antibodies to recombinant hiv-1 vif, tat, and nef proteins in human sera

11
Med Microbiol Immunol (1990) 179:1-11 Springer-Verlag 1990 Antibodies to recombinant HIV-1 vif, tat, and nef proteins in human sera Ulrike Wieland l, Joachim E. Kiihn 1, Christian Jassoy 1, Helga Riibsamen-Waigmann 2, Vera Wolber 3, and Riidiger W. Braun 1 1Institut fiir Medizinische Virologic, Universit~it Heidelberg, Im Neuenheimer Feld 324, D-6900 Heidelberg, Federal Republic of Germany 2 Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42-44, D-6000 Frankfurt, Federal Republic of Germany 3 Max-Planck Institut ftir Medizinische Forschung, Jabnstrasse 29, D-6900 Heidelberg, Federal Republic of Germany Abstract. The prevalence of antibodies against HIV-1 regulatory proteins in sera of HIV-infected patients from different stages of disease was investigated. HIV-1 vif, tat, and nef genes were cloned in procaryotic vectors and were expressed as MS-2 fusion proteins (vif and nef) or as a non-fusion protein (tat). These recombinant proteins were employed in immunoblot experiments. The specifity of the recognition was confirmed by competition experiments and with control sera from HIV-negative patients. Analysis of 136 serum samples revealed a high percentage of antibodies against nef, irrespective of the stage of disease. Antibodies against tat were found less frequently and increased from 16 % to 40 % with disease progression. Vif antibodies were detected only in a low percentage in early stages of disease, but their prevalence increased to 36 % and 72% with progression of disease to AIDS-related complex and AIDS. Our data suggest that the detection of antibodies against nef may represent an additional and useful marker for the diagnosis of HIV infection, whereas the detection of vif antibodies may indicate disease progression. Introduction The serological diagnosis of HIV infection is based on the demonstration of antibodies against viral structural proteins detected by enzyme-linked immunosor- bent assay or by immunoblot. From several previous publications, however, it is evident that nonstructural viral antigens may also elicit a humoral immune response (Kfihn et al. 1987; Allan et al. 1985). This may be valuable for the diagnosis of HIV infection. Kan et al. (1986), Lee et al. (1986) and Sodroski et al. (1986) have demonstrated antibodies against vif in HIV-infected patients and Offprint requests to: U. Wieland

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Page 1: Antibodies to recombinant HIV-1 vif, tat, and nef proteins in human sera

Med Microbiol Immunol (1990) 179:1-11

�9 Springer-Verlag 1990

Antibodies to recombinant HIV-1 vif, tat, and nef proteins in human sera

Ulrike Wieland l, Joachim E. Kiihn 1, Christian Jassoy 1, Helga Riibsamen-Waigmann 2, Vera Wolber 3, and Riidiger W. Braun 1

1 Institut fiir Medizinische Virologic, Universit~it Heidelberg, Im Neuenheimer Feld 324, D-6900 Heidelberg, Federal Republic of Germany 2 Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42-44, D-6000 Frankfurt, Federal Republic of Germany 3 Max-Planck Institut ftir Medizinische Forschung, Jabnstrasse 29, D-6900 Heidelberg, Federal Republic of Germany

Abstract. The prevalence of antibodies against HIV-1 regulatory proteins in sera of HIV-infected patients from different stages of disease was investigated. HIV-1 vif, tat, and nef genes were cloned in procaryotic vectors and were expressed as MS-2 fusion proteins (vif and nef) or as a non-fusion protein (tat). These recombinant proteins were employed in immunoblot experiments. The specifity of the recognition was confirmed by competition experiments and with control sera from HIV-negative patients. Analysis of 136 serum samples revealed a high percentage of antibodies against nef, irrespective of the stage of disease. Antibodies against tat were found less frequently and increased from 16 % to 40 % with disease progression. Vif antibodies were detected only in a low percentage in early stages of disease, but their prevalence increased to 36 % and 72% with progression of disease to AIDS-related complex and AIDS. Our data suggest that the detection of antibodies against nef may represent an additional and useful marker for the diagnosis of HIV infection, whereas the detection of vif antibodies may indicate disease progression.

