JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1991, p. 488-492
Vol. 29, No. 3
0095-1137/91/030488-05$02.00/0 Copyright C) 1991, American Society for Microbiology
Immune Response to a Major Epitope of p24 during Infection with Human Immunodeficiency Virus Type 1 and Implications for Diagnosis and Prognosis BLANDINE JANVIER,1 ARMELLE BAILLOU,1 PHILIPPE ARCHINARD,2 MARCELLE MOUNIER,3 BERNARD MANDRAND,2 ALAIN GOUDEAU,1 AND FRANCIS BARIN1* Laboratoire de Virologie, Departement de Microbiologie Me'dicale et Moleculaire, URA CNRS 1334, CHRU Bretonneau, 37044 Tours,' UM 103 CNRS-BIOMERIEUX, 69364 Lyon,2 and Laboratoire de Bacteriologie- Virologie, CHR U Dupuytren, 87000 Limoges, 3 France Received 29 May 1990/Accepted 14 November 1990
A sequential inhibition enzyme-linked immunoassay (SIEIA) using a peroxidase-conjugated monoclonal antibody reacting to the sequence AAEWDRVHP of p24HIV-1 (amino acids 209 to 217 of p55) was developed in order to detect and determine the titer of antibody to this epitope in various populations of human immunodeficiency virus type 1 (HIV-1)-positive patients. There was a good correlation between SIEIA and a commercially available competition assay that uses recombinant p24 protein and polyclonal antibody to HIV-1 antigen, demonstrating the importance of the described epitope. Analysis of sera from French patients showed a decline of antibody to the AAEWDRVHP sequence associated with the progression of AIDS. No decrease was observed with serum samples from African patients. An immune response to the epitope was detected by SIEIA early in the course of seroconversion. Although our SIEIA uses a single p24 epitope, these data are in accordance with previously published studies in which antibodies to the whole p24 were analyzed. Sera reacting to p24 only (indeterminate profiles by Western blot [immunoblot]) did not bind to AAEWDRVHP. This epitope, which is conserved between HIV-1 and HIV-2/simian immunodeficiency virus, appears to be a major antigenic domain of p24. The area containing the sequence AAEWDRVHP and the corresponding monoclonal antibody may serve as a convenient alternative to whole purified p24 and polyclonal antibody in diagnostic and prognostic assays.
The major core protein p24 of human immunodeficiency virus type 1 (HIV-1) is a structural protein useful in the diagnosis of HIV infection. Antibody to p24 appears rapidly after primary infection (7, 10) and usually disappears when HIV-1-associated immune deficiency progresses, making it an important prognosis marker (4, 5, 8, 9, 14, 17, 18). In a previous study, major epitopes of p24HIV` and p26HIV-2 were identified by using a panel of nine murine monoclonal antibodies (MAb) directed to p24HIV-4 and by using the Pepscan procedure (12). One of the epitopes with the sequence AAEWDRVHP is conserved in p24HIV-1 as well as p26HIV-2. This sequence or the area containing it appeared to be the more regularly antigenic domain recognized during natural HIV infection. In order to detect and determine the titer of antibody to this epitope, we have developed a sequential inhibition enzyme-linked immunoassay (SIEIA) which uses a peroxidase-conjugated MAb reacting to AAEWDRVHP. Several populations comprising French patients at various stages of HIV-1 infection and African patients suffering from HIV-1-related AIDS were studied. Results were compared with those obtained with a commercially available enzyme assay which allows the determination of antibody titer to the whole HIV-1 core protein.
patients were tested twice, at two different CDC stages: they were initially tested at either the CDC II or the CDC III stage, and the second samples were collected between 2 and 5 years later, when the patients evolved to stage CDC III or CDC IV. The patients belonged to various known risk groups (Table 1). Serum samples were taken from 50 HIV1-positive patients from central Africa at stage CDC IV (2). In addition, 21 sequential serum specimens obtained from eight French patients at the early stage of seroconversion to HIV-1 were also tested. The specificity of the assay was evaluated with HIV-1negative serum specimens collected from 262 French subjects and from 22 subjects from central Africa and with 25 serum samples from French subjects which reacted only to p24 by Western blot (immunoblot) (nonspecific reactivities). All the negative sera were collected from healthy blood donors. SIEIA. (i) Preparation of the solid phase. A rabbit was immunized with five 100-rig injections of protein p24HIV` purified by electroelution. The antiserum was positive at a 10-6 dilution in an in-house enzyme-linked immunosorbent assay using microtitration plates coated with HIV-1 viral lysate (3). The immunoglobulins G were purified by ionexchange chromatography; they reacted in Western blot to the gag precursor p55HIV- and to p24HIV-1. Two hundred microliters of this immunoglobulin G preparation was used to coat 96-well microtitration plates at a concentration of 18 ,ug/ml in 50 mM bicarbonate buffer, pH 9.6. After 20 h at 4°C, the plates were washed four times with 0.05 M sodium phosphate-buffered saline (PBS) (pH 7.4) containing 0.1% Tween 20 (PBS-T) and then quenched with 250 ,ul of PBS containing 5% bovine serum albumin (PBS-BSA) for 30 min
MATERIALS AND METHODS Patients. A total of 145 serum specimens from 123 HIV-1positive French patients at various stages of infection were tested; 71, 29, and 45 serum samples were collected at stages CDC II, CDC III, and CDC IV, respectively (6). Twenty-two *
Corresponding author. 488
IMMUNE RESPONSE TO A MAJOR EPITOPE of p24HIVd
VOL. 29, 1991
TABLE 2. Distribution of percentages of inhibition obtained by SIEIA
