Fusion was performed as previously described,14 briefly, 107 immune spleen cells were mixed with 106 NSO/1 myeloma cells and fusion was allowed to proceed for 1 minute at 37°C in 0.5 ml of 50% polyethylene glycol-1450 (SERVA) in RPMI-1640 medium. After 10 days culture in the presence of hypoxanthineaminopterin-thymidine (HAT) selective medium, those cell populations reacting in RIA test with nef peptide-BSA conjugate, were cloned by limiting dilution in hypoxanthine-thymidine (HT) medium. /Ve/peptide-positive clones were expanded and injected in pristane-treated mice in order to obtain large

quantities of monoclonal antibodies. Monoclonal antibodies were purified to homogeneity from ascitic preparations by both Protein A affinity chromatography and hydroxylapatite chromatography on a MAPS-100 HPLC system (BioRad). Isotype determination was performed by streptavidin-biotin system/ Mono Ab-ID ElA Mouse Kit (Zymed Lab, Inc.). Monoclonal

antibody titration

In order to establish the reactivity of different hybridoma clones on nef native protein, titration curves have been per-

nef PROTEIN STUDIES

formed on microtiter plates coated with HIV-1-infected H9 cell lysates. Serial dilutions of purified F14.il immunoglobulin or immunoglobulin with irrelevant specificity were incubated for 1 h at 37°C, followed by three washes in PBS/0.05% Tween-20. Immunoglobulin binding was shown by the incubation for 1 h at 37°C with a secondary 125I-labeled goat antimouse immunoglo-

bulin

(8

x

317

BY MONOCLONAL ANTIBODIES

104cpm/well).

Antibody cross-reaction with 3-8 thymosin

a¡

peptide

To investigate the eventual cross-reaction of F14.11 immunoglobulin with 3-8 thymosin a, peptide, competition curves have been performed on microtiter plates coated with 83-88 nef peptide-BSA (2pg/well). 125I-labelled F14-11 monoclonal antibody (1 x 105 cpm/

was incubated for 2 h at 37CC in the presence of serial dilutions of 83-88 nef peptide-BSA or 3-8 thymosin a¡ peptideBSA or BSA alone.

well)

Immunoprecipitation HIV-1-infected H9 cell lines were metabolically labeled with [32P]orthophosphoric acid (NEN) and detergent solubilized. Cell lysates were precleared by overnight incubation with rabbit antimouse Ig/Protein A-sepharose beads (Pharmacia). Next, 10 x 106cpmof the precleared supernatant was incubated with 20 p,L of anti-ne/monoclonal antibody ascitic fluid or with 20 u,g of either F14.il purified immunoglobulins or aspecific monoclonal antibody. The immunocomplexes were precipitated by rabbit antimouse Ig Protein-A sepharose beads and washed 10 times with RIPA washing buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.03% NaN3, 0.25% NP40, 1% SDS, 0.1% Na deossicolate, 0.1% BSA). The bound proteins were boiled in electrophoresis sample buffer and analyzed by 12.5% polyacrylamide gel electrophoresis; the gel was dried and autoradiographed.

Immunocytochemical detection of p27 nef protein Peripheral blood mononuclear cells (PBMCs) from six antiHIV-1 seropositive subjects (enzyme-linked immunosorbent assay and Western blot confirmed) and their seronegative regular heterosexual partners, previously found to be HIV-1 infected by "in situ" hybridization with DNA probe,1516 have been studied. Peripheral blood mononuclear cells were separated from heparinized blood samples on Ficoll-Hypaque gradients. Samples of 105 cells were cytocentrifuged onto microscope slides, air-dried, fixed in acetone for 5 minutes, and stored at -20°C until use. The cells were incubated for 1 h at room temperature with blocking buffer (50 mM Tris-HCl pH 7.5, EDTA 1 mM, Tween-20 0.3%, and powdered skimmed milk 25%) to prevent nonspecific binding, washed for 5 minutes with washing solution (0.09% Brij and 0.5 M NaCl) and incubated for 1 h at room temperature with 2.5 pg of anti-ne/monoclonal antibody in 25 pL of blocking buffer. Cells were then washed three times for 5 minutes with washing solution to remove the antibody excess and 25 pL of alkaline phosphatase-conjugate antimouse immunoglobulin (1:100 in blocking buffer) was added. After 1 h at room temperature, cells were washed three times for 5 minutes with washing solution and twice with a buffer pH 9.5 (100 mM Tris-HCl, 1 M NaCl and 5 mM MgCl2). Immunostaining was performed using a mixture of nitro blue tetrazolium and 5-bromo-4-cloro-3-indolyl phosphate. A solution of 5 mM levamisole was added to the chromogenic substrate solution to inhibit the activity of endogenous cellular alkaline phosphatase. HIV-1-infected and noninfected H9 T-cell lines and PBMCs from six healthy blood donors were used as positive and negative controls.

