JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1990, p. 2107-2110 0095-1137/90/092107-04$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 28, No. 9
Immunoprecipitation of Human Immunodeficiency Virus Type 2 Glycoproteins by Sera Positive for Human Immunodeficiency Virus Type 1 ROMILIO T. ESPEJO* AND PAULINA URIBE Centro de Estudios Cientificos de Santiago, Casilla 16443, Santiago 9, and Virology Section, Faculty of Medicine, University of Chile, Santiago, Chile Received 27 November 1989/Accepted 29 May 1990
Analysis by radioimmunoprecipitation of serum samples from 27 different human immunodeficiency virus type 1 (HIV-l)-infected individuals residing in Chile showed that the sera of 26% of these individuals also react with glycoprotein gpl25 of HIV type 2 (HIV-2). This cross-reaction seems to reflect a qualitative difference among infected individuals, because the titer of antibodies against gpl20 of HIV-1 in the cross-reacting samples did not differ significantly from that in the non-cross-reacting samples. Most of the HIV-l-seropositive sera, including many that did not react with gp125 of HIV-2, reacted with gpl40, the precursor of HIV-2 glycoproteins. The observed cross-reactions allowed us to distinguish three groups of HIV-l-infected individuals: (i) those whose sera react with both gpl40 and gp125, (ài) those whose sera react with gpl40, and (mii) those whose sera react with neither of these glycoproteins. The possible cause and significance of these
differences is under study. It is accepted that at least two immunodeficiency virus cause acquired immunodeficiency syndrome (AIDS) in humans, human immunodeficiency virus type 1 (HIV-1) and HIV type 2 (HIV-2). Sera from HIV-infected individuals contain different amounts of antibodies to a number of HIV proteins encoded by the HIV genes gag, pol, and env (for a review, see reference 8). It was originally thought that cross-reactivity between the two HIV types was restricted to the proteins encoded by the gag and pol genes, because cross-reactions were not observed between env glycoproteins in the early characterization of HIV-2 (3). Hence, reaction of a serum with the glycoproteins of both HIV types was considered, until recently, strongly suggestive of a dual infection (5). However, since 1987 (7) increasing evidence has been published indicating that some HIV-l-infected individuals may have antibodies that react with the glycoproteins of HIV-2 (1, 2, 6, 10, 14). Also, evidence of reaction with HIV-2 env proteins detected by enzyme-linked immunosorbent assay and Western immunoblot assay in sera from patients infected with HIV-1 alone, according to DNA analysis after amplification by polymerase chain reaction, was presented at the 4th International Conference on AIDS (cited in reference 2). Besides the structural glycoproteins gp125 and gp36, two glycoproteins with apparent molecular weights of 140,000 and 300,000 (gpl4O and gp300, respectively), which are the monomeric and dimeric forms of the precursor for the virion glycoproteins, have been detected in HIV-2-infected cells (12). In this article, we describe the cross-reactivity with HIV-2 glycoproteins observed in some sera from HIV-1seropositive individuals and discuss its possible causes and types can
consequences.
Sera were obtained during the years 1987 through 1989 from HIV-infected individuals, with or without AIDS or AIDS-related complex, residing in Chile. HIV infection was diagnosed by enzyme-linked immunosorbent assay and sub-
*
Corresponding author.
