VIROLOGY 176,458~466 (1990)

Antigenic Epitopes of NEF Proteins from Different HIV-1 Strains as Recognized by Sera from Patients with Manifest and Latent HIV Infection

FRANK OTTO GOMBERT,* WOLFRAM BLECHA,t MARJATb;HTINEN,+ ANNAMARI RANK@ SIGRUN PFEIFER,t WILFRIED TRijGER,* RijDlGER BRAUN,% NIKOLAUS MijLLER-LANTZSCH,” GUNTHER JUNG,’

HELGA RUBSAMEN-WAIGMANNJ AND KAI KROHN+’

*Eberhard-Karls-Universittit Tijbingen, lnstitut fijr Organische Chemie, D-7400 Tiibingen, Federal Republic of Germany; tGeorg-Speyer-Haus, Paul-Ehrlich Institut, Frankfurt am Main, Federal Republic of Germany; *University of Tampere, Institute of Biomedical Sciences, Tampere,

fin/and; BDepartment of Dermatology, Helsinki University Central Hospital, Helsinki, Finland; Tlnstitut ftir Medizinische Virology, Universit$ Heidelberg, D-6900 Heidelberg, Federal Republic of Germany; and %stitut fijr Medizinische

Mikrobiologie und Hygiene, D-6650 Homburg/Saar, Federal Republic of Germany

Received September 18, 1989; accepted February 8. 1990

Human immunodeficiency virus (HIV) infection that generally causes a strong antibody response toward HIV may sometimes occur in a latent form, characterized by seronegativity in assays based on structural HIV proteins. Latently infected individuals, however, often have an antibody response against the nonstructural regulatory HIV-l protein NEF, a factor implicated in down-regulation of viral expression. In order to define the specificity of NfFantibodies, we looked for antibody response against more than 600 overlapping nonapeptides representing the total NEF sequence of three different HIV-1 isolates BRU, SF2, and MAL. Nine distinct homologous antigenic epitopes were recognized by sera from seropositive HIV-1 -infected individuals by the peptide ELISA. We further demonstrated that sera from “at risk” individuals, with no antibodies to HIV structural proteins but reacting with the recombinant NfF protein in Western blot, recognize the same epitopes. Immunological assays based on the defined NfF epitopes can therefore be used to diagnose early or latent HIV infection. o 1990Academic PR+SS. I~C.

INTRODUCTION

Individuals infected with human immunodeficiency viruses type 1 and 2 (HIV-l, HIV-2) usually have high titer antibodies to the structural viral proteins, coded for by the gag, pal, and env genes (Schiipbach et a/., 19841, but less frequently against the nonstructural products of the HIV genes vif, tat, rev, nef, vpu, and vpxthat are present in HIV-infected cells (Haseltine and Wong-Staal, 1988). The latter display regulatory func- tions at various levels of the virus life cycle, ranging from early transcriptional activation to maturation and release of infectious virus particles (Haseltine, 1989). In addition, these same proteins may modulate certain cellular genes, such as those coding for interleukin-2 (IL-2) and IL-2 receptor (Green et a/., 1989) or for sur- face markers, such as CD4 (Guy et a/., 1987).

Among the antibodies reacting with HIV regulatory proteins, those directed against NEF are of special in- terest as they arise early in infection and may even pre- cede the occurrence of antibodies toward the viral structural proteins (Ranki et al., 1987a; Ameisen et a/., 1989). Functionally, NEF seems to suppress viral ex- pression (Luciw et a/., 1987) through its down-regula-

’ To whom requests for reprints should be addressed.

tory effect on the viral promoter activity (Ahmad and Venkatesan, 1988) and perhaps on certain cellular genes as well, e.g., CD4 (Guy et al., 1987). Thus, anti- bodies towards NEF, as indicative of NEF protein ex- pression, may be associated with low virus production and a state of latency (Ranki et al., 1987a; Haseltine, 1989; Ameisen eta/., 1989).