RESULTS Several peptide-reactihg monoclonal antibodies have been obtained by PEG-mediated somatic hybridization of immune spleen cells and NSO/1 myeloma cells. In order to establish the reactivity of different clones on nef viral protein, titration curves of purified monoclonal antibody or purified normal mouse immunoglobulin have been performed on microtiter plates coated with HIV-infected cell lysates. The binding of antibodies is detected by a secondary 125I-labeled goat antimouse immunoglobulin. The results shown in Figure 2 indicate that the nef peptidespecific F14.il clone exhibits the ability to specifically react in a dose-dependent manner on HIV-1-infected cell lysates. To further investigate the F14.11 reactivity, we tested it in a RIPA assay. HIV-1 -infected cells were metabolically labeled by 32P, lysed and incubated with antibody; this was either 20 pL of ascitic fluid or 20 p,g of purified F14.11. A monoclonal antibody with irrelevant specificity was used for negative control experiments. Lanes 1 and 2 of Figure 3 show a protein band of approximately 27 kD immunoprecipitated by purified F14.il (lane 1) or F14.il ascitic fluid (lane 2) while any band is immunoprecipitated by the purified aspecific monoclonal antibody (lane 3). F14.11 has been used for the immunocytochemical study of either HIV-1-infected/uninfected T-cell lines or PBMCs from HIV-1-infected seropositive subjects and from their seronegative regular sexual partners. Strong intracytoplasmatic granular staining has been observed in HIV-1-infected H9 cell lines (Fig. 4a) as well as in PBMCs from three HIV-1-infected seropositive subjects and from two seronegative regular sexual partners. Typical nef+ PBMCs from seropositive subjects and their seronegative partners, exhibit morphological detail consistent with that of lymphocytes and are given in Figure 4b and c,

respectively. F14.il immunocytochemical reactivity appears to be specific since it reacts with neither HIV-1 noninfected cell lines (Fig. 4d) nor with several cytocentrifuged PBMCs from healthy notat-risk donors. Monocytelike-positive cells were observed only in the three seropositive subjects (Fig. 4e). To investigate the eventual F14.il antibody cross-reactivity with thymosin a, peptide, competition curves were performed on microtiter plates coated with nef peptide-BSA. The results shown in Figure 5 indicate that the F14.11 monoclonal antibody does not recognize thymosin otl, since its binding on 83-88 nef peptide is not inhibited by nef-homologous 3-8 thymosin a¡ peptide.

318

DE SANTIS ET AL. 4000

-i

D

F14 11

•

Aspecific

Mab

>-

10

Ig ug/ml FIG. 2.

Titration

curve

of F14.11

o or

aspecific

DISCUSSION It is known that HIV-1 establishes a persistent infection among infected subjects. On the basis of clinical and epidemiológica! studies, it has been observed that the "window" period

69

-

-

46

30

-

21.5

-

14.3

monoclonal antibodies

(lane 3).

HIV-infected cell

lysate.

between the appearance of detectable specific antibodies and the production of high levels of HIV-1 core p24 antigen is currently projected to be in the range of 4 years.17 As a consequence of this, the routinary diagnosis of HIV-1 infection in high-risk individuals, based on demonstration of specific serum antibodies against structural viral proteins, may be not fully predictive of the infectious state. The recent evidence of silent HIV-1 infection for up to 36 months after positive culture, in a cohort of homosexual men18 and the transmission of HIV-1 from blood donors screened as seronegative at the time of donation19 suggests that silent infection seems to pose a very important problem in both public health and medical ethics. In such very high-risk groups as homosexual men and regular sexual partners of seropositive subjects, the absence of specific antibody is not fully predictive of the HIV-1 infectious state.20 In this situation, when screening high-risk individuals for HIV-1 infection, it may be necessary to use high-resolution molecular techniques as "in situ" hybridization and gene amplification by polymerase chain reaction for the detection of HIV-1 genotype.21 Conflicting observations concerning the role of HIV-1 nef gene have been reported. Several groups reported that nef exerts a down-regulatory effect upon virus replication.5-8 Others have shown evidence against the negative regulatory role of nef on HIV-1 transcription and viral growth,10- ' ' while others reported a possible modulatory effect of nef on HIV-1 replication, supporting the hypothesis that rce/could be important in the establishment of HIV-1 latency.12 In order to better understand the role of nef in HIV-1 pathogenetic mechanisms we have generated a nef peptidespecific monoclonal antibody found to be able to specifically recognize the natural viral protein in RIA, RIPA, and immuno-

cytochemical analysis.