sequent immunoblot, indirect immunofluorescence, or radioimmunoprecipitation. An HIV-2 human serum reference panel was obtained from the AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, Bethesda, Md. The radioimmunoprecipitation assay (RIPA) was performed as previously described (4): either HIV-lBRU or HIV-2ROD, obtained from L. Montagnier, was used to infect CEM cells in RPMI 1640 containing 10% fetal calf serum and 2 ,ug of Polybrene per ml. After 7 to 10 days, when the cytopathic effect was clearly noticeable, the infected cells were transferred to minimum essential-Dulbecco medium without cysteine and labeled by the addition of 0.25 mCi of [35S]cysteine. After 24 h, the medium was clarified of cells by centrifugation and the virus was precipitated by the addition of 30% polyethylene glycol in 0.4 M NaCI to a final concentration of 10% polyethylene glycol. After 16 h at 4°C, the precipitated virus was collected by centrifugation for 1 h at 6,000 x g and suspended in RIPA buffer (4). This preparation was cleared of debris by centrifugation at 6,000 x g for 5 min. About 100,000 cpm of labeled proteins in 50 ,ul of RIPA buffer was incubated overnight with 5 pul of serum at 4°C. After the addition of 70 ,ul of protein A-Sepharose (0.1 g/ml of RIPA buffer) and further incubation at 4°C for 3 h with occasional shaking, the protein A-Sepharose with bound immunoglobulin G was centrifuged and washed four times with RIPA buffer. The pellet was then suspended in disrupting buffer (9), heated for 3 min at 100°C, and centrifuged, and the released proteins were subjected to electrophoresis in 10 or 12.5% polyacrylamide gels (4). After electrophoresis, the gels were treated with Amplify (Amersham) and exposed for autoradiography by using an intensifying screen. Immunoprecipitation of HIV-1 gpl6O or HIV-2 gpl4O was tested with antigens obtained from cells labeled and infected as described above. The labeled cells were centrifuged and subsequently lysed by incubation overnight in RIPA buffer at 4°C. After lysis, the cell debris was centrifuged down at 600 x g for 10 min and the supernatant with the labeled proteins was stored at -20°C. 2107
J. CLIN. MICROBIOL.
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FIG. 1. RIPA of sera from different HIV-1-seropositive individuals with either HIV-1BRU or HIV-2RoD [35S]cysteine-labeled proteins. Sera k and b reacted with the gp125 glycoprotein of HIV-2. Viral polypeptides are indicated on the left. Electrophoresis was performed in 12.5% polyacrylamide gels.
Each serum sample used in this study was also tested for cross-reactivity with the LiaTek HIV1+2, based on the line immunoassay technique of Innogenetics N. V., Brussels, Belgium, and distributed by Organon Teknika B. V., Boctel, The Netherlands. Analysis of HIV-1-positive serum samples from 27 individuals showed that 7 of these patients had antibodies that reacted to different extents with gp125 of HIV-2, while all reacted to similar extents with gpl20 of HIV-1. However, analysis of these doubly reactive sera by both immunoblot and indirect fluorescence failed to confirm HIV-2 infection (6; further analysis of additional patients). Furthermore, none of these samples reacted with the synthetic peptide of the HIV-2 transmembrane protein included in the LiaTek HIV1+2 line immunoassay (data not shown). Figure 1 shows some of the results obtained with different serum samples after radioimmunoprecipitation and subsequent gel electrophoresis (RIPA); sera k and b show the described crossreactivity. However, cross-reactivity is a relative term, and its observation depends on the sensitivity of the procedure used. Hence, its detection in some samples by RIPA could be simply due to the presence of a much higher content of antibodies against gpl20 in the cross-reactive samples. To explore this possibility, the titers against gpl20 of HIV-1 of two doubly and five singly reactive sera were compared. No significant differences between these two groups was observed when the RIPA was performed with serially diluted samples. Figure 2 shows the results obtained for a doubly reactive serum (b) and a singly reactive serum (r). These results indicate that the observed cross-reaction (also shown in Fig. 2, lanes 1 and 2) is not due to a significantly higher amount of antibodies against gpl20 in the cross-reactive sample. The structural glycoproteins of HIV are derived from larger glycoprotein precursors, gpl6O and gpl40 in HIV'BRU and HIV-2ROD, respectively (12, 15). To test the reaction of the two groups of sera with gpl40, a RIPA was performed with labeled antigens obtained from disrupted infected cells, which contain a large amount of the glycoprotein precursor. Figure 3A shows that both singly and doubly reactive sera were able to precipitate gpl4O of HIV-2. Unexpectedly, the serum sample r that did not react with
FIG. 2. RIPA using serial dilutions of serum b (doubly reactive) and serum r (singly reactive) with HIV-1. Lanes 1 and 2 show the patterns obtained with HIV-2 using 1/10 dilutions of sera b and r, respectively. The indicated dilutions correspond to those obtained in the serum-virus reaction mixture. Electrophoresis was performed in 12.5% polyacrylamide gels.