The concept of latent (Ranki et al., 1987a) or silent HIV infection characterized by seronegativity toward the structural viral proteins initially raised controversy but has recently been confirmed. In a study of 133 se- ronegative persons belonging to a high risk group for AIDS, virus isolation was successful from 31 cases (lmagawa et al., 1989). Similarly, antibodies toward NEF have been found in HIV-l -infected individuals be- fore seroconversion to structural proteins and also in individuals who have remained seronegative during a prolonged follow-up (Ameisen eta/., 1989). In AIDS risk groups, such as sexually active homosexual men, a solitary antibody response toward HIV NEF can there- fore be taken as an indication of ongoing seroconver- sion or, if the antibody pattern remains similar for a longer period of time, of true HIV latency. Recently, we and others (Ranki et a/., 1990; Deinhardt, personal communication) have found, however, that solitary re- sponses toward HIV-l NEF can be seen in individuals

0042-6822190 $3.00 458 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction I” any form reserved.

ANTIGENIC EPITOPES OF Nff PROTEINS FROM HIV-1 STRAINS 459

not belonging to any HIV risk group and being unlikely to have been exposed to HIV. The finding of solitary anti-NEFantibodies, therefore, resembles that of a soli- tary response toward HIV-l GAG p24 protein that has been reported to occur in up to 1% of normal healthy blood donors (Biberfeld et al., 1986).

In order to define the specificities of the antibodies recognizing recombinant HIV-l NEFprotein first in gen- uinely HIV-infected individuals and second in individu- als unlikely to have been exposed to HIV, we undertook an effort to identify the antigenic NEF epitopes recog- nized by these two types of sera using the epitope anal- ysis method (Geysen et al., 1987). Three sets of >200 overlapping nonapeptides were synthesized according to a modified procedure, representing the total se- quences of three HIV-1 isolates, namely BRU, MAL, and SF2. The nonapeptides were covalently linked to a matrix of polyethylene pins fitting into a 8 X 12-well microtiter plate containing dilutions of the test sera. The presence of antibodies recognizing any of the non- apeptides was then monitored by the use of a modified enzyme-linked immunosorbent assay (ELISA) (Geysen et a/., 1987). The NEF epitopes recognized by the sera of HIV-infected individuals also reacting with a recom- binant NEF protein in Western blot were compared to those recognized by HIV ELISA-negative risk group and nonrisk group sera showing only NEFpositivity in West- ern blot.

MATERIALS AND METHODS

Sera

Ten randomly selected sera (cases l-l 0) were from HIV-1 -infected individuals, who in Western blot showed antibody reactivity toward all HIV-1 structural proteins as well as toward a recombinant NEF protein, produced in Escherichia co/i (Guy et al., 1987). Six sera (cases 1 l-l 6) from sexually active homosexual men were HIV-1 antibody negative toward HIV-1 structural proteins in Western blot and ELISA assays (Envicor, Abbott, Chicago, IL or Wellcozyme anti-HTLV III, Well- come Diagnostics), but reacted with the recombinant NEF protein in Western blot. This reactivity pattern has remained similar in repeatedly drawn serum samples for more than 6 months and none of the subjects has seroconverted. Eight sera (cases 17-24) were derived from a cohort of 1819 dermatological patients, volun- teered for HIV-1 antibody screening at the Department of Dermatology, Helsinki University Central Hospital, out of which 93 were tested by Western blot against recombinant NEFprotein as well. Only one of the 1819 patients proved to be genuinely HIV-l infected, whereas 14 of the 93 sera showed a positive reaction

a 1234567

,. 1234567

p27- p14-

FIG. 1. (a) Western blot analysis of HIV-1 ELISA-positive and nega- tive sera with disrupted HIV virion proteins. Lanes 1 and 2: HIV ELISA-positive sera. Lanes 3 and 4: HIV-1 ELISA-negative sera from homosexual men. Lane 5: case 22, a patient with autoimmune thy- reoiditrs not belonging to any HIV risk groups. Lanes 6 and 7: HIV-l ELISA-negative sera from healthy control individuals. (b) Western blot analysis of test sera using recombinant NEFas antigen. Lanes 1 to 7 contained the same sera as In legend to a. The band with a molecular mass of 14 kDa (p 14) represents degradation product of NEF.

to NEF protein. Finally, three healthy laboratory work- ers were used as negative controls.

Western blot analysis

Antibodies to HIV-1 NEF protein were detected by Western blot as previously described (Ranki et al., 198713, 1990). As antigen source, we used recombi- nant NEF protein, expressed in E. co/i (Guy et al., 1987) and kindly provided by Dr. M.-P. Kieny and Dr. J.-P. Le- cocq (Transgene, Strasbourg, France).