Introduction

The serological diagnosis of HIV infection is based on the demonstration of antibodies against viral structural proteins detected by enzyme-linked immunosor- bent assay or by immunoblot. From several previous publications, however, it is evident that nonstructural viral antigens may also elicit a humoral immune response (Kfihn et al. 1987; Allan et al. 1985). This may be valuable for the diagnosis of HIV infection. Kan et al. (1986), Lee et al. (1986) and Sodroski et al. (1986) have demonstrated antibodies against vif in HIV-infected patients and

Offprint requests to: U. Wieland

Page 2: Antibodies to recombinant HIV-1 vif, tat, and nef proteins in human sera

2 u. Wieland et al.

Krone et al. (1988) found antibodies against tat. Antibody responses against vif, tat, and nef were previously described by Arya and Gallo (1986) and by Franchini et al. (1987). Recently, Ameisen et al. (1989a,b) have shown that antibodies against nef may precede overt seroconversion of HIV-infected patients and, thus, may serve as an early marker for HIV infection. Only few attempts have been made to correlate the occurrence of antibodies against several nonstructural proteins with the different stages of HIV infection in a large collective of patients.

To evaluate the diagnostic significance of antibodies against three HIV-1 nonstructural proteins, we have compared the prevalence of antibodies against recombinant vif, tat, and nef in 95 sera of HIV-infected patients in different stages of disease.

Materials and methods

Cloning and expression of HIV-1 vif, tat and nef in recombinant plasmids. The vif gene was derived from the recombinant plasmid pBH10-R3 (kindly provided by R. C. Gallo, NIH, Bethesda, USA). The 1.1 kb EcoRI fragment (bp 4,228-5,323) containing the vif gene was inserted into the pUC18 polylinker, resulting in a plasmid named pUC18-S1. Digesting the EcoRI fragment with Sau3AI resulted in a 440-bp fragment comprising bp 4,608 to 5,047. This fragment was inserted in the BamHI polylinker site of pUC18, which resulted in a loss of the respective 5' BamHI site. The construct containing the correct orientation of the fragment was recut with BamHI and EcoRI and, to obtain the complete vif gene, the 276-bp Sau3AI/EcoRI fragment of pUC18-S1 was inserted, resulting in pUC18-S3. Finally the SalI/EcoRI fragment of the latter construct was inserted into the expression vector pEx34C: the complete vif gene was expressed as a fusion protein, containing the first 99 amino acids of the MS-2 polymerase at its amino terminus. Expression occurred in the E.coli K537 after a temperature shift from 29~ overnight to 42~ for 3h, thus activating the lambda-pL promotor of pEx34 by inactivating its thermolabile repressor cI (Klinkert et al. 1988; Remaut et al. 1981; Strebel et al. 1986) (Fig. 1A).

A nef gene comprising fragment was excised via the BamHI/Hind-III restriction sites from pNL4-3 (Adachi et al. 1986) and inserted into pUC18. The insert was shortend at its 5' end by cleavage with BamHI/XhoI and blunt-end ligation. The EcoRI/Hind-III nef fragment, lacking 102 nucleotides at the 5' end was cloned into the above-mentioned expression vector pEx34B. Nef was expressed as a truncated protein, missing the first 34 amino-terminal amino acids (Fig. 1B).

Tat coding DNA was derived from the c-DNA clone pCV-1 (Arya et al. 1985). pCV-1 was cut with Sau3A and the 301-bp tat fragment was ligated into EcoRI/ BamHI cut M13mp9. The missing bp of the tat sequence were regenerated with synthetic oligonucleotides, so that the sequence upstream of the ATG was identical to the p21 expression vector ptac ras 32 (Tucker et al. 1986). Tat was excised from M13mp9 with EcoRI/PstI and ligated into EcoRI/PstI cut ptac ras 32 to produce ptac-tat. IPTG induction of ptac-tat lead to the expression of a complete non- fusion protein (Fig. 1C).