gp125 was able to precipitate its precursor, gpl40. The titer of antibodies against this last protein was, however, much lower in the singly reactive serum r (Fig. 3B). A significant amount of label which was retained on top of the resolving gel and that precipitated proportionally to the amount of immunoprecipitated gpl40 was consistently observed with the preparations of lysed HIV-2-infected cells. Further testing with antigen obtained from disrupted infected cells of the serum samples that did not react with gp125 showed that most reacted with gpl40 of HIV-2 (Fig. 4A). As expected, all the samples that precipitated gp125 also reacted with gpl40 (results not shown). The type of reaction with the HIV-2 glycoproteins has remained invariable in the sequential samples from the five individuals followed up to date, from 6 to 18 months (results not shown). To determine the possible occurrence of the observed cross-reactivity among HIV-2-positive sera, the six samples included in the HIV-2 serum reference panel of the AIDS Research and Reference Reagent Program were examined by the method described above. Only one of these samples (Guinea Bissau 7396) reacted, but very feebly, with the HIV-1 glycoprotein precursor (Fig. 4B), while samples Senegal 4D, Senegal 3C, Ivory Coast 051, Ivory Coast 541, and Guinea Bissau 7395 showed no detectable reaction, as illustrated for the Ivory Coast 541 sample in Fig. 4B. To determine the extent of the observed cross-reaction, the titers against both HIV-1 and HIV-2 glycoproteins in seven different serum samples reacting with gp125 and/or gpl40 from HIV-1-infected individuals were determined by performing a RIPA with serial dilutions of the sera. Titers against HIV-1 glycoproteins gpl20 and gpl6O of 6,250 and 31,250, approximately, were obtained for all the samples, while the titers against the corresponding proteins of HIV-2, gp125 and gpl40, were always at least 25 times smaller. A
VOL. 28, 1990
NOTES
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FIG. 3. Reactions observed by RIPA of serum b (doubly reactive) and serum r (singly reactive) with the env precursor and viral proteins of either HIV-1 or HIV-2. (A) Lanes: 1 and 1', reactions with HIV-1 antigens released into the medium; 2 and 2', reactions with HIV-1 antigens obtained from lysed infected cells; 3 and 3', reactions with HIV-2 antigens released into the medium; 4 and 4', reactions with HIV-2 antigens obtained from lysed infected cells. (B) Reactions with HIV-2 antigens obtained from lysed infected cells by using 1/5 serial dilutions. Dilutions are from right to left. Electrophoresis was performed in 10% polyacrylamide gels.
similar observation was obtained with the sample of the HIV-2 serum reference panel showing a faint cross-reaction; while the sample reacted with the HIV-2 glycoproteins to a titer of 1,250, it did not react with gpl20 or gpl6O at a dilution of 1/50. The results presented in this work indicate that the puta-
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FIG. 4. (A) Reactions with the glycoprotein precursor of HIV-2, gpl40, observed by RIPA with serum samples that did not react with glycoprotein gp125 of HIV-2. Reactions were performed by incubating 1/10 dilutions of the serum samples with labeled antigens obtained from cells infected with either HIV-1 or HIV-2. Electrophoresis was performed in 10% polyacrylamide gels. (B) Reactions with the glycoprotein precursors of HIV-1 (gpl6O) and HIV-2 (gpl4O) observed by RIPA in both HIV-1- and HIV-2-positive serum samples. GB and IC stand for samples Guinea Bissau 7396 and Ivory Coast 541 of the HIV-2 human serum reference panel, respectively. Reactions were performed as indicated for Fig. 4A. Only the upper portions of the gels are shown.
tive common epitopes in the larger glycoproteins of HIV-1 and HIV-2 can be detected with greater sensitivity by RIPA than by Western immunoblot assay and that sera from different HIV-1-infected individuals can react with these epitopes to different extents. This cross-reactivity was, however, never higher than 1/25 of the titer obtained with the homologous virus. The observed large differences in the extent of reaction with gp125 of HIV-2 among the tested sera reflect a difference in the quality of antibodies in the individual, since these differences do not depend on the titer of antibodies directed against gpl20 of HIV-1. The cause of this difference is uncertain; it might be due to differences either in the HIV-1 infecting strain or in the individual serological response. Since common epitopes may represent conserved regions with essential functions, the presence of antibodies against these epitopes might have prognostic value, especially if this is due to a particular serological response rather than to differences in the infecting strain. According to our results, it is possible to distinguish three groups of HIV-1positive individuals: (i) those whose sera react with HIV-2 gpl40 and gp125, (ii) those whose sera react with HIV-2 gpl40, and (iii) those whose sera react with neither of these proteins. The results of the analysis of sequential serum samples suggest that the type of cross-reactivity is fairly constant. Attempts to relate the observed type of crossreactivity with the stage of evolution of infection have failed to show a simple relationship. We are following up individuals in each of these groups to better determine the constancy or variability of the cross-reactivity with both time and evolution of the infection. The present results also show that immunoprecipitation of the gpl40 precursor of the HIV-2 glycoproteins by sera of HIV-1-infected individuals is quite common and hence not a fair indication of a possible HIV-2 infection when HIV-1positive sera are being tested. This larger extent of crossreaction with the precursor could be due either to the