TABLE 1. Distribution of French patients according to risk groups and clinical stages
No. of patients per serum category
No. of patients per
Risk group
clinical stage CDC II CDC III CDC IV
Homosexual or bisexual men Intravenous drug users Transfusion recipients Hemophiliacs Heterosexual contacts Patients originating from endemic area Patients with no known risk
27 13 6 16 5 1 3
11 9 1 8 0 0 0
23 10 1 6 1 2 2
Total
71
29
45
at 37°C. After one cycle of washing with PBS-T, 150 RIl of HIV-1 viral lysate prepared as previously described (3) and diluted in PBS containing 2.5% BSA and 0.05% Tween 20 (PBS-BSA-T) was dispensed in the wells (0.3 jig of viral lysate per well). The plates were left for 20 h at room temperature. Four cycles of washing were carried out prior to use of the plates. (ii) Test procedure. The sequential inhibition assay was carried out with MAb 23A5G4, which was selected during a previous study of nine MAbs to p24HtVd (12). This MAb is directed to an epitope spanning the sequence AAEWD RVHP (amino acids 209 to 217 of p55). MAb 23A5G4 was conjugated to horseradish peroxidase by using the periodic method of Nakane and Kawaoi (16). A 70-jil volume of PBS-BSA-T and a 20-,u volume of serum sample were incubated for 15 min at 37°C in each well of the plates prepared as described above, before 50 pI of an appropriate dilution (1:500) of the conjugated MAb 23A5G4 in PBS-BSA-T was added. Dilution was chosen to give half-maximum binding that is an optical density of approximately 0.8 with negative sera. After 90-min at 37°C, followed by four cycles of washing, inhibition was revealed with 100 ,ul of a 2-mg/ml solution of ortho-phenylenediamine (Sigma) in 0.05 M sodium phosphate-0.02 M citric acid buffer, pH 5, to which 3 ,ul of hydrogen peroxide per ml was added extemporaneously. The color development was stopped after 30 min with 50 p.l of 4 N sulfuric acid, and plates were read at 492 nm. The percentage of inhibition and the antibody titer of every sample were evaluated by using a standard curve drawn from doubling dilutions from 1:4 to 1:512 of a highly positive control serum included in every plate; the absorbance value obtained with the 1:8 dilution of the positive control had to be less than 0.1. Three negative control serum samples were included; their absorbance values had to be between 0.5 and 1. When positive or negative standard values were not within the fixed ranges, the tests were not valid. The percentage of inhibition was calculated for every sample as follows: percent inhibition = {1-[(ODa - ODpc)/ (ODnc - ODpc)]} x 100, where ODa is the absorbance value of the sample, ODpc is absorbance value of the 1:8 dilution of the positive control, and ODnc is the mean absorbance value of the negative control. Sample optical densities were directly transformed into reciprocal antibody titers by using a titration curve drawn on a semilogarithmic scale from absorbance values of positive control dilutions. Correlation with a commercially available assay. SIEIA
489
% Inhibition
Central African
French
CDC CDC CDC HIV-1 CDC II IV negative IV III
HIV-1 negative
p24 reactive only
81-90 91-100
197 51 12 2 0 0 0 0 0 0
25 0 0 0 0 0 0 0 0 0
11 3 1 0 5 1 6 4 4 36
10 2 2 1 0 1 1 1 1 10
23 5 5 0 0 3 3 1 1 4
22 0 0 0 0 0 0 0 0 0
7 1 0 1 2 1 1 1 1 35
Total
262
25
71
29
45
22
50
0-10 11-20 21-30 31-40 41-50 51-60 61-70
71-80
results were correlated with those of a commercially available assay (HIV-1 anti-core EIA; Abbott) by using a panel of 97 unselected HIV-1-positive serum specimens collected from patients at various clinical stages. In the Abbott competition assay, beads coated with whole recombinant p24 protein are incubated with a mixture of a serum sample and a polyclonal human antibody to HIV-1 core conjugated with horseradish peroxidase. Antibody titers were calculated as previously described (1). Correlation was studied by using the Spearman rank test (19). RESULTS All 284 HIV-negative serum samples showed less than 40% inhibition (Table 2). Of the negative samples, 95.1 and 99.3% showed less than 20 and 30% inhibition, respectively; only two negative samples showed between 30 and 40% inhibition. Samples with more than 40% inhibition were considered positive for antibody to p24. None of 25 serum samples reacting only to p24 by Western blot prevented the binding of the 23A5G4 conjugate. All these samples showed less than 10% inhibition and prove the specificity of the assay, since it is now well recognized that sera reacting only to core protein p24 and p17 by Western blot do not indicate HIV exposure (11, 13, 15). Among the French serum samples, there was a clear decrease of the immune response to the p24 epitope AAEW DRVHP associated with the progression of AIDS. Seventynine percent of samples from CDC II patients, 48% of samples from CDC III patients, and only 27% of samples from CDC IV patients were positive in the assay (Fig. la through c). Statistical differences between CDC II and CDC IV patients were significant (P < 0.01). As has been described previously for a commercially available assay (2), antibody to p24 persisted in 82% of CDC IV patients from central Africa (Fig. ld), a significantly higher prevalence than that observed in CDC IV French patients (P < 10-7). French serum samples also showed a decrease of antibody titer associated with advancing stages of AIDS. For instance, 61% of CDC II patients and 65% of CDC III patients who were positive for antibody to p24 in the SIEIA presented an anti-p24 titer equal to or greater than 1:8, compared with only 32% of CDC IV patients (Fig. le through g). The median antibody titers were 1:32, 1:8, and 1:1 for CDC II, CDC III, and CDC IV patients, respectively. As de-
490
J. CLIN. MICROBIOL.
JANVIER ET AL.