Page 3: Antibodies to recombinant HIV-1 vif, tat, and nef proteins in human sera

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Page 4: Antibodies to recombinant HIV-1 vif, tat, and nef proteins in human sera

4 u. Wieland et al.

To assure correct transcription and translation of the inserted open reading frames, the joint sequences between vector and insert as well as the inserted fragments were controlled by dideoxy-chain-termination sequencing after subclo- ing in M13 (Sanger et al. 1977). Sequence analysis revealed correct transition from the bacterial sequences to the inserted open reading frames, and no base changes which would affect the amino acid sequence of the expressed proteins were observed.

Protein Purification. After expression the vif and nef fusion proteins were partially purified from bacterial proteins by differential urea extraction (Kfipper et al. 1982; Klinkert et al. 1988). Both fusion proteins were enriched in the supernatant after 7 M urea extraction, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie blue staining (Neuhoff et al. 1985). The vif fusion protein was not as soluble as the nef fusion protein and, thus, was also found in the non-soluble fraction after urea extraction.

The tat protein was partially purified by applying the supernatant of the cell lysate from a 61 culture to a 300 ml Q-sepharose column (Pharmacia, Freiburg, FRG). The column was washed with 0.35 M NaC1 and the tat-protein was eluted with 1.5 M NaC1.

SDS-PAGE andimmunoblotting. SDS-PAGE was performed basically as described previously (Thomas and Kornberg 1975). Proteins were seperated on discontinu- ous non-gradient 17% SDS-polyacrylamide gels of 0.75 mm thickness that were run at 4~ to a total of approximately 1,200 volt hours (Vh). As a molecular weight marker a low-molecular weight calibration kit that contains six standard proteins from 14 to 94 kDa was used (Pharmacia).

For immunoblotting the proteins seperated by SDS-PAGE were electropho- retically transferred to polyvinylidene difluoride membranes (Immobilon PVDF, Millipore, Eschborn, FRG) at 80 V for 2 h (Towbin et al. 1979; Kfihn et al. 1988a,b). Following transfer, the PVDF-membranes were blocked with 4% bovine serum albumin (Sigma, Deisenhofen, FRG), 2% non-fat dried milk powder (Fink/Frema, Herrenberg, FRG), 1 mM phenylmethylsulfonyl-fluoride (PMSF), 0.01% merthiolate in 50 mM Tris-HCL, 150 mM NaC1, pH 7.4 (TBS) for 2 h at 37~ At the same time patient sera were preabsorbed to E. coli K537 tysate (1:100) at 37~ After blocking the PVDF-membranes were washed with TBS containing 0.5% BSA, 0.01% merthiolate, 1 mM PMSF and incubated with the preabsorbed patient sera at a final dilution of 1:200 at 4~ overnight. Binding of the first antibody was detected by incubation (37~ 1 h) with a biotinylated goat anti-human IgG (Fc-specific) (Medac, Hamburg, FRG) following a 1-h incubation with horseradish peroxidase-streptavidin complexes (Amersham, Braunschweig, FRG) at room temperature at a dilution of 1:400, respectively (Guesdon et al. 1979). Finally membrane-bound peroxidase activity was visual- ized by adding substrate solution containing 0.5 mg/ml 4-chloro-l-naphtol (Sigma, Deisenhofen, FRG) and 0.03% H202 in TBS pH 7.4. The reaction was allowed to proceed until background staining developed. Between all above- mentioned steps washing with 0.5% BSA, 1 mM PMSF, 0.01% merthiolate in TBS, pH 7.4 was performed.

