- Email: [email protected]
AIDS: an Immune Response against the Immune System. Role of a Precise Tridimensional Molecular Mimicry
doi:10.1006/jaut.2000.0500, available online at http://www.idealibrary.com on
Journal of Autoimmunity (2001) 16, 287–291
AIDS: an Immune Response against the Immune System. Role of a Precise Tridimensional Molecular Mimicry Pierre-Franc¸ois Serres HIPPOCAMPE (Research and Development, Human and Veterinary Medicine), 50–52 Avenue Chanoine Cartellier, 69230 Saint Genis Laval, France
Key words: AIDS, cross reactivity, IL-2, molecular mimicry, transmembrane envelope protein
This study describes some of the expected autoimmune consequences of a recently described virus–host molecular mimicry in patients infected with human immunodeficiency virus (HIV). Some physical and structural analogies have been recently identified between some precise antigenic sites of the trimeric ectodomain of the transmembrane envelope protein of AIDS-associated lentiviruses (HIV, Simian immunodeficiency virus and feline immunodeficiency virus) and some precise and crucial interleukin 2 (IL-2) antigenic sites of the corresponding infected species (man, monkey, cat). As expected, HIV-positive sera recognize human IL-2, and a cross-reactivity was found between the structurally and physically analogous antigenic sites of GP41 (HIV1) and human IL-2. The possible impact on AIDS-associated disorders of this tridimensional GP41 (HIV1)/human IL-2 molecular mimicry is suggested. © 2001 Academic Press
Introduction
observations must be underlined, from which two autoimmune consequences can be expected:
A precise molecular mimicry between the trimeric ectodomain of the transmembrane protein of immunosuppressive lentiviruses [human immunodeficiency virus (HIV); simian immunodeficiency virus (SIV); feline immunodeficiency virus (FIV)] and interleukin 2 (IL-2) of the corresponding infected species has been identified recently [1]. It was observed that, in all the cases examined (man, monkey, cat), because of their trimeric ‘coiledcoil’ assembly, the ectodomain of GP41 (HIV-1 and SIV) [2–6] and GP36 (FIV) [1] have the same aminoacid clusters in an identical spatial configuration as the IL-2 amino-acid clusters, interacting with two or even three of the , and subunits of the IL-2 receptor [1]. Moreover, it was observed that, in all the species affected by AIDS, the ‘IL-2 like’ sites of the viral transmembrane envelope (HIV, SIV, FIV) are highly conserved among the viral strains, indicating that this molecular host–virus mimicry is probably most useful to the virus. In addition, it is worth noting that the structurally analogous amino-acid clusters of GP41 (HIV1) and human IL-2 show strong physical similarities [1]. All the observed physical similarities (antigenicity, hydrophobia, solvent accessibility and surface electrostatic potential) indicate a very good prognosis for the recognition of the analogous sites of the two molecules by the same antibodies. Regarding the observed molecular mimicry between GP41 (HIV) and human IL-2, two important
(1) as previously mentioned, GP41 (HIV1) and human IL-2 analogous clusters are both antigenic. From this observation, a cross-reactivity between the analogous and antigenic clusters of the two molecules can be expected. (2) ‘IL-2 like’ sites of GP41 (HIV1) are immunodominant and correspond to the described immunodominant clusters of GP41 found in HIV-infected patients [7]. Therefore, it can be expected that HIV-positive sera recognizing the ‘IL-2-like’ immunodominant sites of GP41 will also recognize IL-2, and more precisely, the crucial sites on IL-2 involved in the interaction with , and subunits of the IL-2 receptor. In the present study, we looked for these two expected autoimmune consequences of the precise molecular mimicry between the viral transmembrane envelope protein GP41 (HIV1) and human IL-2.
Materials and Methods Protein structures The protein structures (X-ray diffraction and NMR) obtained from atomic coordinates (protein data bank: PDB) were visualized with RASMOL (version 2–6) [8] and GRASP [9].
Correspondence to: P.-F. Serres. Fax: 33 4 72 39 53 11. E-mail: [email protected] 287 0896–8411/01/030287+05 $35.00/0
© 2001 Academic Press
288
P.-F. Serres
Figure 2. Front view of trimeric GP41 (HIV1) and human IL-2. IL-2 amino acid clusters interacting with the (blue), (red), (green) subunits of the IL-2 receptor found to be identical and in the same positions on trimeric GP41 (HIV1). Colour code—K, R: blue/I, L: green/Q: yellow/E: brown/ T: orange/N: pink/ D: purple.
Figure 1. IL-2 amino acid clusters interacting with the (blue), (red), (green) subunits of the IL-2 receptor.
Pepscans Two types of Pepscans were used: a classical approach (8–12 mers) for mapping linear epitopes [10] and a conformational approach (30 mers simple, double and triple helix constructs) for mapping discontinuous epitopes. For each serum and antibody tested, 910 synthetized peptides (8, 12, 30 mers) were used.