a
e CDC E1/FRANCE
CDC I / FRANCE 80
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60
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b
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20
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2
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32
64
128
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CDC mI/FRANCE 80
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60
440
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-10 20 30 40 50 60 70 80 90 100
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128
CDC IV IFRANCE
80
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20
20
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0 40 So 60 70 80 90 100
100%
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CDC IV /CENTRAL AFRICA
CDC IV /CENTRAL AFRICA
80
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60
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40
20
20
0
32
100%
100%
CDCIV/FRANCE
d
16
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80 90 100 PERCENT INIMION
0-10 20 30 40 50 60 7
4
8
16
32
64
128
ANTIBODY TITER
FIG. 1. Distribution of percentages of inhibition among various populations of HIV-1-positive patients (a through d) and distribution of antibody titer to AAEWDRVHP by category of serum reactive to this epitope (e through h). Results are shown for sera from French patients at stages CDC II (a and e), CDC III (b and f), and CDC IV (c and g) and sera from central African patients at stage CDC IV (d and h). The percentages of sera are indicated on the ordinate.
scribed above for qualitative results, anti-p24-positive Afripatients, even at stage CDC IV, had high anti-p24 titers: 86% of them had antibody titers equal to or greater than 1:8 (Fig. lh). Of the 22 patients tested twice, 17 tested positive by the SIEIA with the initial blood samples. Of these, nine patients were monitored for 4 to 5 years, and eight showed an evolution of their clinical status and concurrently a clear decrease or disappearance of their antibody titer; antibody to p24 declined in patients with high initial titers and disappeared in patients with low initial titers. Eight patients were monitored for 2 to 3 years. Although they presented a similar clinical evolution, their antibody titers remained stable. This seems to indicate that a minimal period of time is necessary before a diminution of anti-p24 titer can be observed. The five patients who were initially negative by SIEIA (one CDC can
II and four CDC III) remained negative and evolved to stage CDC IV in less than 3 years. Anti-p24 titers from the SIEIA were compared with results obtained with the HIV-1 anti-core EIA from Abbott for 97 HIV-1-positive serum samples (Fig. 2). Although the Abbott assay appeared slightly more sensitive than the SIEIA, there was a good correlation between the two assays, as assessed by a Spearman rank correlation coefficient of rs = 0.89 (P < 0.001). These data suggest that the antibody titer to AAEWDRVHP has almost the same significance as the antibody titer to the whole p24, indicating that the described epitope is a major antigenic domain of this protein. Twenty-one serum samples were collected sequentially from eight seroconverters. Profiles of the first serum samples are typical of early seroconversion, i.e., show the presence
IMMUNE RESPONSE TO A MAJOR EPITOPE of p24HIV-1
VOL. 29, 1991 S I E IA 0
0
2
1
4
8
case except one (case 5), antibody titers by SIEIA in subsequent serum samples rose.
16 32 >32
DISCUSSION In a previous study, we described a conserved epitope on the major core proteins of HIV-1 and HIV-2 that appeared to be immunogenic during natural infection (12). This epitope, identified with MAb and the Pepscan procedure, corresponds to the sequence AAEWDRVHP in p24HIV-1. We used a peroxidase-conjugated MAb (23A5G4) directed to this epitope to generate an SIEIA in order to detect and determine the titer of antibody to p24HIV-1 in human sera. Results obtained with sera from French patients confirmed the correlation between an absence or decrease of antibody to p24 and the advancing stage of HIV-1 infection. For instance, the prevalence of antibody to AAEWDRVHP fell from 79 to 48 and 27% in CDC II, CDC III, and CDC IV patients, respectively. Among patients who were positive for antibody to AAEWDRVHP, titers were also higher in patients with mild symptoms (CDC II and CDC III). Twentytwo patients who clinically evolved to advanced stages of the disease were monitored for 2 to 5 years. Five who were initially negative for antibody to AAEWDRVHP developed overt AIDS in less than 3 years. Of the 17 patients whose first serum samples were positive by SIEIA at the initial sampling, 10 presented a decrease in or a disappearance of antibody to AAEWDRVHP in correlation with the progression of the disease. Detection of antibody to AAEW DRVHP by the SIEIA in the earliest Western-blot-positive samples from HIV-1 seroconversions further indicated that
massse
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32 >32
FIG. 2. Comparison of the antibody titers to p24 obtained for 97 HIV-1-positive serum specimens with the SIEIA and the Abbott HIV-1 anti-core EIA.
of antibody to gpl60 only or antibody to gp160 and p24 (Fig. 3). Four of eight early samples were positive for p24 in the Western blot and positive in our SIEIA, with titers between 1:2 and 1:4. Two of the four initial samples that were negative in our SIEIA were also negative (case 1) or very weakly positive (case 7) in the Western blot assay. The four patients who initially tested negative by SIEIA had positive subsequent blood samples (cases 1, 2, 3, and 7). In every
+ 1
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0
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491
Antibody titer FIG. 3. Evolution of serological profiles in sequentially collected sera from eight seroconverters. In every case, the first Western blot shown corresponded to the first serum sample found positive for antibody to HIV-1. The dates (day-month-year) of blood sampling and the antibody titers to AAEWDRVHP are indicated below the corresponding Western blots. 0
1
8 32
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492
JANVIER ET AL.