Page 5: Antibodies to recombinant HIV-1 vif, tat, and nef proteins in human sera

Antibodies to HIV-1 vif, tat, and nef 5

For competition experiments lysates of mock-infected or HIV-l-infected H9 cells were prepared as follows: 5 Xl07 cells were washed three times in PBS (pH 7.2), resuspended in a small amount of PBS, briefly boiled and finally frozen (-20 ~ C) and thawn three times. Sera were preabsorbed with the above lysates for 2 h at 37~ After centrifugation the supernatants were employed in immunoblot experiments as described above.

Serum samples. Ninety-five serum samples were obtained from HIV-l-infected patients who attended the University Clinics of Heidelberg (FRG) or the Georg- Speyer-House in Frankfurt (FRG) between 1987 and 1989. The sera of the HIV-1- infected patients were divided in four major groups, according to the individual patient's stage of disease: asymptomatic HIV infection, lymphadenopathy syn- drome (LAS), AIDS-related-complex (ARC) and AIDS. Additionaly six serum samples from HIV-2-infected patients were investigated. Thirty-five sera of randomly selected patients without risk factors and without clinical and laboratory findings indicative of HIV infection served as negative controls. All sera were routinely screened for the presence of antibodies against HIV-1 and HIV-2 (Abbott Recombinant HIV-1/HIV-2 EIA, Abbott Diagnostic Products, Wiesbaden, FRG) and the ELISA-positive results were confirmed by Western Blot (HTLV-III Western Blot IgG, Du Pont, Bad Homburg, FRG).

Results

Cloning of the HIV-1 open reading frames of vif and nef in pEx34 resulted in the expression of fusion proteins, which contained the first 99 amino acids of the MS-2 polymerase at their amino terminus. In the case of vif, the MS-2 sequence was followed by the complete vif open reading flame as deduced from sequence analysis of pEx34 vif in M 13. In the case of nef, the MS-2 protein was followed by the nef open reading frame except for the 34 amino-terminal amino ac ids (Fig. 1A,B). As shown in Fig. 2, both proteins migrated in SDS-PAGE in close agreement with their predicted molecular weight at 33 kDa (vif) and 35 kDa (nef). Tat was cloned as a nonfnsion protein in the vector ptac and migrated at 14.5 kDa (Figs. 1C,2).

The recombinant vif, tat, and nef proteins were used to screen 136 human serum samples from HIV-infected and negative control patients for antibodies against vif, tat, and nef. Sera were divided in six groups: set 1 comprised 35 sera from seronegative patients, serving as a negative control. Set 2 comprised 6 sera from HIV-2-positive patients, and sets 3 to 6 comprised 95 serum samples from HIV-1- infected patients in the different stages of disease: asymptomatic HIV infection (37 sera), LAS (22 sera), ARC (11 sera), or AIDS (25 sera). All sera were screened in immunoblot experiments with the three recombinant proteins. In addition, every blot contained one lane with MS-2 protein only, which served as an internal negative control. Examples of such blots are depicted in Fig. 3. In general no reactivity with the fusion proteins was detected with HIV-negative sera, whereas sera from HIV-positive patients showed an individual reaction pattern, recogniz- ing one, two, or all three recombinant proteins. A reactivity with the MS-2 protein

Page 6: Antibodies to recombinant HIV-1 vif, tat, and nef proteins in human sera

6 U. Wieland et al.

Fig. 2. Expression of recombinant HIV-1 vif, tat and nef. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Coomassie staining) of partially purified vif (lane 2), tat (lane 3) and nef (lane 4) proteins. In lane 1 pEx34 negative control lysate is shown. Arrows indicate the positions of the respective proteins

Fig. 3. Immunoblot experiments: reactivity of three serum samples from HIV-infected patients with recombinant vif, tat and nef proteins. Lanes represent reactivity with pEx34 control lysate (1), with vif (2), with tat (3) and with nef (4). From left to right the serum samples were obtained from an asymptomatic patient, a patient with AIDS-related complex and a patient with AIDS