Purified NEF(240 pg/ml, 97% purity) or concentrated HIV-1 (HTLV-IIIB) virus preparation (5 mg/mI) was boiled in sample buffer containing 1% SDS and 1% 2- mercaptoethanol for 3 min, and was then run on 109/o PAGE gel and subsequently transferred onto a 0.45- pm nitrocellulose paper (Schleicher & Schuell, Keene, NH) as previously described (Ranki eta/., 1987b). Strips were preincubated in blocking buffer (Tris-HCI, 2% bo- vine serum albumin, 1% normal goat serum, and 5%, w/v, defatted dried milk powder) and thereafter incu- bated with patient sera diluted 1 :lOO in the blocking buffer. Binding of immunoglobulins to HIV proteins was

460 GOMBERT ET AL

L

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2500

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:, 1500 0

IO00

500

0 0

PEPTIDE N”rlBER

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1000

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El 0 x 600

z 400

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0 0 20 40 60 80 100 120 140 160 180 200 220 240

PEPTIDE NUMBER

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PEPTIDE NUMBER

3500.

3000. D

2500.

8 0 2000.

=, 1500. 0

1000.

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0 20 40 60 80 100 120 140 160 180 200 220 240 PEPTIDE NUMBER

FIG. 2. ELISA results against peptides derived from HIV-l Nff protein from isolate SF2 or MAL. (A) Sequence from isolate SF2 and serum .-. ^ , ^_^ from a homosexual man, who is HIV-1 positive in ELISA and shows anti-NEFantibodies in Western blot (case 1). (B) Sequence trom rsolate Sk2 and serum from a homosexual man, who is HIV-1 ELISA and Western blot negative, but NEf Western blot positive (case 1 1). (C) Sequence from isolate MAL and serum from a dermatological patient, who is HIV-1 ELISA and Western blot negative, but NEFWestern blot positive (case 22). (D) Sequence from isolate SF2 and serum from HIV-l ELISA and NEFWestern blot negative control serum.

detected with biotinylated anti-human IgG (Vector, Bur- lingame, CA), horseradish peroxidase-avidin-P (Vec- tor), and 4-chloro-1 -naphthol (Sigma, St. Louis, MO) as substrate.

Peptide synthesis

A simultaneous synthesis of 601 nonapeptides spanning the whole sequences of HIV-1 protein iso- lates BRU, SF2, and MAL was carried out by the Pep- scan method (Geysen et al., 1984). The chemical cou- pling onto the polyethylene rods (CRB, Cambridge) was carried out with the IO-fold excess of g-fluorenyl- methoxycarbonyl - L - amino acid, 1 - hydroxybenzotri azole, 2-(1 H-benzotriazol-1 -yl)-1,1,3,3-tetramethyl- uronium hexafluorophosphate, and a 14-fold excess of N-ethyldiisopropylamine in /V,/V-dimethylformamide in 1 hr coupling time. The coupling yield and the racemi- zation was analyzed by gas-liquid chromatography of

hydrolyzed peptide samples from several pins on a Chirasil-Val glass capillary column to prove the correct synthesis.

ELISA method

The polyethylene rods were blocked by incubating them with agitation in 96-well microtiter plates contain- ing 1 O/O ovalbumin, 1% BSA, 1 Yo Tween 20 in PBS (pH 7.2) for 1 hr at room temperature. After blocking, pins were incubated with serum samples (diluted 1:900 or 1: 1200) for 2 hr at 20” while shaking. To remove un- bound serum proteins, the pin plates were washed three times for 5 min with 300 ml 1% Tween 20-PBS (pH 7.2) and once with doubly distilled water. Peroxida- se-conjugated rabbit anti-human IgG was used at a di- lution of 1:500 in blocking media. The pins were incu- bated in diluted conjugate for 1 hr at 20” with agitation. The washing of pins after conjugation was performed