this epitope is recognized early in the course of natural infection. It has been previously reported that high anti-p24 titers are usually conserved in HIV-1-positive patients from Africa even at advanced stages of the disease (2). Results obtained with the SIEIA confirmed these data and indicated furthermore that epitope AAEWDRVHP is conserved among HIV-1 strains presently circulating in Africa and is highly immunogenic during natural infection in central African residents. There was a good correlation between our SIEIA, based on a conjugated MAb, and a commercially available assay that uses a polyclonal antibody to the whole HIV-1 core protein, indicating that the area containing the sequence AAEWDRVHP is a major antigenic domain of p24HIWl. This epitope is also conserved in HIV-2/simian immunodeficiency virus strains (12). Peptide AAEWDRVHP and its corresponding MAb may thus serve as a convenient alternative to purified viral p24 and polyclonal antibody in developing diagnostic and prognostic assays. ACKNOWLEDGMENTS We are grateful to M. F. Maitre, L. Hervioux, P. Choutet, and C. Guerois for the clinical information on sera from HIV-1-positive French patients, to P. Kocheleff and E. Petat for the African sera, and to M. H. Charles for technical help. We thank A. Pompidou for encouragement and help at the initiation of our program. This work was supported by grant 86C-0926 from the Ministere de la Recherche et de l'Enseignement Superieur and by funds from Universite Francois Rabelais and bioMerieux. Blandine Janvier was supported by a doctoral fellowship from the Fondation Merieux, Lyon, France. REFERENCES 1. Allain, J. P., Y. Laurian, D. A. Paul, F. Verroust, M. Leuther, C. Gazengel, D. Senn, M. J. Larrieu, and C. Bosser. 1987. Long-term evaluation of HIV antigen and antibodies to p24 and gp4l in patients with hemophilia. N. Engl. J. Med. 317:11141121. 2. Baillou, A., F. Barin, J. P. Allain, E. Petat, P. Kocheleff, P. Kadende, and A. Goudeau. 1987. Human immunodeficiency virus antigenemia in patients with AIDS and AIDS-related disorders: a comparison between European and central African populations. J. Infect. Dis. 156:830-833. 3. Barin, F., S. M'Boup, F. Denis, P. Kanki, J. S. Allan, T. H. Lee, and M. Essex. 1985. Serological evidence for virus related to simian T-lymphotropic virus III in residents of West Africa. Lancet ii:1387-1389. 4. Biggar, R. J., M. Melbye, P. Ebbesen, S. Alexander, J. 0. Nielsen, P. Sarin, and V. Faber. 1985. Variation in human T lymphotropic virus III (HTLV-III) antibodies in homosexual men: decline before onset of illness related to acquired immune deficiency syndrome (AIDS). Br. Med. J. 291:997-998. 5. Burke, D. S., R. R. Redfield, P. Putman, and S. S. Alexander. 1987. Variations in Western blot banding patterns of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus. J. Clin. Microbiol. 25:81-84.
J. CLIN. MICROBIOL. 6. Centers for Disease Control. 1986. Classification system for human T-lymphotropic virus type III/lymphadenopathy-associated virus infections. Ann. Intern. Med. 105:234-237. 7. Chou, M. J., T. H. Lee, A. Hatzakis, T. Mandalaki, M. F. McLane, and M. Essex. 1988. Antibody response in early human immunodeficiency virus type I infection in hemophiliacs. J. Infect. Dis. 157:805-811. 8. de Wolf, F., J. M. A. Lange, J. T. M. Houweling, R. A. Coutinho, P. T. Schellekens, J. Van Der Noordaa, and J. Goudsmit. 1988. Numbers of CD4 cells and the levels of core antigens and of antibodies to the human immunodeficiency virus as predictors of AIDS among seropositive homosexual men. J. Infect. Dis. 158:615-622. 9. Forster, M. S., L. M. Osborne, R. Chiengson-Popov, C. Kenny, R. Burnell, D. J. Jeffries, A. J. Pinching, J. R. W. Harris, and J. N. Weber. 1987. Decline of anti-p24 antibody precedes antigenemia as correlate to prognosis in HIV1 infection. AIDS 1:235-240. 10. Gaines, H., A. Sonnerborg, J. Czajkowski, F. Chiodi, E. M. Fenyo, M. von Sydow, P. 0. Pehrson, L. Moberg, B. Asjo, and M. Forsgren. 1987. Antibody response in primary human immunodeficiency virus infection. Lancet i:1249-1253. 11. Jackson, J. B., K. L. MacDonald, J. Cadwell, C. Sullivan, W. E. Kline, M. Hanson, K. J. Sannerud, S. L. Stramer, N. J. Fildes, S. Y. Kwok, J. J. Sninsky, R. J. Bowman, H. F. Polesky, H. H. Balfour, and M. T. Osterholm. 1990. Absence of HIV infection in blood donors with indeterminate western blot tests for antibody to HIV1. N. Engl. J. Med. 322:217-222. 12. Janvier, B., P. Archinard, B. Mandrand, A. Goudeau, and F. Barin. 1990. Linear B-cell epitopes of the major core protein of human immunodeficiency virus types 1 and 2. J. Virol. 64:42584263. 13. Josephson, S. L., N. S. Swack, M. T. Ramirez, and W. J. Hausler, Jr. 1989. Investigation of atypical Western blot (immunoblot) reactivity involving core proteins of human immunodeficiency virus type 1. J. Clin. Microbiol. 27:932-937. 14. Lange, J. M. A., D. A. Paul, H. G. Huisman, F. de Wolf, H. Van Den Berg, R. A. Coutinho, S. A. Danner, J. Van Der Noordaa, and J. Goudsmit. 1986. Persistent HIV antigenemia and decline of HIV core antibodies associated with transition to AIDS. Br. Med. J. 293:1459-1462. 15. Lefrere, J. J., A. M. Courouce, G. Lucotte, C. Boitard, C. Kaplan, J. C. Nicolas, F. Bricout, P. Lambin, C. Doinel, J. Y. Muller, J. F. Bach, and C. Salmon. 1988. Follow-up of subjects with isolated and persistent anti-core (anti-p24 or anti-p17) antibodies to HIV. AIDS 2:287-290. 16. Nakane, P. K., and A. Kawaoi. 1974. Peroxidase labelled antibody: a new method of conjugation. J. Histochem. Cytochem. 22:1084-1091. 17. Pedersen, C., C. M. Nielsen, B. F. Vestergaard, J. Gerstoft, K. Krogsgaard, and J. 0. Nielsen. 1987. Temporal relation of antigenemia and loss of antibodies to core antigens to development of clinical disease in HIV infection. Br. Med. J. 295:567569. 18. Schupbach, J., M. Popovic, R. V. Gilden, M. A. Gonda, M. G. Sarngadharan, and R. C. Gallo. 1984. Serological analysis of a subgroup of human T-lymphotropic (HTLV-III) associated with AIDS. Science 224:503-505. 19. Siegel, S. 1956. Nonparametric statistics for the behavioural sciences, p. 202-213. McGraw-Hill Book Co., New York.