Page 7: Antibodies to recombinant HIV-1 vif, tat, and nef proteins in human sera

Antibodies to HIV-1 vif, tat, and nef 7

Fig. 4. Antigen competition of recombinant vif, tat and nef. Panel 1 shows the reactivity of recombinant HIV-1 vif (lane 1), tat (lane 2) and nef (lane 3) with a serum sample collected from an AIDS patient. Preabsorption of the same serum to lysates of mock-infected H9 cells did not change the signal intensity (panel 2), whereas preabsorption of the serum to lysates of HIV- 1-infected H9 cells significantly reduced antibody binding in immunoblot experiments (panel 3)

alone could not be observed in the control or in the HIV-posit ive sera. To confi rm the specificity o f the immunob lo t reaction, compet i t ion experiments were per- formed. As shown in Fig. 4, preabsorpt ion o f sera to lysates of mock-infected H9 cells did not alter the reactivity with the recombinant proteins (panel 2). In contrast , preabsorpt ion o f sera to lysates o f HIV-l - infec ted H9 cells significantly reduced the observed an t ibody binding in immunob lo t experiments (panel 3).

In Table 1 the results o f the immunob lo t experiments are shown in detail: 86 % of the sera f rom HIV-posit ive patients reacted with the nef protein, regardless o f the stage of disease. Antibodies against the tat protein were found less frequently and increased f rom 16% to 40% with disease progression f rom the asymptomat ic stage to AIDS. Vif antibodies were detected only in 3% of the asymptomat ic patients, in 9% of the patients with LAS, but in 36% of the A R C patients and finally in 72% of the patients with AIDS. Of the six HIV-2 positive sera, one reacted with the nef protein. None of the 35 control sera reacted with the vif or tat protein, but 2 o f these sera showed a reactivity with the nef protein. The latter sera were collected f rom healthy children under the age of 4 wi thout any risk factors. This indicates the occurrence of antibodies against nef or a closely related prote in in non-HIV-infected individuals, as has been described elsewhere (Gomber t et al. 1989).

Page 8: Antibodies to recombinant HIV-1 vif, tat, and nef proteins in human sera

8

Table 1. Reactivity of 136 sera with recombinant nef, tat and vif proteins

U. Wieland et al.

No. of Reactivity Reactivity Reactivity sera with nef with tat with vif

HIV-neg. 35 2 ( 5.7%) HIV-2 6 1 (16.7%)

Asymptomatic 37 29 (78.4%) LAS 22 18 (81.8%) ARC 11 11 (100%) AIDS 25 24 (96%)

No. of HIV-l-positive 95 82 (86.3%) sera tested

Total No. of sera tested 136

0 (0%) 0(0%)

6(16.2%) 6 (27.3%) 3 (27.3%)

10 (40%)

25 (26.3%)

0 0%) 0 0%)

1 2.7%) 2 9.1%) 4 (36.4%)

18 (72%)

25 (26.3%)

LAS = Lymphadenopathy syndrome; ARC = AIDS-related complex

Discussion

Employing recombinant HIV-1 vif, tat, and nef proteins in immunoblot experi- ments, we could demonstrate the presence of the corresponding antibodies in serum samples of HIV-l-infected patients. The specificity of the reactions was confirmed by competi t ion experiments with lysates of HIV-l-infected H9 cells. Reactivity of sera with the MS-2 fusion protein itself was not observed. This is in accordance with a previous publication (Hess et al. 1988) which demonstrated the absence of MS-2 reactivity in 156 human serum samples.

The occurrence of antibodies against viral regulatory proteins has been described previously (Kiihn et al. 1987; Franchini et al. 1987; Ranki et al. 1987; Arya and Gallo 1986). Some of these studies, however, were performed either with oligopeptides (McPhee et al. 1988), which do not necessarily represent the complete antigenic profile of the respective protein, or were performed with recombinant proteins representing only parts of the respective genes (Kan et al. 1986; Franchini et al. 1986).