2110
J. CLIN. MICROBIOL.
NOTES
existence of a larger cross-reaction between some regions of the transmembrane proteins, also contained in the precursor, or to the presentation of common epitopes in the precursor that are masked in the end product. A recent report on the different antigenicities observed with the glycoproteins of feline leukemia virus according to their degrees of glycosylation or processing (11) favors the latter possibility; however, cross-reactivity of some human sera containing antibodies to HIV-1 with HIV-2 peptides derived from the transmembrane protein of HIV-2 was also recently reported (13). The label present in the HIV-2-infected cell lysates, which is immunoprecipitated but retained on top of the resolving gel, probably consists of the glycoprotein precursor oligomer gp300. These studies were supported in part by the Office for Scientific and Technical Cooperation of the Ministry of Foreign Relations of the Government of France, the Office in Chile of the Commission of the European Communities, and the Fondo Nacional para el Desarrollo Cientifico y Tecnologico, project 88/0093, Santiago, Chile. We thank Cecilia Sepulveda of the Laboratory of Immunology, Hospital Clinico, Universidad de Chile, for supplying different serum samples and for valuable comments and Cecilia Hidalgo for critical reading of the manuscript. It is a pleasure to acknowledge the kind gift of HIV-lBRU, HIV-2ROD, and CEM cells from Luc Montagnier and the receipt of the HIV-2 serum reference panel from S. Osmanov, World Health Organization, through the AIDS Research and Reference Reagent Program. LITERATURE CITED 1. Butto, S., P. Verani, F. Titti, G. Rezza, L. Sernicola, and G. B. Rossi. 1988. Simultaneous seropositivity to HIV-1 and HIV-2 in Italian drug abusers. AIDS 2:139-140. 2. Castro, B. A., C. Ceng-Mayer, L. A. Evans, and J. A. Levy. 1988. HIV heterogeneity and viral pathogenesis. AIDS 2(Suppl. 1):517-527. 3. Clavel, F., D. Guetard, F. Brun-Vezinet, S. Chamaret, M.-A. Rey, M. O. Santos-Ferreira, A. G. Laurent, C. Dauguet, C. Katlama, C. Rouzioux, D. Klatzmann, J. L. Champalimaud, and L. Montagnier. 1986. Isolation of a new human retrovirus from West African patients with AIDS. Science 233:343-346. 4. Clavel, F., K. Mansinho, S. Chamaret, D. Guetard, V. Favier, J.
5.
6. 7.
8. 9.
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Nina, M. O. Santos-Ferreira, J. L. Champalimaud, and L. Montagnier. 1987. Human immunodeficiency virus type 2 infection associated with AIDS in West Africa. N. Engl. J. Med. 316: 1180-1185. Cortes, E., R. Detels, D. Aboulafia, X. L. Li, T. Moudgil, M. Alam, C. Bonecker, A. Gonzaga, L. Oyafuso, M. Tondo, C. Boite, N. Hammershlak, C. Capitani, D. J. Slamon, and D. D. Ho. 1989. HIV-1, HIV-2, and HTLV-1 infection in high-risk groups in Brazil. N. Engl. J. Med. 320:953-958. Espejo, R. T., P. Uribe, and C. Sepulveda. 1989. HIV infection in Brazil. N. Engl. J. Med. 321:830-831. Foucault, C., O. Lopez, G. Jourdan, J. J. Fournel, P. Perret, and J. C. Gluckman. 1987. Double HIV-1 and HIV-2 seropositivity among blood donors. Lancet ii:165-166. Jackson, J. B., and H. H. Balfour, Jr. 1988. Practical diagnostic testing for human immunodeficiency virus. Clin. Microbiol. Rev. 1:124-138. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:68"685. O'Brien, T. R., W. Hayward, S. D. Holmberg, C. A. Schable, G. Schochetman, and J. W. Curran. 1989. HIV infection in Brazil. N. Engl. J. Med. 321:831. Poss, M. L., J. I. Mullins, and E. A. Hoover. 1989. Posttranslational modifications distinguish the envelope glycoprotein of the immunodeficiency disease-inducing feline leukemia virus retrovirus. J. Virol. 63:189-195. Rey, M.-A., B. Krust, A. G. Laurent, L. Montagnier, and A. G. Hovanessian. 1989. Characterization of human immunodeficiency virus type 2 envelope glycoproteins: dimerization of the glycoprotein precursor during processing. J. Virol. 