Vol. 29, No. 3
0095-1137/91/030488-05$02.00/0 Copyright C) 1991, American Society for Microbiology
Immune Response to a Major Epitope of p24 during Infection with Human Immunodeficiency Virus Type 1 and Implications for Diagnosis and Prognosis BLANDINE JANVIER,1 ARMELLE BAILLOU,1 PHILIPPE ARCHINARD,2 MARCELLE MOUNIER,3 BERNARD MANDRAND,2 ALAIN GOUDEAU,1 AND FRANCIS BARIN1* Laboratoire de Virologie, Departement de Microbiologie Me'dicale et Moleculaire, URA CNRS 1334, CHRU Bretonneau, 37044 Tours,' UM 103 CNRS-BIOMERIEUX, 69364 Lyon,2 and Laboratoire de Bacteriologie- Virologie, CHR U Dupuytren, 87000 Limoges, 3 France Received 29 May 1990/Accepted 14 November 1990
A sequential inhibition enzyme-linked immunoassay (SIEIA) using a peroxidase-conjugated monoclonal antibody reacting to the sequence AAEWDRVHP of p24HIV-1 (amino acids 209 to 217 of p55) was developed in order to detect and determine the titer of antibody to this epitope in various populations of human immunodeficiency virus type 1 (HIV-1)-positive patients. There was a good correlation between SIEIA and a commercially available competition assay that uses recombinant p24 protein and polyclonal antibody to HIV-1 antigen, demonstrating the importance of the described epitope. Analysis of sera from French patients showed a decline of antibody to the AAEWDRVHP sequence associated with the progression of AIDS. No decrease was observed with serum samples from African patients. An immune response to the epitope was detected by SIEIA early in the course of seroconversion. Although our SIEIA uses a single p24 epitope, these data are in accordance with previously published studies in which antibodies to the whole p24 were analyzed. Sera reacting to p24 only (indeterminate profiles by Western blot [immunoblot]) did not bind to AAEWDRVHP. This epitope, which is conserved between HIV-1 and HIV-2/simian immunodeficiency virus, appears to be a major antigenic domain of p24. The area containing the sequence AAEWDRVHP and the corresponding monoclonal antibody may serve as a convenient alternative to whole purified p24 and polyclonal antibody in diagnostic and prognostic assays.
The major core protein p24 of human immunodeficiency virus type 1 (HIV-1) is a structural protein useful in the diagnosis of HIV infection. Antibody to p24 appears rapidly after primary infection (7, 10) and usually disappears when HIV-1-associated immune deficiency progresses, making it an important prognosis marker (4, 5, 8, 9, 14, 17, 18). In a previous study, major epitopes of p24HIV` and p26HIV-2 were identified by using a panel of nine murine monoclonal antibodies (MAb) directed to p24HIV-4 and by using the Pepscan procedure (12). One of the epitopes with the sequence AAEWDRVHP is conserved in p24HIV-1 as well as p26HIV-2. This sequence or the area containing it appeared to be the more regularly antigenic domain recognized during natural HIV infection. In order to detect and determine the titer of antibody to this epitope, we have developed a sequential inhibition enzyme-linked immunoassay (SIEIA) which uses a peroxidase-conjugated MAb reacting to AAEWDRVHP. Several populations comprising French patients at various stages of HIV-1 infection and African patients suffering from HIV-1-related AIDS were studied. Results were compared with those obtained with a commercially available enzyme assay which allows the determination of antibody titer to the whole HIV-1 core protein.
patients were tested twice, at two different CDC stages: they were initially tested at either the CDC II or the CDC III stage, and the second samples were collected between 2 and 5 years later, when the patients evolved to stage CDC III or CDC IV. The patients belonged to various known risk groups (Table 1). Serum samples were taken from 50 HIV1-positive patients from central Africa at stage CDC IV (2). In addition, 21 sequential serum specimens obtained from eight French patients at the early stage of seroconversion to HIV-1 were also tested. The specificity of the assay was evaluated with HIV-1negative serum specimens collected from 262 French subjects and from 22 subjects from central Africa and with 25 serum samples from French subjects which reacted only to p24 by Western blot (immunoblot) (nonspecific reactivities). All the negative sera were collected from healthy blood donors. SIEIA. (i) Preparation of the solid phase. A rabbit was immunized with five 100-rig injections of protein p24HIV` purified by electroelution. The antiserum was positive at a 10-6 dilution in an in-house enzyme-linked immunosorbent assay using microtitration plates coated with HIV-1 viral lysate (3). The immunoglobulins G were purified by ionexchange chromatography; they reacted in Western blot to the gag precursor p55HIV- and to p24HIV-1. Two hundred microliters of this immunoglobulin G preparation was used to coat 96-well microtitration plates at a concentration of 18 ,ug/ml in 50 mM bicarbonate buffer, pH 9.6. After 20 h at 4°C, the plates were washed four times with 0.05 M sodium phosphate-buffered saline (PBS) (pH 7.4) containing 0.1% Tween 20 (PBS-T) and then quenched with 250 ,ul of PBS containing 5% bovine serum albumin (PBS-BSA) for 30 min
MATERIALS AND METHODS Patients. A total of 145 serum specimens from 123 HIV-1positive French patients at various stages of infection were tested; 71, 29, and 45 serum samples were collected at stages CDC II, CDC III, and CDC IV, respectively (6). Twenty-two *
Corresponding author. 488
IMMUNE RESPONSE TO A MAJOR EPITOPE of p24HIVd
VOL. 29, 1991
TABLE 2. Distribution of percentages of inhibition obtained by SIEIA
TABLE 1. Distribution of French patients according to risk groups and clinical stages