ANTIGENIC EPITOPES OF NEF PROTEINS FROM HIV-l STRAINS 461

TABLE 1

REACTIVITYOF HIV+NEF+ SERAWITH NEF EPITOPESFROM DIFFERENTHIVSTRAINS

Sample Isolate 1 2 3a 3b 3c 3d 4 5 6

Case 1

Case 3

Case 7

Case 9

Case 10

Case 25

Case 26

Case 27

SF2 5.3 SF2 5.8 BRU 6.8 MAL 5.7

SF2 3.2 BRU 8.8 MAL 7.2

SF2 4.4 BRU 10.5 MAL 7.8

SF2 1.3 BRU 2.8 MAL 2.5

SF2 1.7 BRU 2.5 MAL 2.1

SF2 1.3 BRU 1.3 MAL 1.6

SF2 1.4 BRU 1.6 MAL 1.7

SF2 1.3 BRU 2.2 MAL 1.7

HIV-l and NEFantibody positive patients

8.9 7.5 5.0 8.0

4.2 5.9 3.9 5.8 6.4 9.0 7. 1 6.5

4.1 3.0 7. 1 6.3

12.5 10.5

2.3 3.9 4.6 9. 1

10.9 9.2

1.1 1.1 2.1 2.1 2.5 1.9

1.6 1.5 1.9 1.6 3.3 1.9

Negative controls

1.5 1.4 1.5 1.6 1.5 1.2

4.0 1.7 1.4 1.9 1.7 1.5

1.3 1.3 1.2 1.2 1.6 1.5

15.0 9.2 2.9 6.9 13.9 9. 1 6.6 2.4 5.2 9. 1 7.3 7. 1 3.6 7.6 8.9 6.6 5.3 7.4 4.8 7.5

3.9 7.3 4.9

12. 1 5. 1 1.5 5.3 8.3 14.7 4.9 2.3 9.2 8.9 16.8 10.8 6.0 8.5 12.5

6.8 11.2 12.4

1.3 3.5 3.8

1.5 4.6 3.2

6.4 4.5 1.5 3.6 5.6 9.8 5.9 6.2 11.1 11.3

11. 1 5.7 9.3 8.9 nt

1.2 1.2 3.4 3.0 2.4 3.5

1.7 1.4 2.4 3.9 2.8 4.6

1.2 1.2 1.4

1.4 1.4 1.1 1.3 1.1 1.6 1.7 3.1 1.6 1.6 1.4 1.4 3.4 2.3 1.6

1.2 1.1 1.4

1.5 1.6 1.3 1.6 1.4 1.7 1.7 1.3 1.7 1.4 2.4 1.8 1.6 2.4 1.8

1.4 1.4 1.8

1.9 1.9 1.3 1.2 1.2 1.8 1.8 1.3 1.7 1.5 1.7 1.8 1.9 1.9 1.3

1.1 2.7 4.0

1.7 1.7 4.4

1.0 1.9 3.6 2.5 2.4 2.3

1.3 1.5 3.3 2.7 2.4 2.1

as after sample incubation. As substrate, 50 mg of 2,2’- azino-bis-(3-ethylbenzthiazolinesulfonic acid) (ABTS) in 100 ml of 0.1 M Na,HPO,-0.08 M citric acid (pH 4.0) with 30 ~1 H202 was used. The pins were incubated with substrate for 30-45 min in the dark at 20” while shaking. The photometric determination was carried out in ELISA-reader at 405 nm. Bound antibodies were removed from the pins between each test by sonicat- ing pin plates once for 30 min in water bath containing 1% SDS and 0.1% 2-mercaptoethanol in 0.1 n/l NaH,PO, (pH 7.2) twice for 10 min in doubly distilled water, and finally for 3 min with methanol.

RESULTS

Sera showing positive or negative reaction in HIV-l ELISA assays were first tested with Western blotting against disrupted HIV-1 virion preparation and against

recombinant HIV-l NEF protein. ELISA reactive sera recognized most HIV-1 structural proteins (Fig. la, lanes 1 and 2). Reactivity toward the recombinant NEF was seen in Western blotting with the HIV-l-positive sera (Fig. 1 b, lanes 1 and 2) but also with some HIV-l - negative sera coming from sexually active homosexual men (Figs. 1 a and 1 b, lanes 3 and 4) or from the cohort of dermatological patients not belonging to any HIV risk group (Figs. 1 a and 1 b, lane 5). Representative sera from each of the aforementioned groups and control sera which in Western blot were negative both against HIV-1 and NfF were then subjected to the epitope analysis.