Enzyme-linked immunosorbent assay (ELISA) anti-IL-2 Diluted (1/100) HIV-positive sera (CDC stage II, III, IV) (Institut Fournier, Paris) and diluted (1/100) HIV-negative sera (negative blood donors, CRTS, St Maurice de Beynost) were incubated in ELISA microplates coated with recombinant human IL-2. After washing, the recognition of human IL-2 was classically detected by enzyme-labelled anti-human IgG. Briefly, 20 g human-carrier-free recombinant human IL-2 (R & D Systems, 202, IL/CF) were adjusted to a concentration of 2 g/ml in phosphatebuffered saline (PBS). One hundred micrograms of the 2 g/ml dilution were incubated overnight at 4°C in ELISA microplates Nunc-Immuno Module Star Well Maxisorp (Ref. 13479, Polylabo). Microplates were saturated for 2 h at
Figure 3. Top view of trimeric GP41 (HIV1) and human IL-2. IL-2 amino acid clusters interacting with the (blue), (red), (green) subunits of the IL-2 receptor found to be identical and in the same positions on trimeric GP41 (HIV1). Colour code—K, R: blue/I, L: green/Q: yellow/E: brown/N: pink/D: purple.
room temperature in 0.5% PBS–BSA (100 l per well) and washed three times in PBS–Tween 20 (0.01%) before reading. Reading of the enzymatic reaction was performed on a microplate reader Dynatech MR 5000, using a 450 nm filter.
IL1 GP1 IL2 GP2 IL1 GP1*
IL2 GP2 IL GP IL-*
GP-
KPMYFKFTLMRTLKPNKYNNIGN TVWGIKQLQARILAVERYLKDQQ ELKHLQCLEEELKPLEEVLNLAQ IWNNMTWMEWEREIDNYTSLIYS TQLQLEHLLLDLQMILNGI LLRAIEAQQHLLQLTVG SLIYSLIEESQNQQEK NFHLRPRDLISNINVIVLELKGS MEWEREIDNYTSLIYSLIEESQN TATIVEFLNRWITFCQSIISTLT MEWEREIDNYTSLIYSLIEESQN INNYKNPKLTRMLTFKFYMP DETATIVEFLNRWITFCQSII KSLEQIWNNMTWMEWEREIDNYTS
Positive controls were obtained from diagnostic HIV kits: Behring, Diagnostic Pasteur, Biotrol, Abott, and from a pool of 15 HIV+ sera (stage III–IV–CDC) (Institut Fournier, Paris). 0 WITFCQSIISTL
LNRWITFCQSII
VEFLNRWITFCQ
ATIVEFLNRWIT
DETATIVEFLNR
EYADETATIVEF
FMCEYADETATI
ETTFMCEYADET
KGSETTFMCEYA
LELKGSETTFMC
VIVLELKGSETT
NINVIVLELKGS
LISNINVIVLEL
PRDLISNINVIV
HLRPRDLISNIN
KNFHLRPRDLIS
AQSKNFHLRPRD
LNLAQSKNFHLR
EEVLNLAQSKNF
KPLEEVLNLAQS
EELKPLEEVLNL
CLEEELKPLEEV
HLQCLEEELKPL
ELKHLQCLEEEL
KATELKHLQCLE
MPKKATELKHLQ
KFYMPKKATELK
LTFKFYMPKKAT
TRMLTFKFYMPK
PKLTRMLTFKFY
YKNPKLTRMLTF
INNYKNPKLTRM
LNGINNYKNPKL
QMILNGINNYKN
LDLQMILNGINN
HLLLDLQMILNG
QLEHLLLDLQMI
TQLQLEHLLLDL
TKKTQLQLEHLL
SSSTKKTQLQLE
Table 1. Sequences of the structurally and physically analogous amino acid clusters of GP41 (HIV1) and human IL-2
APTSSSTKKTQL
Figure 4. Front view of the fit of the two structures GP41 (HIV1) (PDB: 1ENV) and human IL-2 (PDB: 1IRL) showing the spatial superposition of identical amino acids within the analogous clusters of the two molecules. Each coloured block contains two identical amino acids. The first amino acid belongs to the first molecule and the second to the second molecule.