No. of patients per serum category
No. of patients per
Risk group
clinical stage CDC II CDC III CDC IV
Homosexual or bisexual men Intravenous drug users Transfusion recipients Hemophiliacs Heterosexual contacts Patients originating from endemic area Patients with no known risk
27 13 6 16 5 1 3
11 9 1 8 0 0 0
23 10 1 6 1 2 2
Total
71
29
45
at 37°C. After one cycle of washing with PBS-T, 150 RIl of HIV-1 viral lysate prepared as previously described (3) and diluted in PBS containing 2.5% BSA and 0.05% Tween 20 (PBS-BSA-T) was dispensed in the wells (0.3 jig of viral lysate per well). The plates were left for 20 h at room temperature. Four cycles of washing were carried out prior to use of the plates. (ii) Test procedure. The sequential inhibition assay was carried out with MAb 23A5G4, which was selected during a previous study of nine MAbs to p24HtVd (12). This MAb is directed to an epitope spanning the sequence AAEWD RVHP (amino acids 209 to 217 of p55). MAb 23A5G4 was conjugated to horseradish peroxidase by using the periodic method of Nakane and Kawaoi (16). A 70-jil volume of PBS-BSA-T and a 20-,u volume of serum sample were incubated for 15 min at 37°C in each well of the plates prepared as described above, before 50 pI of an appropriate dilution (1:500) of the conjugated MAb 23A5G4 in PBS-BSA-T was added. Dilution was chosen to give half-maximum binding that is an optical density of approximately 0.8 with negative sera. After 90-min at 37°C, followed by four cycles of washing, inhibition was revealed with 100 ,ul of a 2-mg/ml solution of ortho-phenylenediamine (Sigma) in 0.05 M sodium phosphate-0.02 M citric acid buffer, pH 5, to which 3 ,ul of hydrogen peroxide per ml was added extemporaneously. The color development was stopped after 30 min with 50 p.l of 4 N sulfuric acid, and plates were read at 492 nm. The percentage of inhibition and the antibody titer of every sample were evaluated by using a standard curve drawn from doubling dilutions from 1:4 to 1:512 of a highly positive control serum included in every plate; the absorbance value obtained with the 1:8 dilution of the positive control had to be less than 0.1. Three negative control serum samples were included; their absorbance values had to be between 0.5 and 1. When positive or negative standard values were not within the fixed ranges, the tests were not valid. The percentage of inhibition was calculated for every sample as follows: percent inhibition = {1-[(ODa - ODpc)/ (ODnc - ODpc)]} x 100, where ODa is the absorbance value of the sample, ODpc is absorbance value of the 1:8 dilution of the positive control, and ODnc is the mean absorbance value of the negative control. Sample optical densities were directly transformed into reciprocal antibody titers by using a titration curve drawn on a semilogarithmic scale from absorbance values of positive control dilutions. Correlation with a commercially available assay. SIEIA
489
% Inhibition
Central African
French
CDC CDC CDC HIV-1 CDC II IV negative IV III
HIV-1 negative
p24 reactive only
81-90 91-100
197 51 12 2 0 0 0 0 0 0
25 0 0 0 0 0 0 0 0 0
11 3 1 0 5 1 6 4 4 36
10 2 2 1 0 1 1 1 1 10
23 5 5 0 0 3 3 1 1 4
22 0 0 0 0 0 0 0 0 0
7 1 0 1 2 1 1 1 1 35
Total
262
25
71
29
45
22
50
0-10 11-20 21-30 31-40 41-50 51-60 61-70
71-80
results were correlated with those of a commercially available assay (HIV-1 anti-core EIA; Abbott) by using a panel of 97 unselected HIV-1-positive serum specimens collected from patients at various clinical stages. In the Abbott competition assay, beads coated with whole recombinant p24 protein are incubated with a mixture of a serum sample and a polyclonal human antibody to HIV-1 core conjugated with horseradish peroxidase. Antibody titers were calculated as previously described (1). Correlation was studied by using the Spearman rank test (19). RESULTS All 284 HIV-negative serum samples showed less than 40% inhibition (Table 2). Of the negative samples, 95.1 and 99.3% showed less than 20 and 30% inhibition, respectively; only two negative samples showed between 30 and 40% inhibition. Samples with more than 40% inhibition were considered positive for antibody to p24. None of 25 serum samples reacting only to p24 by Western blot prevented the binding of the 23A5G4 conjugate. All these samples showed less than 10% inhibition and prove the specificity of the assay, since it is now well recognized that sera reacting only to core protein p24 and p17 by Western blot do not indicate HIV exposure (11, 13, 15). Among the French serum samples, there was a clear decrease of the immune response to the p24 epitope AAEW DRVHP associated with the progression of AIDS. Seventynine percent of samples from CDC II patients, 48% of samples from CDC III patients, and only 27% of samples from CDC IV patients were positive in the assay (Fig. la through c). Statistical differences between CDC II and CDC IV patients were significant (P < 0.01). As has been described previously for a commercially available assay (2), antibody to p24 persisted in 82% of CDC IV patients from central Africa (Fig. ld), a significantly higher prevalence than that observed in CDC IV French patients (P < 10-7). French serum samples also showed a decrease of antibody titer associated with advancing stages of AIDS. For instance, 61% of CDC II patients and 65% of CDC III patients who were positive for antibody to p24 in the SIEIA presented an anti-p24 titer equal to or greater than 1:8, compared with only 32% of CDC IV patients (Fig. le through g). The median antibody titers were 1:32, 1:8, and 1:1 for CDC II, CDC III, and CDC IV patients, respectively. As de-
490
J. CLIN. MICROBIOL.
JANVIER ET AL.