In order to check the validity of the method and to see if similar epitopes occur in the three HIV strains chosen for the synthesis of peptides, we first tested five HIV-1 positive sera which in Western blots showed antibodies to all structural HIV-l proteins as well as to-

462 GOMBERT ET AL.

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PEPTlDE NUMBER

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200 I I

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PEPTIDE NUMBER

3000

I

f3 2500

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0 0 20 40 60 loo 120 140

PEPTIDE NUMBER 160 100 200 220 240

-1501 . . . , . , 0 20 40 60 00 100 120 140 160 180 200 220 240

PEPTIDE NUMBER

3500

3000 I

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PEPTIDE NVMBER

250,

FIG. 3. Upper panels: Epitope analysis with peptides from the three different NEF isolates BRU (a), SF2 (b), and MAL (c). Serum from a homosexual man, who is HIV-l positive in ELISA and shows anti-NEFantibodies in Western blot (case 1) serum dilution 1: 1500. Lower panels: Computer-assisted epitope prediction of the three different NEF isolates BRU (a), SF2 (b), and MAL (c). The program combines the predictions of conformation (Garnier ef al., 1978) wtth various prediction methods for epitopes and computes a werghted sum indicating probable epitopes.

ANTIGENIC EPITOPES OF NEF PROTEINS FROM HIV-1 STRAINS 463

lpitopc I

10 20 30 40

?Ipitope 2 Lpitope 3a Lpitope 3b

Lpitope 3c rpitope 3.3

Lpitopa I rpitope 5 rpitope 6

160 170 180 190 ZOO

FIG. 4. Amino acid alignment of HIV-1 isolates BRU, SF2, and MAL. The central regions of the epitopes recognized by the tested sera are boxed.

ward the recombinant NEF protein. Three sera from healthy laboratoryworkers were used as control. Three of the five HIV-positive sera reacted strongly with sev- eral HIV-l NEF-related nonapeptides (Fig. 2A, Table I), while two sera were either negative or gave a weak re- action with one or two regions only. We defined a posi- tive reaction with an epitope having an index (epitope OD,,,/background OD,,,,) 2 4.0. When the sera were run against peptides from all three strains, we recog- nized clearly defined epitopes (Figs. 3A-3C and 4, Ta- ble 1), which were very similar in different HIV-l strains. However, epitope 4 seemed to be present in the MAL isolate only. Repetitive testing gave comparable results (e.g., Table 1, case 1).

The amino acid sequences of the epitopes recog- nized by the positive sera are collected in Fig. 4. Obvi- ously, while some of the sera (Table 1, cases 1, 3, and 7) recognized several epitopes and thus can be classi- fied as clearly NEF positive, others (Table 1, cases 9 and 10) only recognized one or two epitopes. The three control sera which were negative against HIV-l and NEF in Western blotting usually showed no reactivity

against the NEF-derived peptides (Fig. 2D); a border- line reactivity against epitope 3a was seen with one control serum, however.

To further investigate the recognition of the epitopes defined in Fig. 4, a larger set of sera was tested. Be- cause of the finding that the identified epitopes (except No. 4) were present in all three isolates, each serum was now tested only against one of the three isolates. Five additional randomly selected sera were tested which were positive for antibodies to HIV structural proteins and NEF. Six sera were from sexually active homosexual men, who were negative for antibodies against HIV-1 structural proteins (Fig. la, lanes 3 and 4) but reacted with the recombinant NEF protein (Fig. 1 b, lanes 3 and 4), and eight sera were derived from a cohort of dermatological patients. The reactivity of the panel of sera is shown in Table 2. The epitopes defined by the results in Table 1 could be found using peptides from all three viral strains. However, Table 2 clearly in- dicates that several sera from HIV-l -infected individu- als positive in NEF Western blots only recognized one or two of the epitopes, suggesting that their anti-NEF reactivity was due to unspecific binding or to cross-re- actions with some unknown cellular or viral protein. A third possibility for the discrepancy in results obtained with Western blot and epitope analysis would be a re- activity against noncontinuous epitopes.

Cases 1 l-16 in Table 2 were from HIV-1 antibody negative individuals belonging to a HIV-risk group and thus having possibility to have been exposed to HIV-l. Four sera reacted with most of the identified epitopes within the NEF sequence (usually epitopes 1, 3a, 3c, 3d, 5, 6) and the general pattern of reactivity was indis- tinguishable from that seen with the HIV-l antibody positive sera (Fig. 2B, Table 2). One serum showed a borderline reactivity towards one of the epitopes.