O.D. (ccd)
ERYLKDQQLLGIWG&&TTAVPWNASWSNKSL
LAVERYLKDQQLLG&&VPWNASWSNKSLEQI
ARILAVERYLKDQQ&&NASWSNKSLEQIWNN
QLQARILAVERYLK&&WSNKSLEQIWNNMTW
GIKQLQARILAVER&&KSLEQIWNNMTWMEW
TVWGIKQLQARILA&&EQIWNNMTWMEWERE
LQLTVWGIKQLQAR&&WNNMTWMEWEREIDN
QHLLQLTVWGIKQL&&MTWMEWEREIDNYTS
EAQQHLLQLTVWGI&&MEWEREIDNYTSLIY
RAIEAQQHLLQLTV&&EREIDNYTSLIYSLI
NLLRAIEAQQHLLQ&&IDNYTSLIYSLIEES
QQNNLLRAIEAQQH&&YTSLIYSLIEESQNQ
IVQQQNNLLRAIEA&&LIYSLIEESQNQQEK
LSGIVQQQNNLLRA&&SLIEESQNQQEKNEQ
RQLLSGIVQQQNNL&&EESQNQQEKNEQELL
VQARQLLSGIVQQQ&&QNQQEKNEQELLELD
O.D. (ccd)
IDNYTSLIYSLIEE&&NQQEKNEQELLELDK
EREIDNYTSLIYSL&&ESQNQQEKNEQELLE
MEWEREIDNYTSLI&&LIEESQNQQEKNEQE
MTWMEWEREIDNYT&&IYSLIEESQNQQEKN
WNNMTWMEWEREID&&TSLIYSLIEESQNQQ
EQIWNNMTWMEWER&&DNYTSLIYSLIEESQ
KSLEQIWNNMTWME&&REIDNYTSLIYSLIE
WSNKSLEQIWNNMT&&EWEREIDNYTSLIYS
NASWSNKSLEQIWN&&TWMEWEREIDNYTSL
VPWNASWSNKSLEQ&&NNMTWMEWEREIDNY
TTAVPWNASWSNKS&&QIWNNMTWMEWEREI
LICTTAVPWNASWS&&SLEQIWNNMTWMEWE
SGKLICTTAVPWNA&&SNKSLEQIWNNMTWM
WGCSGKLICTTAVP&&ASWSNKSLEQIWNNM
LGIWGCSGKLICTT&&PWNASWSNKSLEQIW
QQLLGIWGCSGKLI&&TAVPWNASWSNKSLE
LKDQQLLGIWGCSG&&ICTTAVPWNASWSNK
ERYLKDQQLLGIWG&&GKLICTTAVPWNASW
LAVERYLKDQQLLG&&GCSGKLICTTAVPWN
ARILAVERYLKDQQ&&GIWGCSGKLICTTAV
QLQARILAVERYLK&&QLLGIWGCSGKLICT
GIKQLQARILAVER&&KDQQLLGIWGCSGKL
TVWGIKQLQARILA&&RYLKDQQLLGIWGCS
LQLTVWGIKQLQAR&&AVERYLKDQQLLGIW
QHLLQLTVWGIKQL&&RILAVERYLKDQQLL
EAQQHLLQLTVWGI&&LQARILAVERYLKDQ
RAIEAQQHLLQLTV&&IKQLQARILAVERYL
NLLRAIEAQQHLLQ&&VWGIKQLQARILAVE
QQNNLLRAIEAQQH&&QLTVWGIKQLQARIL
IVQQQNNLLRAIEA&&HLLQLTVWGIKQLQA
LSGIVQQQNNLLRA&&AQQHLLQLTVWGIKQ
RQLLSGIVQQQNNL&&AIEAQQHLLQLTVWG
VQARQLLSGIVQQQ&&LLRAIEAQQHLLQLT
O.D. (ccd)
Role of a host–virus molecular mimicry in AIDS 289
3500
3000
2500
2000
1500
1000 500 0
3500
3000
2500
2000
1500
1000 500 0
Figure 5. Recognition of the site GP (–) on GP41 (HIV1) by an anti-human IL-2 antibody recognizing the site IL (–) on IL-2. The corresponding sequences are indicated in Table 1.
3000
2500
2000
1500
1000
500
Figure 6. Recognition of the sites IL 1 and IL 2 on IL-2 by a monoclonal anti GP41 antibody recognizing sites GP 1 and GP 2 on GP41 (HIV1). The corresponding sequences are indicated in Table 1.
*Conformational site involving the two sequences.
Results
Molecular mimicry between the trimeric ectodomain of GP41 (HIV1) and human IL-2
Figures 1, 2 and 3 summarize the structural analogies found between some highly conserved and antigenic sites of GP41 (HIV1) and the precise human IL-2 sites
290
P.-F. Serres
Table 2. Characteristics of the developed anti IL-2 ELISA
(1) (2) (3) (4) Total
Serum samples (n)
True positive (TP)
True negative (TN)
False positive (FP)
False negative (FN)
Sensitivity
Specificity
PPV
NPV
95 84 90 2083 2352
30 14 36
41 67 53 2005 2166
2 3 1 78 84
22 — —
58% 100% 100%
95% 96% 98% 96%
94% 82% 97%
65% 100% 100%
80
22
(1): ELISA anti-IL2−R h IL-2 with carrier; (2) (3) (4): ELISA anti-IL2−R h IL-2 without carrier. PPV, Positive Predictive Value=TP/(TP+FP); NPV, Negative Predictive Value=TN/(TN+FN); Sensitivity, TP/(TP+FN); Specificity, TN/(TN+FP).