63:647-658. Schulz, T. F., W. Oberhuber, J. M. Hofbauer, P. Hengster, C. Larcher, L. C. Gurler, R. Tedder, H. Wachter, and M. P. Dierich. 1989. Recombinant peptides derived from the env-gene of HIV-2 in the serodiagnosis of HIV-2 infections. AIDS 3:165172. Tedder, R. S., T. O. O'Connor, H. Hughes, H. N'jie, T. Corrah, and H. Whittle. 1988. Envelope cross-reactivity in Western blot for HIV-1 and HIV-2 may not indicate dual infection. Lancet ii:927-930. Veronese, F. M., A. De Vico, T. Copeland, S. Oroszlan, R. C. Gallo, and M. Sarngadharan. 1985. Characterization of gp4l as the transmembrane protein coded by the HTLV-III/Lav envelope gene. Science 229:1402-1405.
Vol. 28, No. 9
Immunoprecipitation of Human Immunodeficiency Virus Type 2 Glycoproteins by Sera Positive for Human Immunodeficiency Virus Type 1 ROMILIO T. ESPEJO* AND PAULINA URIBE Centro de Estudios Cientificos de Santiago, Casilla 16443, Santiago 9, and Virology Section, Faculty of Medicine, University of Chile, Santiago, Chile Received 27 November 1989/Accepted 29 May 1990
Analysis by radioimmunoprecipitation of serum samples from 27 different human immunodeficiency virus type 1 (HIV-l)-infected individuals residing in Chile showed that the sera of 26% of these individuals also react with glycoprotein gpl25 of HIV type 2 (HIV-2). This cross-reaction seems to reflect a qualitative difference among infected individuals, because the titer of antibodies against gpl20 of HIV-1 in the cross-reacting samples did not differ significantly from that in the non-cross-reacting samples. Most of the HIV-l-seropositive sera, including many that did not react with gp125 of HIV-2, reacted with gpl40, the precursor of HIV-2 glycoproteins. The observed cross-reactions allowed us to distinguish three groups of HIV-l-infected individuals: (i) those whose sera react with both gpl40 and gp125, (ài) those whose sera react with gpl40, and (mii) those whose sera react with neither of these glycoproteins. The possible cause and significance of these
differences is under study. It is accepted that at least two immunodeficiency virus cause acquired immunodeficiency syndrome (AIDS) in humans, human immunodeficiency virus type 1 (HIV-1) and HIV type 2 (HIV-2). Sera from HIV-infected individuals contain different amounts of antibodies to a number of HIV proteins encoded by the HIV genes gag, pol, and env (for a review, see reference 8). It was originally thought that cross-reactivity between the two HIV types was restricted to the proteins encoded by the gag and pol genes, because cross-reactions were not observed between env glycoproteins in the early characterization of HIV-2 (3). Hence, reaction of a serum with the glycoproteins of both HIV types was considered, until recently, strongly suggestive of a dual infection (5). However, since 1987 (7) increasing evidence has been published indicating that some HIV-l-infected individuals may have antibodies that react with the glycoproteins of HIV-2 (1, 2, 6, 10, 14). Also, evidence of reaction with HIV-2 env proteins detected by enzyme-linked immunosorbent assay and Western immunoblot assay in sera from patients infected with HIV-1 alone, according to DNA analysis after amplification by polymerase chain reaction, was presented at the 4th International Conference on AIDS (cited in reference 2). Besides the structural glycoproteins gp125 and gp36, two glycoproteins with apparent molecular weights of 140,000 and 300,000 (gpl4O and gp300, respectively), which are the monomeric and dimeric forms of the precursor for the virion glycoproteins, have been detected in HIV-2-infected cells (12). In this article, we describe the cross-reactivity with HIV-2 glycoproteins observed in some sera from HIV-1seropositive individuals and discuss its possible causes and types can
consequences.