Of the eight sera from dermatological patients with- out any known exposure to HIV but who reacted with the NEF protein in Western blot (Figs. 1 a and 1 b, lane 5) one (case 22) showed a strong reactivity to the same epitopes that were recognized by sera from individuals with proven HIV infection (Fig. 2C). The patient, a 25- year-old heterosexual man with no known history of HIV exposure, had a newly recognized autoimmune thyreoiditis. With four sera of this group, weak reactivi- ties were seen against a single NEF epitope in each case (epitope 3d or 5).

DISCUSSION

Our findings confirm the high immunogenicity of HIV- 1 NEF protein. Several antigenic epitopes, recognized by sera derived from HIV-l -infected individuals, could

464 GOMBERT ET AL.

TABLE 2

REACTIVIPIOFSERAFROM DIFFERENTGROUPSOFPATIENTSAGAINSTTHEIDENTIFIEDNEFEPITOPES (EXPRESSEDASEPITOPEOD,,,/BACKGROUND OD,,,,)

Samole Isolate 1 2 3a 3b 3c 3d 4 5 6

HIV+NfF+ 1 2 3

4 5 6 7

8 9

10

SF2 5.3 8.9 4.2 5.9 SF2 2.4 2.4 2.0 2.8 SF2 3.2 3.9 4.1 3.0

BRU 8.0 2.0 4.7 6. 1 BRU 2.6 2.0 1.8 2.6 BRU 2.9 2.4 2.1 2.8 BRU 10.5 11.2 4.6 9. 1

MAL 6.6 1.0 5. 1 5.8 MAL 2.5 3.8 2.5 1.9 MAL 2.1 3.2 3.3 1.9

HIV-NW+ 11 12

13

14 15 16

SF2 4.6 SF2 1.4

BRU 7.3

MAL 4.0 MAL 3.6 MAL 1.9

HIV-NEf+ 17 18

SF2 2.0 SF2 1.8

19 BRU 3.0 20 BRU 3.4 21 BRU 2.0

22 MAL 9.8 23 MAL 2.8 24 MAL 3.9

7.3 1.4

2.5

1.8 2.2 1.1

HIV-infected cases

HIV- 1 -exposed partners

3.4 5.3 1.5 1.8

4.2 2.8

4.1 3.3 2.7 4.0 2.4 2.0

15.0 3.4

12.1

4.0 3.6 4.9 9.8

11.4 2.4 2.8

16.9 3.0

3.8

5. 1 5.2 2.2

Nonexposed dermatological patients

1.9 2.3 2.2 3.4 1.5 1.3 1.8 2.5

2.2 2.8 3.4 3.4 1.7 2.3 1.3 2.8 1.8 1.7 1.9 2.8

1.8 5.3 6.3 9.9 1.9 2.8 1.9 3.2 1.7 2.5 2.2 2.2

9.2 2.9 6.9 13.9 3.4 2.2 2.8 2.6 5. 1 1.5 5.3 8.3

2.4 2.0 5.6 nta 2.4 1.8 3.6 nt 2.6 2.2 3.4 nt 5.9 6.2 11. 1 11.3

9.1 4.0 8.2 5.4 3.5 4.0 2.4 2.3 4.6 4.4 2.4 2.1

8.6 1.7 5.9 13.5 2.2 1.3 1.8 1.8

4.2 2.0 5.0 nt

3.4 2.5 8.9 5.8 5.2 3.5 4.7 4.0 1.9 1.7 4.0 2.3

3.1 1.7 2.8 2.7 5.0 1.0 3.0 2.3

2.8 2.0 3.6 nt 3.4 2.6 4.2 nt 2.3 1.8 3.3 nt

6.9 4.7 7.3 4. 1 1.9 1.9 4.1 3.4 2.2 2.1 5.9 2.8

a Not tested.

be identified and the antibody response toward these regions was generally strong and easily detectable. Such sera recognized five to nine clearly defined epi- topes (Tables 1 and 2, Fig. 4) within the NEFsequence, while sera likely to have nonspecific or non-HIV-related anti-NW response did not recognize these epitopes or reacted with only one or two of the epitopes. Further- more, sera from HIV at risk individuals recognized the same NEFepitopes, if any, as HIV-infected sera.