Analyzing 95 serum samples from HIV-l-infected patients we have found that the majority (86%) of these sera reacted with the nef protein. This percentage is considerably higher than previously thought (Ranki et al. 1987; Franchini et al. 1987; Allan et al. 1985). One reason for the relatively high frequency of nef antibodies observed in this study is possibly the high sensivity of the immunoblot procedure used (Kiihn et al. 1988 b). Reactivity to nefwas observed already in early stages of infection in 78 % of the patients. In accordance with the results of Ameisen et al. (1989a,b) this finding supports the demand to employ nefin routine screening tests for the early diagnosis of HIV infection.

Tat antibodies were found in our study in 16% to 40% of HIV-1 infected patients with a slight increase with disease progression to AIDS. This is in accordance with previous results reported by different authors (Krone et al. 1988; McPhee et al. 1988; Aldovini et al. 1986; Barone et al. 1986).

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Antibodies to HIV-1 vif, tat, and nef 9

Whereas the d e m o n s t r a t i o n of nef an t ibod ies may serve as an ear ly d iagnos t i c marke r , vif an t ibod ies were f o u n d to increase with disease progress ion . In on ly 1 out of 37 (2.7%) a s y m p t o m a t i c pa t ien ts , bu t in 72% of A I D S pa t ien ts cou ld an t ibod ies agains t vif be d e m o n s t r a t e d in our s tudy. This resul t might e i ther indicate tha t p r o l o n g e d immune exposure to vif induces an immune response aga ins t a p ro t e in with p o o r immunogen ic i t y or might indicate tha t i m m u n i t y agains t vif or express ion of vif leads to disease progress ion . Our f indings on the a n t i b o d y response aga ins t vif ex tend the results o f previous publ ica t ions : K a n et al. (1986) found an t ibod ies to vif in HIV- infec ted pa t ien ts wi thou t co r re l a t ion to the s tage of disease in 30 % to 50 %. F r a n c h i n i et al. (1987) r epo r t ed s imi lar results bu t f o u n d a sl ight increase o f an t ibod ies aga ins t vif with p rogress ion to A I D S . R a n k i et al. (1987) descr ibe the presence o f an t ibod ies agains t vif in ear ly H I V infect ion; s imilar i ly , A r y a (1987) f o u n d in a l imi ted a m o u n t o f sera vif an t ibod ie s less f requent ly in A I D S pa t ien ts than in hea l thy H I V carr iers . This d ivergence m a y par t i a l ly be exp la ined by the fact tha t the r e c o m b i n a n t vif p ro t e in used in our s tudy represented the comple te a m i n o ac id sequence o f vif and tha t the p rov i r a l vif D N A was ob t a ined f rom a di f ferent H I V clone than in some o f the a b o v e - m e n t i o n e d repor ts . F r anch in i et al. (1987), A r y a and Ga l lo (1986) and K a n et al. (1986) cou ld detect an t ibod ies agains t the vif p ro te in in sera f rom HIV-nega t ive , non- r i sk indiv iduals , which was no t obse rved in the s tudy o f Lee et al. (1986) and in our s tudy. In conclus ion , the d e m o n s t r a t i o n o f v i f an t ibod ies m a y represent a sufficient m a r k e r for advanced stages o f disease.

Acknowledgements. The authors express their gratitude to Martin Hartrnann, Department of Dermatology, University of Heidelberg (FRG), for providing the sera from HIV-infected patients. We furthermore gratefully acknowledge the expert technical assistance of M. Schrenk. Finally we would like to thank Wolfgang Wieland for elaborating Fig. 1A and B. This project was supported by the German Ministry of Research and Technology (BMFT), grant no. II-058-88.

References

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Aldovini A, Debouck C, Feinberg MB, Rosenberg M, Arya SK, Wong-Staal F (1986) Synthesis of the complete trans-activation gene product of human T lymphotropic virus type III in Escherichia col# demonstration of immunogenicity in vivo and expression in vitro. Proc Natl Acad Sci USA 83:6672-6676

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Received October 9, 1989