Sera were obtained during the years 1987 through 1989 from HIV-infected individuals, with or without AIDS or AIDS-related complex, residing in Chile. HIV infection was diagnosed by enzyme-linked immunosorbent assay and sub-
*
Corresponding author.
sequent immunoblot, indirect immunofluorescence, or radioimmunoprecipitation. An HIV-2 human serum reference panel was obtained from the AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, Bethesda, Md. The radioimmunoprecipitation assay (RIPA) was performed as previously described (4): either HIV-lBRU or HIV-2ROD, obtained from L. Montagnier, was used to infect CEM cells in RPMI 1640 containing 10% fetal calf serum and 2 ,ug of Polybrene per ml. After 7 to 10 days, when the cytopathic effect was clearly noticeable, the infected cells were transferred to minimum essential-Dulbecco medium without cysteine and labeled by the addition of 0.25 mCi of [35S]cysteine. After 24 h, the medium was clarified of cells by centrifugation and the virus was precipitated by the addition of 30% polyethylene glycol in 0.4 M NaCI to a final concentration of 10% polyethylene glycol. After 16 h at 4°C, the precipitated virus was collected by centrifugation for 1 h at 6,000 x g and suspended in RIPA buffer (4). This preparation was cleared of debris by centrifugation at 6,000 x g for 5 min. About 100,000 cpm of labeled proteins in 50 ,ul of RIPA buffer was incubated overnight with 5 pul of serum at 4°C. After the addition of 70 ,ul of protein A-Sepharose (0.1 g/ml of RIPA buffer) and further incubation at 4°C for 3 h with occasional shaking, the protein A-Sepharose with bound immunoglobulin G was centrifuged and washed four times with RIPA buffer. The pellet was then suspended in disrupting buffer (9), heated for 3 min at 100°C, and centrifuged, and the released proteins were subjected to electrophoresis in 10 or 12.5% polyacrylamide gels (4). After electrophoresis, the gels were treated with Amplify (Amersham) and exposed for autoradiography by using an intensifying screen. Immunoprecipitation of HIV-1 gpl6O or HIV-2 gpl4O was tested with antigens obtained from cells labeled and infected as described above. The labeled cells were centrifuged and subsequently lysed by incubation overnight in RIPA buffer at 4°C. After lysis, the cell debris was centrifuged down at 600 x g for 10 min and the supernatant with the labeled proteins was stored at -20°C. 2107
J. CLIN. MICROBIOL.
NOTES
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FIG. 1. RIPA of sera from different HIV-1-seropositive individuals with either HIV-1BRU or HIV-2RoD [35S]cysteine-labeled proteins. Sera k and b reacted with the gp125 glycoprotein of HIV-2. Viral polypeptides are indicated on the left. Electrophoresis was performed in 12.5% polyacrylamide gels.
Each serum sample used in this study was also tested for cross-reactivity with the LiaTek HIV1+2, based on the line immunoassay technique of Innogenetics N. V., Brussels, Belgium, and distributed by Organon Teknika B. V., Boctel, The Netherlands. Analysis of HIV-1-positive serum samples from 27 individuals showed that 7 of these patients had antibodies that reacted to different extents with gp125 of HIV-2, while all reacted to similar extents with gpl20 of HIV-1. However, analysis of these doubly reactive sera by both immunoblot and indirect fluorescence failed to confirm HIV-2 infection (6; further analysis of additional patients). Furthermore, none of these samples reacted with the synthetic peptide of the HIV-2 transmembrane protein included in the LiaTek HIV1+2 line immunoassay (data not shown). Figure 1 shows some of the results obtained with different serum samples after radioimmunoprecipitation and subsequent gel electrophoresis (RIPA); sera k and b show the described crossreactivity. However, cross-reactivity is a relative term, and its observation depends on the sensitivity of the procedure used. Hence, its detection in some samples by RIPA could be simply due to the presence of a much higher content of antibodies against gpl20 in the cross-reactive samples. To explore this possibility, the titers against gpl20 of HIV-1 of two doubly and five singly reactive sera were compared. No significant differences between these two groups was observed when the RIPA was performed with serially diluted samples. Figure 2 shows the results obtained for a doubly reactive serum (b) and a singly reactive serum (r). These results indicate that the observed cross-reaction (also shown in Fig. 2, lanes 1 and 2) is not due to a significantly higher amount of antibodies against gpl20 in the cross-reactive sample. The structural glycoproteins of HIV are derived from larger glycoprotein precursors, gpl6O and gpl40 in HIV'BRU and HIV-2ROD, respectively (12, 15). To test the reaction of the two groups of sera with gpl40, a RIPA was performed with labeled antigens obtained from disrupted infected cells, which contain a large amount of the glycoprotein precursor. Figure 3A shows that both singly and doubly reactive sera were able to precipitate gpl4O of HIV-2. Unexpectedly, the serum sample r that did not react with
FIG. 2. RIPA using serial dilutions of serum b (doubly reactive) and serum r (singly reactive) with HIV-1. Lanes 1 and 2 show the patterns obtained with HIV-2 using 1/10 dilutions of sera b and r, respectively. The indicated dilutions correspond to those obtained in the serum-virus reaction mixture. Electrophoresis was performed in 12.5% polyacrylamide gels.