Table 3. Comparison of the means (DO ELISA anti-IL2) of two populations (HIV+ and HIV−) Serum samples (n)
(1) (2) (3) Total
Mean (DO)
Standard deviation (sd)
HIV+
HIV−
HIV+
HIV−
HIV+
HIV−
52 14 36 102
43 70 54 167
0·93 0·92 2·14
0·33 0·23 0·25
0·12 0·04 0·44
0·05 0·01 0·01
dof
Student’s t test
Critical T Bilateral
P(T
88 14 36
10·32 12·95 16·90
1·99 2·14 2·03
7·84E-17 3·49E-09 1·06E-18
(1): ELISA anti-IL2−R h IL-2 with carrier; (2) (3): ELISA anti-IL2−R h IL-2 without carrier. dof, degrees of freedom.
interacting with the , and subunits of the IL-2 receptor. Figure 4 shows the fit of the two structures GP41 (HIV1)/IL-2 and the spatial superposition of identical amino acids of the analogous clusters within the two molecules. The results presented in Figures 2, 3 and 4 show that the molecular mimicry between the trimeric ectodomain of GP41 (HIV1) and human IL-2 is extremely precise. Table 1 summarizes the sequences of the structurally and physically analogous amino acid clusters of GP41 (HIV1) and human IL-2.
Cross-reactivity GP41 (HIV1)–IL-2 Mapped sequences are: GP41 (HIV1): NH2-VQARQLLSGIVQQQNNLLRAIE AQQHLLQLTVWGIKQLQARILAVERYLKDQQLLG IWGCSGKLICTTAVPWNASWSNKSLEQIWNNMTW MEWEREIDNYTSLIYSLIEESQNQQEKNEQELLELD K-COOH IL2: NH2-APTSSSTKKTQLQLEHLLLDLQMILNGIN NYKNPKLTRMLTFKFYMPKKATELKHLQCLEEE LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK GSETTFMCEYADETATIVEFLNRWITFCQSIISTLTCOOH Some results are given in Figures 5 and 6. Figure 5 shows the recognition of the site GP (–) on GP41 (HIV1) by an anti-human IL-2 antibody recognizing
the analogous site IL (–) on IL-2 (antibody AF 202-NA-R & D Systems). Figure 6 shows the recognition of the sites IL 1 and IL 2 on IL-2 by a monoclonal anti GP41 antibody recognizing the analogous sites GP 1 and GP 2 on GP41 (HIV1) (monoclonal anti GP41 antibody; clone M25 [11]. The results indicated in Figures 5 and 6 give some examples of the expected cross-reactivity between the physically and structurally antigenic analogous sites of GP41 (HIV1) and human IL-2.
ELISA anti-human IL2 2352 HIV+ and HIV− sera were dosed. Table 2 shows that the developed ELISA anti-human IL-2, which uses a carrier free recombinant human IL-2, gives an excellent sensitivity (100%) and a good specificity (96%). Table 3 and Figure 7 show that DO ELISA anti-IL-2 of the two sera populations (HIV+, HIV−) are statistically very different.
Discussion From these results it can be strongly assumed that the anti-IL-2 autoantibodies found in HIV infected patients are one of the consequences of a precise and functional molecular mimicry between some antigenic and immunodominant sites of GP41 (HIV1) and some antigenic sites of human IL-2. It must be underlined
Role of a host–virus molecular mimicry in AIDS
291
M. B. Foster for her help in the preparation of the manuscript. Current research carried out and managed by Hippocampe, in collaboration with private and public teams, is supported by ANRS (National Agency for Aids Research).
4
DO à 450 nm
3
References 2
1
0 0
1
2
3
4
4
3,5
DO à 450 nm
3
2,5
2
1,5
1
0,5
97
93
89
85
81
77
73
69
65
61
57
53
49
45
41
37
33
29
25
21
17
9
13
5
1
0
Figure 7. DO ELISA anti-IL-2 comparison of the means of two populations of sera (HIV+, HIV−) m+2 sd: mean+(standard deviation×2).
that the ‘IL-2 like’ sites of GP41 (HIV1) are immunodominant. Therefore, in the immune response against GP41 (HIV1): Immunodominant=Immunosuppressive. This paradoxical finding probably means that the more the immune system fights against the virus, the more it fights against IL-2, i.e. against itself. In fact, IL-2 is really the maestro of the immune response. Further studies will be necessary to determine the plausible impact of the GP41/IL-2 cross-reactivity on HIV pathogenesis and AIDS associated disorders.