FIG. 3. Radioimmunoprecipitation of nef protein by purified F14.11 (lane 1), F14.11 ascitic fluid (lane 2), or aspecific M Ab

on

Moreover, F14.il monoclonal antibody shows no crossreactivity with ««/-homologous 3-8 thymosin a, peptide. It has been previously shown that «e/-specific antibodies are present in HIV-1-infected subjects, confirming that nef is immunogenic in

nef PROTEIN STUDIES

BY MONOCLONAL ANTIBODIES

E

319

WÊÊBKÊÊÊÊÊÊÊÊÊÊÊIÊÊIÊÊSÊÊÊÊÊÊÊÊÊÊÊÊÊÊ

FIG. 4A.

Immunocytochemical staining of HIV-1-infected H9 cell line with F14.11 MAb (x 1000).

FIG. 4B.

Immunocytochemical staining of PBMCs from a HIV-1-infected seropositive patient with F14.11 MAb (x 1000).

FIG. 4C.

Immunocytochemical staining of PBMCs from a seronegative HIV-1-infected patient with F14.11 MAb (xlOOO).

FIG. 4D.

Immunocytochemical staining of HIV-1 noninfected cell line with F14.il MAb ( x 1000).

FIG. 4E.

Immunocytochemical staining of a monocytelike cell from a HIV-1-infected seropositive patient with F14.11

(X1000).

MAb

320

DE SANTIS ET AL.

40000

n

__« 30000 --

20000

-•-

(M

- -©-

10000 H

BSA 3-8

pept.-BSA 83-88 pept.-BSA F14.11 Ig

Q. Ü

-i-1-1-1-1-1-1-1-1-1-1-1

2

4

6

12

10

8

Ig ug/ml FIG. 5.

Competition curve of 125I F14.11 with nefpeptide

the natural host22 and suggesting its possible role in the progression to AIDS through the inactivation of the HIV-1 negative regulatory protein during the early stage of infection. This hypothesis is also supported by the recent observation that HIV-1-infected seronegative subjects exhibit higher ne/immunoreactivity than seropositive subjects.23 Since F14.il ne/spécifie monoclonal antibody is able to identify the protein in the cytoplasm of PBMCs from HIV1-infected seronegative subjects at risk for AIDS, we believe it could represent a valuable tool for monitoring the expression of nef, especially during the period of silent infection. The search for nonstructural proteins during silent infection could be a further tool for developing new diagnostic strategies.

ACKNOWLEDGMENTS We thank Dr. Maurizio Federico, Virology Istituto Superiore di Sanità, Rome, for the kind 1-infected cells.

Laboratory, gift of HIV-

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B

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BSA

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Ig

O.

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1987;330:266-269.

7. Ahmad N and Venkatesan S: Nef protein of HIV-1 is a transcriptional repressor of HIV-1 LTR. Science 1988;241:1481-1485. 8. Rosen CA, Sodroski JG, Haseltine WA: The location of eis acting regulatory sequence in the human T cell lymphotropic virus type III (HTLV III)/LAV long terminal repeat. Cell 1985;41:813-823. 9. Franchini G, Robert-Guroff M, Wong-Staal F, Ghrayeb J, Kato I, and Chang TW: Expression of the protein encoded by the 3' open reading frame of human T cell lymphotropic virus type HI in bacteria: demonstration of its immunoreactivity with human sera. Proc Nati Acad Sei (USA) 1986;83:5282-5285. 10. Kim S, Ikeuchi K, Byrn R, Groopman J, and Baltimore D: Lack of a negative influence on viral growth by the nef gene of human immunodeficiency virus type 1. Proc Nati Acad Sei (USA)

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11. Hammes

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transcriptional

inhibitor. Proc Nati Acad Sei

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Differential effects of nef on HIV replicatiomimplications for viral pathogenesis in the host. Science 1989;246:1629-1632. 13. Merrifield RB: The synthesis of tetrapeptide. Jam Chem Soc

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to:

Dr. Rita De Santis Menarini Ricerche Sud

Department of Biotechnology Via Tito Sped, 10 00040 Pomezia

Rome, Italy