gp125 was able to precipitate its precursor, gpl40. The titer of antibodies against this last protein was, however, much lower in the singly reactive serum r (Fig. 3B). A significant amount of label which was retained on top of the resolving gel and that precipitated proportionally to the amount of immunoprecipitated gpl40 was consistently observed with the preparations of lysed HIV-2-infected cells. Further testing with antigen obtained from disrupted infected cells of the serum samples that did not react with gp125 showed that most reacted with gpl40 of HIV-2 (Fig. 4A). As expected, all the samples that precipitated gp125 also reacted with gpl40 (results not shown). The type of reaction with the HIV-2 glycoproteins has remained invariable in the sequential samples from the five individuals followed up to date, from 6 to 18 months (results not shown). To determine the possible occurrence of the observed cross-reactivity among HIV-2-positive sera, the six samples included in the HIV-2 serum reference panel of the AIDS Research and Reference Reagent Program were examined by the method described above. Only one of these samples (Guinea Bissau 7396) reacted, but very feebly, with the HIV-1 glycoprotein precursor (Fig. 4B), while samples Senegal 4D, Senegal 3C, Ivory Coast 051, Ivory Coast 541, and Guinea Bissau 7395 showed no detectable reaction, as illustrated for the Ivory Coast 541 sample in Fig. 4B. To determine the extent of the observed cross-reaction, the titers against both HIV-1 and HIV-2 glycoproteins in seven different serum samples reacting with gp125 and/or gpl40 from HIV-1-infected individuals were determined by performing a RIPA with serial dilutions of the sera. Titers against HIV-1 glycoproteins gpl20 and gpl6O of 6,250 and 31,250, approximately, were obtained for all the samples, while the titers against the corresponding proteins of HIV-2, gp125 and gpl40, were always at least 25 times smaller. A
VOL. 28, 1990
NOTES
*
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FIG. 3. Reactions observed by RIPA of serum b (doubly reactive) and serum r (singly reactive) with the env precursor and viral proteins of either HIV-1 or HIV-2. (A) Lanes: 1 and 1', reactions with HIV-1 antigens released into the medium; 2 and 2', reactions with HIV-1 antigens obtained from lysed infected cells; 3 and 3', reactions with HIV-2 antigens released into the medium; 4 and 4', reactions with HIV-2 antigens obtained from lysed infected cells. (B) Reactions with HIV-2 antigens obtained from lysed infected cells by using 1/5 serial dilutions. Dilutions are from right to left. Electrophoresis was performed in 10% polyacrylamide gels.
similar observation was obtained with the sample of the HIV-2 serum reference panel showing a faint cross-reaction; while the sample reacted with the HIV-2 glycoproteins to a titer of 1,250, it did not react with gpl20 or gpl6O at a dilution of 1/50. The results presented in this work indicate that the puta-
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FIG. 4. (A) Reactions with the glycoprotein precursor of HIV-2, gpl40, observed by RIPA with serum samples that did not react with glycoprotein gp125 of HIV-2. Reactions were performed by incubating 1/10 dilutions of the serum samples with labeled antigens obtained from cells infected with either HIV-1 or HIV-2. Electrophoresis was performed in 10% polyacrylamide gels. (B) Reactions with the glycoprotein precursors of HIV-1 (gpl6O) and HIV-2 (gpl4O) observed by RIPA in both HIV-1- and HIV-2-positive serum samples. GB and IC stand for samples Guinea Bissau 7396 and Ivory Coast 541 of the HIV-2 human serum reference panel, respectively. Reactions were performed as indicated for Fig. 4A. Only the upper portions of the gels are shown.