Acknowledgements We wish to thank Dr W. Weissenhorn (Harvard University) for the trimeric GP41 (HIV1) structure and Dr
1. Serres P.F. 2000. Molecular mimicry between the trimeric ectodomain of the transmembrane protein of immunosuppressive lentiviruses (HIV–SIV–FIV) and interleukin 2. C.R. Acad. Sci. Paris, Sciences de la vie/Life Sciences 323: 1019–1029 2. Weissenhorn W., Dessen A., Harrison S.C., Skehel J.J., Wiley D.C. 1997. Atomic Structure of the Ectodomain from HIV-1 Gp41. Nature 387: 426–430 3. Tan K., Liu J.H., Wang J.H., Shen S., Lu M. 1997. Atomic Structure of a Thermostable Subdomain of HIV-1 Gp41. Proc. Nat. Acad. Sci. USA 94: 12303–12308 4. Chan D.C., Fass D., Berger J.M., Kim P.S. 1997. Core Structure of Gp41 from the HIV Envelope Glycoprotein. Cell 89: 263–273 5. Yang Z.N., Mueser T.C., Kaufman J., Stahl S.J., Wingfield P.T., Hyde C.C. 1999. The Crystal Structure ˚ Resolution. of the Siv Gp41 Ectodomain at 1.47 A J. Struct. Biol. 126: 131–144 6. Caffrey M., Cai M., Kaufman J., Stahl S.J., Wingfield P.T., Covell D.G., Gronenborn A.M., Clore G.M. 1998. Three-dimensional solution structure of the 44 kDa ectodomain of SIV gp41. Embo J. 17: 4572–4584 7. Binley J.M., Ditzel H.J., Barbas C.F., III, Sullivan N., Sodroski J., Parren P.W.H.I., Burton D.R. 1996. Human antibody responses to HIV type 1 glycoprotein 41 cloned in phage display libraries suggest three major epitopes are recognized and give evidence for conserved antibody motifs in antigen binding. AIDS Res. Hum. Retroviruses 12(10): 911–924 8. Sayle R.A., Milner-White E.J. 1995. RasMol: Biomolecular graphics for all. Trends Biochem. Sci. 20(9): 333–379 9. Nicholls A., Sharp K.A., Honing B. 1991. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins: Structure, Function and Genetics 11: 281–296 10. Slootstra J.W., Puijk W.C., Ligtvoet G.J., Langeveld J.P., Meloen R.H. 1996. Structural aspects of antibody–antigen interaction revealed through small random peptide libraries. Mol-Divers 1(2): 87–96 11. Veronese F.D., DeVico A.L., Copeland T.D., Oroszlan S., Gallo R.C., Sarngadharan M.G. 1985. Characterization of gp41 as the transmembrane protein coded by the HTLV-III/LAV envelope gene. Science 229: 1402–1405
Journal of Autoimmunity (2001) 16, 287–291
AIDS: an Immune Response against the Immune System. Role of a Precise Tridimensional Molecular Mimicry Pierre-Franc¸ois Serres HIPPOCAMPE (Research and Development, Human and Veterinary Medicine), 50–52 Avenue Chanoine Cartellier, 69230 Saint Genis Laval, France
Key words: AIDS, cross reactivity, IL-2, molecular mimicry, transmembrane envelope protein
This study describes some of the expected autoimmune consequences of a recently described virus–host molecular mimicry in patients infected with human immunodeficiency virus (HIV). Some physical and structural analogies have been recently identified between some precise antigenic sites of the trimeric ectodomain of the transmembrane envelope protein of AIDS-associated lentiviruses (HIV, Simian immunodeficiency virus and feline immunodeficiency virus) and some precise and crucial interleukin 2 (IL-2) antigenic sites of the corresponding infected species (man, monkey, cat). As expected, HIV-positive sera recognize human IL-2, and a cross-reactivity was found between the structurally and physically analogous antigenic sites of GP41 (HIV1) and human IL-2. The possible impact on AIDS-associated disorders of this tridimensional GP41 (HIV1)/human IL-2 molecular mimicry is suggested. © 2001 Academic Press
Introduction
observations must be underlined, from which two autoimmune consequences can be expected:
A precise molecular mimicry between the trimeric ectodomain of the transmembrane protein of immunosuppressive lentiviruses [human immunodeficiency virus (HIV); simian immunodeficiency virus (SIV); feline immunodeficiency virus (FIV)] and interleukin 2 (IL-2) of the corresponding infected species has been identified recently [1]. It was observed that, in all the cases examined (man, monkey, cat), because of their trimeric ‘coiledcoil’ assembly, the ectodomain of GP41 (HIV-1 and SIV) [2–6] and GP36 (FIV) [1] have the same aminoacid clusters in an identical spatial configuration as the IL-2 amino-acid clusters, interacting with two or even three of the , and subunits of the IL-2 receptor [1]. Moreover, it was observed that, in all the species affected by AIDS, the ‘IL-2 like’ sites of the viral transmembrane envelope (HIV, SIV, FIV) are highly conserved among the viral strains, indicating that this molecular host–virus mimicry is probably most useful to the virus. In addition, it is worth noting that the structurally analogous amino-acid clusters of GP41 (HIV1) and human IL-2 show strong physical similarities [1]. All the observed physical similarities (antigenicity, hydrophobia, solvent accessibility and surface electrostatic potential) indicate a very good prognosis for the recognition of the analogous sites of the two molecules by the same antibodies. Regarding the observed molecular mimicry between GP41 (HIV) and human IL-2, two important
(1) as previously mentioned, GP41 (HIV1) and human IL-2 analogous clusters are both antigenic. From this observation, a cross-reactivity between the analogous and antigenic clusters of the two molecules can be expected. (2) ‘IL-2 like’ sites of GP41 (HIV1) are immunodominant and correspond to the described immunodominant clusters of GP41 found in HIV-infected patients [7]. Therefore, it can be expected that HIV-positive sera recognizing the ‘IL-2-like’ immunodominant sites of GP41 will also recognize IL-2, and more precisely, the crucial sites on IL-2 involved in the interaction with , and subunits of the IL-2 receptor. In the present study, we looked for these two expected autoimmune consequences of the precise molecular mimicry between the viral transmembrane envelope protein GP41 (HIV1) and human IL-2.