Our experimental results agree only up to 50-609/o with computer-assisted epitope predictions (Fig. 3). None of our nonapeptides from the region 149-l 59

(extended counting) showing one of the strongest epi- tope propensity reacted with any sera. These findings contradict the results of Ameisen et al., (1989) where a 14-mer peptide 148-l 61 (isolate BRU) selected by computer analyses of MFreacted with 20 of 22 sera in a radioimmunoassay (RIA). One explanation for these different results could be the high degree of charged amino and carboxy groups within this region (9 out of 14 residues), which could lead to higher nonspecific binding of antibodies in RIA. This problem is reduced in our nonapeptide ELISA because the charged N-ter- minii are capped by acetylation and the C-terminii are

ANTIGENIC EPITOPES OF NfF PROTEINS FROM HIV-1 STRAINS 465

covalently linked to the polymer. Another explanation could be the forming of a more stable secondary struc- ture in the 14-mer peptide representing a discontinu- ous epitope, which cannot be found in our assay sys- tem. However, preliminary results with our lipopeptide ELISA system (Baltz et al., 1988) using the icosapep- tides 141-160 and 151-170 (BRU) linked to the lipid compound tripalmitoyl-S-glycerylcysteine (Bessler et a/., 1985; Jung et a/., 1985) did not show a reactivity against these regions. In other investigations (Niedrig eta/., 1989) on HIV-structural proteins, actual epitopes found experimentally and computer predictions did not necessarily correlate, so that it appears that epitopes can only be defined experimentally and that even de- pending on the experimental conditions different re- sults can be obtained. In accordance with our results, in a recent work by Sabatier et a/. (1989) only 4 out of 3 1 NEF antibody positive sera reacted in ELISA assay against a synthetic peptide consisting of amino acids 1 18-l 67 of the BRU isolate. Also, our present findings concerning epitopes 1, 2, 5, and 6 are mostly in agree- ment with the results obtained by Sabatier et a/.

The biological significance of the solitary anti-NEf antibodies remains to be classified. In cases 1 1, 13, 14, and 15 who are homosexual men with a possible previous exposure to HIV-l, it is likely that they are la- tently infected with the virus in spite of a negative find- ing for HIV antibodies or HIV antigen. Their sera re- acted with exactly the same NEF epitopes as the genu- ine anti-HIV-l positive sera. The identical nature of the HIV-antibody positive and negative sera with NEF epi- topes was also confirmed in preliminary adsorption ex- periments using purified recombinant NEF. Case 22 is of special interest, as his reactivity toward the NEFpep- tides was similar to that seen by genuinely HIV-infected sera. We could not verify any known exposure to HIV and he has remained HIV Western blot seronegative for a follow-up of 18 months. However, the association of autoimmune thyreoiditis to NEFantibodies in this pa- tient and the recent finding of HIV-l gag-related se- quences in another autoimmune thyroid disease, thy- reotoxicosis (Grave’s disease) (Ciampolillo et al., 1989) raises the possibilitythat these reactivities might be as- sociated with an infection with another unidentified hu- man retrovirus.

Finally, the low level reactivities toward one or two of the identified NEF epitopes with some of the NEF Western blot positive sera must be taken with caution. As genuine HIV-l infection is extremely rare in Finland (252 recognized cases in 5 million inhabitants), it is hardly likely that these reactivities reflect a latent HIV infection. The definition of antigenic epitopes in NEF that are recognized only by genuinely HIV-infected sera

is of importance, as immunological assays, such as ELISA, based on synthetic peptides representing these defined sequences can be used to distinguish be- tween a nonspecific reaction and genuine HIV-1 infec- tion, even when antibodies against the structural HIV proteins are of low quantity or even lacking.

ACKNOWLEDGMENTS

We thank Kaija J&vinen for excellent laboratory work, Dr. Sirkka- Liisa Valle for some of the sera, and Dr. M. P. Kieny from Transgene (Strasbourg, France) for the gift of recombinant NEF protein. The study was supported by the Academy of Finland and by American Foundation for AIDS Research (AmFAR). The Georg-Speyer-Haus is supported by the German Bundesministerium ffir Gesundheit and the Hessisches Ministerium fiir Wissenschaft und Kunst. This work was supported in part by the German Bundesministerium fiir For- schung und Technologie (Project G. Jung, FKZ II-01 2-86).

REFERENCES

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