tive common epitopes in the larger glycoproteins of HIV-1 and HIV-2 can be detected with greater sensitivity by RIPA than by Western immunoblot assay and that sera from different HIV-1-infected individuals can react with these epitopes to different extents. This cross-reactivity was, however, never higher than 1/25 of the titer obtained with the homologous virus. The observed large differences in the extent of reaction with gp125 of HIV-2 among the tested sera reflect a difference in the quality of antibodies in the individual, since these differences do not depend on the titer of antibodies directed against gpl20 of HIV-1. The cause of this difference is uncertain; it might be due to differences either in the HIV-1 infecting strain or in the individual serological response. Since common epitopes may represent conserved regions with essential functions, the presence of antibodies against these epitopes might have prognostic value, especially if this is due to a particular serological response rather than to differences in the infecting strain. According to our results, it is possible to distinguish three groups of HIV-1positive individuals: (i) those whose sera react with HIV-2 gpl40 and gp125, (ii) those whose sera react with HIV-2 gpl40, and (iii) those whose sera react with neither of these proteins. The results of the analysis of sequential serum samples suggest that the type of cross-reactivity is fairly constant. Attempts to relate the observed type of crossreactivity with the stage of evolution of infection have failed to show a simple relationship. We are following up individuals in each of these groups to better determine the constancy or variability of the cross-reactivity with both time and evolution of the infection. The present results also show that immunoprecipitation of the gpl40 precursor of the HIV-2 glycoproteins by sera of HIV-1-infected individuals is quite common and hence not a fair indication of a possible HIV-2 infection when HIV-1positive sera are being tested. This larger extent of crossreaction with the precursor could be due either to the
2110
J. CLIN. MICROBIOL.
NOTES
existence of a larger cross-reaction between some regions of the transmembrane proteins, also contained in the precursor, or to the presentation of common epitopes in the precursor that are masked in the end product. A recent report on the different antigenicities observed with the glycoproteins of feline leukemia virus according to their degrees of glycosylation or processing (11) favors the latter possibility; however, cross-reactivity of some human sera containing antibodies to HIV-1 with HIV-2 peptides derived from the transmembrane protein of HIV-2 was also recently reported (13). The label present in the HIV-2-infected cell lysates, which is immunoprecipitated but retained on top of the resolving gel, probably consists of the glycoprotein precursor oligomer gp300. These studies were supported in part by the Office for Scientific and Technical Cooperation of the Ministry of Foreign Relations of the Government of France, the Office in Chile of the Commission of the European Communities, and the Fondo Nacional para el Desarrollo Cientifico y Tecnologico, project 88/0093, Santiago, Chile. We thank Cecilia Sepulveda of the Laboratory of Immunology, Hospital Clinico, Universidad de Chile, for supplying different serum samples and for valuable comments and Cecilia Hidalgo for critical reading of the manuscript. It is a pleasure to acknowledge the kind gift of HIV-lBRU, HIV-2ROD, and CEM cells from Luc Montagnier and the receipt of the HIV-2 serum reference panel from S. Osmanov, World Health Organization, through the AIDS Research and Reference Reagent Program. LITERATURE CITED 1. Butto, S., P. Verani, F. Titti, G. Rezza, L. Sernicola, and G. B. Rossi. 1988. Simultaneous seropositivity to HIV-1 and HIV-2 in Italian drug abusers. AIDS 2:139-140. 2. Castro, B. A., C. Ceng-Mayer, L. A. Evans, and J. A. Levy. 1988. HIV heterogeneity and viral pathogenesis. AIDS 2(Suppl. 1):517-527. 3. Clavel, F., D. Guetard, F. Brun-Vezinet, S. Chamaret, M.-A. Rey, M. O. Santos-Ferreira, A. G. Laurent, C. Dauguet, C. Katlama, C. Rouzioux, D. Klatzmann, J. L. Champalimaud, and L. Montagnier. 1986. Isolation of a new human retrovirus from West African patients with AIDS. Science 233:343-346. 4. Clavel, F., K. Mansinho, S. Chamaret, D. Guetard, V. Favier, J.
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