Materials and Methods Protein structures The protein structures (X-ray diffraction and NMR) obtained from atomic coordinates (protein data bank: PDB) were visualized with RASMOL (version 2–6) [8] and GRASP [9].
Correspondence to: P.-F. Serres. Fax: 33 4 72 39 53 11. E-mail: [email protected] 287 0896–8411/01/030287+05 $35.00/0
© 2001 Academic Press
288
P.-F. Serres
Figure 2. Front view of trimeric GP41 (HIV1) and human IL-2. IL-2 amino acid clusters interacting with the (blue), (red), (green) subunits of the IL-2 receptor found to be identical and in the same positions on trimeric GP41 (HIV1). Colour code—K, R: blue/I, L: green/Q: yellow/E: brown/ T: orange/N: pink/ D: purple.
Figure 1. IL-2 amino acid clusters interacting with the (blue), (red), (green) subunits of the IL-2 receptor.
Pepscans Two types of Pepscans were used: a classical approach (8–12 mers) for mapping linear epitopes [10] and a conformational approach (30 mers simple, double and triple helix constructs) for mapping discontinuous epitopes. For each serum and antibody tested, 910 synthetized peptides (8, 12, 30 mers) were used.
Enzyme-linked immunosorbent assay (ELISA) anti-IL-2 Diluted (1/100) HIV-positive sera (CDC stage II, III, IV) (Institut Fournier, Paris) and diluted (1/100) HIV-negative sera (negative blood donors, CRTS, St Maurice de Beynost) were incubated in ELISA microplates coated with recombinant human IL-2. After washing, the recognition of human IL-2 was classically detected by enzyme-labelled anti-human IgG. Briefly, 20 g human-carrier-free recombinant human IL-2 (R & D Systems, 202, IL/CF) were adjusted to a concentration of 2 g/ml in phosphatebuffered saline (PBS). One hundred micrograms of the 2 g/ml dilution were incubated overnight at 4°C in ELISA microplates Nunc-Immuno Module Star Well Maxisorp (Ref. 13479, Polylabo). Microplates were saturated for 2 h at
Figure 3. Top view of trimeric GP41 (HIV1) and human IL-2. IL-2 amino acid clusters interacting with the (blue), (red), (green) subunits of the IL-2 receptor found to be identical and in the same positions on trimeric GP41 (HIV1). Colour code—K, R: blue/I, L: green/Q: yellow/E: brown/N: pink/D: purple.
room temperature in 0.5% PBS–BSA (100 l per well) and washed three times in PBS–Tween 20 (0.01%) before reading. Reading of the enzymatic reaction was performed on a microplate reader Dynatech MR 5000, using a 450 nm filter.
IL1 GP1 IL2 GP2 IL1 GP1*
IL2 GP2 IL GP IL-*
GP-
KPMYFKFTLMRTLKPNKYNNIGN TVWGIKQLQARILAVERYLKDQQ ELKHLQCLEEELKPLEEVLNLAQ IWNNMTWMEWEREIDNYTSLIYS TQLQLEHLLLDLQMILNGI LLRAIEAQQHLLQLTVG SLIYSLIEESQNQQEK NFHLRPRDLISNINVIVLELKGS MEWEREIDNYTSLIYSLIEESQN TATIVEFLNRWITFCQSIISTLT MEWEREIDNYTSLIYSLIEESQN INNYKNPKLTRMLTFKFYMP DETATIVEFLNRWITFCQSII KSLEQIWNNMTWMEWEREIDNYTS
Positive controls were obtained from diagnostic HIV kits: Behring, Diagnostic Pasteur, Biotrol, Abott, and from a pool of 15 HIV+ sera (stage III–IV–CDC) (Institut Fournier, Paris). 0 WITFCQSIISTL
LNRWITFCQSII
VEFLNRWITFCQ
ATIVEFLNRWIT
DETATIVEFLNR
EYADETATIVEF
FMCEYADETATI
ETTFMCEYADET
KGSETTFMCEYA
LELKGSETTFMC
VIVLELKGSETT
NINVIVLELKGS
LISNINVIVLEL
PRDLISNINVIV
HLRPRDLISNIN
KNFHLRPRDLIS
AQSKNFHLRPRD
LNLAQSKNFHLR
EEVLNLAQSKNF
KPLEEVLNLAQS
EELKPLEEVLNL
CLEEELKPLEEV
HLQCLEEELKPL
ELKHLQCLEEEL
KATELKHLQCLE
MPKKATELKHLQ
KFYMPKKATELK
LTFKFYMPKKAT
TRMLTFKFYMPK
PKLTRMLTFKFY
YKNPKLTRMLTF
INNYKNPKLTRM
LNGINNYKNPKL
QMILNGINNYKN
LDLQMILNGINN
HLLLDLQMILNG
QLEHLLLDLQMI
TQLQLEHLLLDL
TKKTQLQLEHLL
SSSTKKTQLQLE
Table 1. Sequences of the structurally and physically analogous amino acid clusters of GP41 (HIV1) and human IL-2
APTSSSTKKTQL
Figure 4. Front view of the fit of the two structures GP41 (HIV1) (PDB: 1ENV) and human IL-2 (PDB: 1IRL) showing the spatial superposition of identical amino acids within the analogous clusters of the two molecules. Each coloured block contains two identical amino acids. The first amino acid belongs to the first molecule and the second to the second molecule.