a
e CDC E1/FRANCE
CDC I / FRANCE 80
SC 3-
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80 90 100 PERCENT INIMION
0-10 20 30 40 50 60 7
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64
128
ANTIBODY TITER
FIG. 1. Distribution of percentages of inhibition among various populations of HIV-1-positive patients (a through d) and distribution of antibody titer to AAEWDRVHP by category of serum reactive to this epitope (e through h). Results are shown for sera from French patients at stages CDC II (a and e), CDC III (b and f), and CDC IV (c and g) and sera from central African patients at stage CDC IV (d and h). The percentages of sera are indicated on the ordinate.
scribed above for qualitative results, anti-p24-positive Afripatients, even at stage CDC IV, had high anti-p24 titers: 86% of them had antibody titers equal to or greater than 1:8 (Fig. lh). Of the 22 patients tested twice, 17 tested positive by the SIEIA with the initial blood samples. Of these, nine patients were monitored for 4 to 5 years, and eight showed an evolution of their clinical status and concurrently a clear decrease or disappearance of their antibody titer; antibody to p24 declined in patients with high initial titers and disappeared in patients with low initial titers. Eight patients were monitored for 2 to 3 years. Although they presented a similar clinical evolution, their antibody titers remained stable. This seems to indicate that a minimal period of time is necessary before a diminution of anti-p24 titer can be observed. The five patients who were initially negative by SIEIA (one CDC can
II and four CDC III) remained negative and evolved to stage CDC IV in less than 3 years. Anti-p24 titers from the SIEIA were compared with results obtained with the HIV-1 anti-core EIA from Abbott for 97 HIV-1-positive serum samples (Fig. 2). Although the Abbott assay appeared slightly more sensitive than the SIEIA, there was a good correlation between the two assays, as assessed by a Spearman rank correlation coefficient of rs = 0.89 (P < 0.001). These data suggest that the antibody titer to AAEWDRVHP has almost the same significance as the antibody titer to the whole p24, indicating that the described epitope is a major antigenic domain of this protein. Twenty-one serum samples were collected sequentially from eight seroconverters. Profiles of the first serum samples are typical of early seroconversion, i.e., show the presence
IMMUNE RESPONSE TO A MAJOR EPITOPE of p24HIV-1
VOL. 29, 1991 S I E IA 0
0
2
1
4
8
case except one (case 5), antibody titers by SIEIA in subsequent serum samples rose.
16 32 >32
DISCUSSION In a previous study, we described a conserved epitope on the major core proteins of HIV-1 and HIV-2 that appeared to be immunogenic during natural infection (12). This epitope, identified with MAb and the Pepscan procedure, corresponds to the sequence AAEWDRVHP in p24HIV-1. We used a peroxidase-conjugated MAb (23A5G4) directed to this epitope to generate an SIEIA in order to detect and determine the titer of antibody to p24HIV-1 in human sera. Results obtained with sera from French patients confirmed the correlation between an absence or decrease of antibody to p24 and the advancing stage of HIV-1 infection. For instance, the prevalence of antibody to AAEWDRVHP fell from 79 to 48 and 27% in CDC II, CDC III, and CDC IV patients, respectively. Among patients who were positive for antibody to AAEWDRVHP, titers were also higher in patients with mild symptoms (CDC II and CDC III). Twentytwo patients who clinically evolved to advanced stages of the disease were monitored for 2 to 5 years. Five who were initially negative for antibody to AAEWDRVHP developed overt AIDS in less than 3 years. Of the 17 patients whose first serum samples were positive by SIEIA at the initial sampling, 10 presented a decrease in or a disappearance of antibody to AAEWDRVHP in correlation with the progression of the disease. Detection of antibody to AAEW DRVHP by the SIEIA in the earliest Western-blot-positive samples from HIV-1 seroconversions further indicated that
massse
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FIG. 2. Comparison of the antibody titers to p24 obtained for 97 HIV-1-positive serum specimens with the SIEIA and the Abbott HIV-1 anti-core EIA.
of antibody to gpl60 only or antibody to gp160 and p24 (Fig. 3). Four of eight early samples were positive for p24 in the Western blot and positive in our SIEIA, with titers between 1:2 and 1:4. Two of the four initial samples that were negative in our SIEIA were also negative (case 1) or very weakly positive (case 7) in the Western blot assay. The four patients who initially tested negative by SIEIA had positive subsequent blood samples (cases 1, 2, 3, and 7). In every
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Antibody titer FIG. 3. Evolution of serological profiles in sequentially collected sera from eight seroconverters. In every case, the first Western blot shown corresponded to the first serum sample found positive for antibody to HIV-1. The dates (day-month-year) of blood sampling and the antibody titers to AAEWDRVHP are indicated below the corresponding Western blots. 0
1
8 32
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492
JANVIER ET AL.