O.D. (ccd)
ERYLKDQQLLGIWG&&TTAVPWNASWSNKSL
LAVERYLKDQQLLG&&VPWNASWSNKSLEQI
ARILAVERYLKDQQ&&NASWSNKSLEQIWNN
QLQARILAVERYLK&&WSNKSLEQIWNNMTW
GIKQLQARILAVER&&KSLEQIWNNMTWMEW
TVWGIKQLQARILA&&EQIWNNMTWMEWERE
LQLTVWGIKQLQAR&&WNNMTWMEWEREIDN
QHLLQLTVWGIKQL&&MTWMEWEREIDNYTS
EAQQHLLQLTVWGI&&MEWEREIDNYTSLIY
RAIEAQQHLLQLTV&&EREIDNYTSLIYSLI
NLLRAIEAQQHLLQ&&IDNYTSLIYSLIEES
QQNNLLRAIEAQQH&&YTSLIYSLIEESQNQ
IVQQQNNLLRAIEA&&LIYSLIEESQNQQEK
LSGIVQQQNNLLRA&&SLIEESQNQQEKNEQ
RQLLSGIVQQQNNL&&EESQNQQEKNEQELL
VQARQLLSGIVQQQ&&QNQQEKNEQELLELD
O.D. (ccd)
IDNYTSLIYSLIEE&&NQQEKNEQELLELDK
EREIDNYTSLIYSL&&ESQNQQEKNEQELLE
MEWEREIDNYTSLI&&LIEESQNQQEKNEQE
MTWMEWEREIDNYT&&IYSLIEESQNQQEKN
WNNMTWMEWEREID&&TSLIYSLIEESQNQQ
EQIWNNMTWMEWER&&DNYTSLIYSLIEESQ
KSLEQIWNNMTWME&&REIDNYTSLIYSLIE
WSNKSLEQIWNNMT&&EWEREIDNYTSLIYS
NASWSNKSLEQIWN&&TWMEWEREIDNYTSL
VPWNASWSNKSLEQ&&NNMTWMEWEREIDNY
TTAVPWNASWSNKS&&QIWNNMTWMEWEREI
LICTTAVPWNASWS&&SLEQIWNNMTWMEWE
SGKLICTTAVPWNA&&SNKSLEQIWNNMTWM
WGCSGKLICTTAVP&&ASWSNKSLEQIWNNM
LGIWGCSGKLICTT&&PWNASWSNKSLEQIW
QQLLGIWGCSGKLI&&TAVPWNASWSNKSLE
LKDQQLLGIWGCSG&&ICTTAVPWNASWSNK
ERYLKDQQLLGIWG&&GKLICTTAVPWNASW
LAVERYLKDQQLLG&&GCSGKLICTTAVPWN
ARILAVERYLKDQQ&&GIWGCSGKLICTTAV
QLQARILAVERYLK&&QLLGIWGCSGKLICT
GIKQLQARILAVER&&KDQQLLGIWGCSGKL
TVWGIKQLQARILA&&RYLKDQQLLGIWGCS
LQLTVWGIKQLQAR&&AVERYLKDQQLLGIW
QHLLQLTVWGIKQL&&RILAVERYLKDQQLL
EAQQHLLQLTVWGI&&LQARILAVERYLKDQ
RAIEAQQHLLQLTV&&IKQLQARILAVERYL
NLLRAIEAQQHLLQ&&VWGIKQLQARILAVE
QQNNLLRAIEAQQH&&QLTVWGIKQLQARIL
IVQQQNNLLRAIEA&&HLLQLTVWGIKQLQA
LSGIVQQQNNLLRA&&AQQHLLQLTVWGIKQ
RQLLSGIVQQQNNL&&AIEAQQHLLQLTVWG
VQARQLLSGIVQQQ&&LLRAIEAQQHLLQLT
O.D. (ccd)
Role of a host–virus molecular mimicry in AIDS 289
3500
3000
2500
2000
1500
1000 500 0
3500
3000
2500
2000
1500
1000 500 0
Figure 5. Recognition of the site GP (–) on GP41 (HIV1) by an anti-human IL-2 antibody recognizing the site IL (–) on IL-2. The corresponding sequences are indicated in Table 1.
3000
2500
2000
1500
1000
500
Figure 6. Recognition of the sites IL 1 and IL 2 on IL-2 by a monoclonal anti GP41 antibody recognizing sites GP 1 and GP 2 on GP41 (HIV1). The corresponding sequences are indicated in Table 1.
*Conformational site involving the two sequences.
Results
Molecular mimicry between the trimeric ectodomain of GP41 (HIV1) and human IL-2
Figures 1, 2 and 3 summarize the structural analogies found between some highly conserved and antigenic sites of GP41 (HIV1) and the precise human IL-2 sites
290
P.-F. Serres
Table 2. Characteristics of the developed anti IL-2 ELISA
(1) (2) (3) (4) Total
Serum samples (n)
True positive (TP)
True negative (TN)
False positive (FP)
False negative (FN)
Sensitivity
Specificity
PPV
NPV
95 84 90 2083 2352
30 14 36
41 67 53 2005 2166
2 3 1 78 84
22 — —
58% 100% 100%
95% 96% 98% 96%
94% 82% 97%
65% 100% 100%
80
22
(1): ELISA anti-IL2−R h IL-2 with carrier; (2) (3) (4): ELISA anti-IL2−R h IL-2 without carrier. PPV, Positive Predictive Value=TP/(TP+FP); NPV, Negative Predictive Value=TN/(TN+FN); Sensitivity, TP/(TP+FN); Specificity, TN/(TN+FP).
Table 3. Comparison of the means (DO ELISA anti-IL2) of two populations (HIV+ and HIV−) Serum samples (n)
(1) (2) (3) Total
Mean (DO)
Standard deviation (sd)
HIV+
HIV−
HIV+
HIV−
HIV+
HIV−
52 14 36 102
43 70 54 167
0·93 0·92 2·14
0·33 0·23 0·25
0·12 0·04 0·44
0·05 0·01 0·01
dof
Student’s t test
Critical T Bilateral
P(T
88 14 36
10·32 12·95 16·90
1·99 2·14 2·03
7·84E-17 3·49E-09 1·06E-18
(1): ELISA anti-IL2−R h IL-2 with carrier; (2) (3): ELISA anti-IL2−R h IL-2 without carrier. dof, degrees of freedom.
interacting with the , and subunits of the IL-2 receptor. Figure 4 shows the fit of the two structures GP41 (HIV1)/IL-2 and the spatial superposition of identical amino acids of the analogous clusters within the two molecules. The results presented in Figures 2, 3 and 4 show that the molecular mimicry between the trimeric ectodomain of GP41 (HIV1) and human IL-2 is extremely precise. Table 1 summarizes the sequences of the structurally and physically analogous amino acid clusters of GP41 (HIV1) and human IL-2.
Cross-reactivity GP41 (HIV1)–IL-2 Mapped sequences are: GP41 (HIV1): NH2-VQARQLLSGIVQQQNNLLRAIE AQQHLLQLTVWGIKQLQARILAVERYLKDQQLLG IWGCSGKLICTTAVPWNASWSNKSLEQIWNNMTW MEWEREIDNYTSLIYSLIEESQNQQEKNEQELLELD K-COOH IL2: NH2-APTSSSTKKTQLQLEHLLLDLQMILNGIN NYKNPKLTRMLTFKFYMPKKATELKHLQCLEEE LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK GSETTFMCEYADETATIVEFLNRWITFCQSIISTLTCOOH Some results are given in Figures 5 and 6. Figure 5 shows the recognition of the site GP (–) on GP41 (HIV1) by an anti-human IL-2 antibody recognizing
the analogous site IL (–) on IL-2 (antibody AF 202-NA-R & D Systems). Figure 6 shows the recognition of the sites IL 1 and IL 2 on IL-2 by a monoclonal anti GP41 antibody recognizing the analogous sites GP 1 and GP 2 on GP41 (HIV1) (monoclonal anti GP41 antibody; clone M25 [11]. The results indicated in Figures 5 and 6 give some examples of the expected cross-reactivity between the physically and structurally antigenic analogous sites of GP41 (HIV1) and human IL-2.
ELISA anti-human IL2 2352 HIV+ and HIV− sera were dosed. Table 2 shows that the developed ELISA anti-human IL-2, which uses a carrier free recombinant human IL-2, gives an excellent sensitivity (100%) and a good specificity (96%). Table 3 and Figure 7 show that DO ELISA anti-IL-2 of the two sera populations (HIV+, HIV−) are statistically very different.
Discussion From these results it can be strongly assumed that the anti-IL-2 autoantibodies found in HIV infected patients are one of the consequences of a precise and functional molecular mimicry between some antigenic and immunodominant sites of GP41 (HIV1) and some antigenic sites of human IL-2. It must be underlined
Role of a host–virus molecular mimicry in AIDS
291
M. B. Foster for her help in the preparation of the manuscript. Current research carried out and managed by Hippocampe, in collaboration with private and public teams, is supported by ANRS (National Agency for Aids Research).
4
DO à 450 nm
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Figure 7. DO ELISA anti-IL-2 comparison of the means of two populations of sera (HIV+, HIV−) m+2 sd: mean+(standard deviation×2).
that the ‘IL-2 like’ sites of GP41 (HIV1) are immunodominant. Therefore, in the immune response against GP41 (HIV1): Immunodominant=Immunosuppressive. This paradoxical finding probably means that the more the immune system fights against the virus, the more it fights against IL-2, i.e. against itself. In fact, IL-2 is really the maestro of the immune response. Further studies will be necessary to determine the plausible impact of the GP41/IL-2 cross-reactivity on HIV pathogenesis and AIDS associated disorders.
Acknowledgements We wish to thank Dr W. Weissenhorn (Harvard University) for the trimeric GP41 (HIV1) structure and Dr
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