this epitope is recognized early in the course of natural infection. It has been previously reported that high anti-p24 titers are usually conserved in HIV-1-positive patients from Africa even at advanced stages of the disease (2). Results obtained with the SIEIA confirmed these data and indicated furthermore that epitope AAEWDRVHP is conserved among HIV-1 strains presently circulating in Africa and is highly immunogenic during natural infection in central African residents. There was a good correlation between our SIEIA, based on a conjugated MAb, and a commercially available assay that uses a polyclonal antibody to the whole HIV-1 core protein, indicating that the area containing the sequence AAEWDRVHP is a major antigenic domain of p24HIWl. This epitope is also conserved in HIV-2/simian immunodeficiency virus strains (12). Peptide AAEWDRVHP and its corresponding MAb may thus serve as a convenient alternative to purified viral p24 and polyclonal antibody in developing diagnostic and prognostic assays. ACKNOWLEDGMENTS We are grateful to M. F. Maitre, L. Hervioux, P. Choutet, and C. Guerois for the clinical information on sera from HIV-1-positive French patients, to P. Kocheleff and E. Petat for the African sera, and to M. H. Charles for technical help. We thank A. Pompidou for encouragement and help at the initiation of our program. This work was supported by grant 86C-0926 from the Ministere de la Recherche et de l'Enseignement Superieur and by funds from Universite Francois Rabelais and bioMerieux. Blandine Janvier was supported by a doctoral fellowship from the Fondation Merieux, Lyon, France. REFERENCES 1. Allain, J. P., Y. Laurian, D. A. Paul, F. Verroust, M. Leuther, C. Gazengel, D. Senn, M. J. Larrieu, and C. Bosser. 1987. Long-term evaluation of HIV antigen and antibodies to p24 and gp4l in patients with hemophilia. N. Engl. J. Med. 317:11141121. 2. Baillou, A., F. Barin, J. P. Allain, E. Petat, P. Kocheleff, P. Kadende, and A. Goudeau. 1987. Human immunodeficiency virus antigenemia in patients with AIDS and AIDS-related disorders: a comparison between European and central African populations. J. Infect. Dis. 156:830-833. 3. Barin, F., S. M'Boup, F. Denis, P. Kanki, J. S. Allan, T. H. Lee, and M. Essex. 1985. Serological evidence for virus related to simian T-lymphotropic virus III in residents of West Africa. Lancet ii:1387-1389. 4. Biggar, R. J., M. Melbye, P. Ebbesen, S. Alexander, J. 0. Nielsen, P. Sarin, and V. Faber. 1985. Variation in human T lymphotropic virus III (HTLV-III) antibodies in homosexual men: decline before onset of illness related to acquired immune deficiency syndrome (AIDS). Br. Med. J. 291:997-998. 5. Burke, D. S., R. R. Redfield, P. Putman, and S. S. Alexander. 1987. Variations in Western blot banding patterns of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus. J. Clin. Microbiol. 25:81-84.
J. CLIN. MICROBIOL. 6. Centers for Disease Control. 1986. Classification system for human T-lymphotropic virus type III/lymphadenopathy-associated virus infections. Ann. Intern. Med. 105:234-237. 7. Chou, M. J., T. H. Lee, A. Hatzakis, T. Mandalaki, M. F. McLane, and M. Essex. 1988. Antibody response in early human immunodeficiency virus type I infection in hemophiliacs. J. Infect. Dis. 157:805-811. 8. de Wolf, F., J. M. A. Lange, J. T. M. Houweling, R. A. Coutinho, P. T. Schellekens, J. Van Der Noordaa, and J. Goudsmit. 1988. Numbers of CD4 cells and the levels of core antigens and of antibodies to the human immunodeficiency virus as predictors of AIDS among seropositive homosexual men. J. Infect. Dis. 158:615-622. 9. Forster, M. S., L. M. Osborne, R. Chiengson-Popov, C. Kenny, R. Burnell, D. J. Jeffries, A. J. Pinching, J. R. W. Harris, and J. N. Weber. 1987. Decline of anti-p24 antibody precedes antigenemia as correlate to prognosis in HIV1 infection. AIDS 1:235-240. 10. Gaines, H., A. Sonnerborg, J. Czajkowski, F. Chiodi, E. M. Fenyo, M. von Sydow, P. 0. Pehrson, L. Moberg, B. Asjo, and M. Forsgren. 1987. Antibody response in primary human immunodeficiency virus infection. Lancet i:1249-1253. 11. Jackson, J. B., K. L. MacDonald, J. Cadwell, C. Sullivan, W. E. Kline, M. Hanson, K. J. Sannerud, S. L. Stramer, N. J. Fildes, S. Y. Kwok, J. J. Sninsky, R. J. Bowman, H. F. Polesky, H. H. Balfour, and M. T. Osterholm. 1990. Absence of HIV infection in blood donors with indeterminate western blot tests for antibody to HIV1. N. Engl. J. Med. 322:217-222. 12. Janvier, B., P. Archinard, B. Mandrand, A. Goudeau, and F. Barin. 1990. Linear B-cell epitopes of the major core protein of human immunodeficiency virus types 1 and 2. J. Virol. 64:42584263. 13. Josephson, S. L., N. S. Swack, M. T. Ramirez, and W. J. Hausler, Jr. 1989. Investigation of atypical Western blot (immunoblot) reactivity involving core proteins of human immunodeficiency virus type 1. J. Clin. Microbiol. 27:932-937. 14. Lange, J. M. A., D. A. Paul, H. G. Huisman, F. de Wolf, H. Van Den Berg, R. A. Coutinho, S. A. Danner, J. Van Der Noordaa, and J. Goudsmit. 1986. Persistent HIV antigenemia and decline of HIV core antibodies associated with transition to AIDS. Br. Med. J. 293:1459-1462. 15. Lefrere, J. J., A. M. Courouce, G. Lucotte, C. Boitard, C. Kaplan, J. C. Nicolas, F. Bricout, P. Lambin, C. Doinel, J. Y. Muller, J. F. Bach, and C. Salmon. 1988. Follow-up of subjects with isolated and persistent anti-core (anti-p24 or anti-p17) antibodies to HIV. AIDS 2:287-290. 16. Nakane, P. K., and A. Kawaoi. 1974. Peroxidase labelled antibody: a new method of conjugation. J. Histochem. Cytochem. 22:1084-1091. 17. Pedersen, C., C. M. Nielsen, B. F. Vestergaard, J. Gerstoft, K. Krogsgaard, and J. 0. Nielsen. 1987. Temporal relation of antigenemia and loss of antibodies to core antigens to development of clinical disease in HIV infection. Br. Med. J. 295:567569. 18. Schupbach, J., M. Popovic, R. V. Gilden, M. A. Gonda, M. G. Sarngadharan, and R. C. Gallo. 1984. Serological analysis of a subgroup of human T-lymphotropic (HTLV-III) associated with AIDS. Science 224:503-505. 19. Siegel, S. 1956. Nonparametric statistics for the behavioural sciences, p. 202-213. McGraw-Hill Book Co., New York.
