Immunology 1991 73 371-376
ADONIS 0019280591001678
Identification of amino acids in the V3 region of gpl20 critical for virus neutralization by human HIV-1-specific antibodies P. A. BROLIDEN,*t$ B. MAKITALO,* L. AKERBLOM,§ J. ROSEN,¶ K. BROLIDEN,*t G. UTTERt M. JONDAL,$ E. NORRBYt & B. WAHREN* *Department of Virology, National Bacteriological Laboratory, tDepartment of Virology, Karolinska Institute, $Department of Immunology, Karolinska Institute, Stockholm, §Department of Veterinary Microbiology, Biomedical Center, Uppsala, Sweden and ¶Johnson & Johnson Biotechnology, La Jolla, California, U.S.A.
Acceptedfor publication 24 April 1991
SUMMARY The importance of the dependence on single amino acids in the V3 region of HIV-1 gpl20 was evaluated for virus neutralization and antibody-dependent cellular cytotoxicity (ADCC). Synthetic overlapping 15-mer peptides and a set of omission peptides covering amino acids 301-317 were used. Sera from 29 HIV-I-infected individuals at different stages of disease were tested for neutralization, ADCC and specific IgG reactivity. Six HIV-1 neutralizing monoclonal antibodies (mAb) acted as controls. All mAb reacted with a region (amino acids 304-318) of gp 120, previously shown to induce neutralizing antibodies. The amino acids essential for reactivity were identified to be within the sequence GPGR (amino acids 312-315). The importance of this region for occurrence of neutralizing antibodies in infected humans was investigated using the same set of peptides. Out of 29 individuals, 21 were found to have neutralizing antibodies in titres between 100 and 1000. Among the neutralization-positive sera, 17/21 (81 %) reacted with amino acids 304-318, compared with only one of eight sera ( 13%) negative in neutralization. When any of the four amino acids G, P. G or R were deleted, the seroreactivity decreased considerably. The conserved sequence GPGR was therefore considered to be the most important for neutralization in this region in human sera as well. Thus, the conserved sequence GPGR in the V3 region of gpl2O is critical for virus neutralization by human HIV-i-specific antibodies. INTRODUCTION The immune response to human immunodeficiency virus (HIV) consists of both cellular and humoral responses. Antibodies which are able to neutralize HIV-l 1.2 have been identified to various regions of gp 120,3-7 gp418 9 and p 17. '0 The importance of neutralizing antibodes in vivo is not yet fully understood. It was recently shown that HIV-1 -infected individuals produce neutralizing antibodies that recognize only the virus that initiated the infection, and are unable to neutralize newly emerging variants." No correlation between the presence of neutralizing antibodies and the clinical stage of disease in HIV-I-infected adults has been found,'2-14 but in paediatric HIV-infection it was correlated with a better clinical status.'5 6 Although immunoglobulins are capable of inhibiting HIV-1 infection in vitro, vaccines and passive immunization have not been able to induce protection in animals,'7"8 until recently when protection in chimpanzees vaccinated with HIV-l gpl20 was reported.'9 Moreoever, protection against simian immunodeficiency virus Correspondence: Dr P. A. Broliden, Dept. of Virology, National Bacteriological Laboratory, S-105 21 Stockholm, Sweden.
371
(SIV) infection in monkeys by immunization with non-replicating SIV202' or modification of disease by pretreatment with infectious HIV-222 has recently been shown. In humans promising results have been reported with passive immunization with high-titred HIV-specific neutralizing plasma in patients with acquired immunodeficiency syndrome (AIDS).23 The principal neutralizing determinant is located within the V3 region of gpI2024 25 including 34-36 amino acids depending upon the HIV strain, and has been shown to form a loop between two cysteins.2627 In the strain HTLV-IIIB, these cysteins are located at positions 296 and 331.2829 Conserved sequences of this region occur around both cysteins and halfway between, while the remainder is variable. The site in the middle between the cysteins has a sequence of GPGR, which is highly conserved between isolates.30 Neutralizing monoclonal antibodies have been found to bind to these four conserved amino acids.63' 34 The region is also one of the few immunodominant parts of gpl2O, with both T- and B-cell activating sites.35 Antibodies against the N- and C-terminal cystein regions appear to be of particular interest as they are possibly protective against transmission of HIV- I to newborns from infected mothers.36
372
P. A. Broliden et al.
Another potentially important mechanism for protection against viral spread is antibody-dependent cellular cytotoxicity (ADCC).37 In this reaction HIV-specific antibodies bind to HIV antigens on the surface of infected cells, which are then killed by Fc-receptor positive effector cells. It has been shown that ADCC to HIV-infected cells is mediated by IgG138 and that the HIV envelope glycoprotein constitutes a target for this reaction.37'39'4' A correlation between the presence of neutralizing and ADCCmediating antibodies in human sera has also been suggested.4' In this study we have analysed the serological response of human sera with or without neutralizing and/or ADCCmediating capacity to the V3 region of gpl2O. MATERIAL AND METHODS
Study population Serum samples from 29 HIV-1 seropositive males at different stages of disease were used. The study included two asymptomatic patients, 16 patients in CDC Group 111,42 seven patients in Group IV:A and four patients with AIDS (Group IV:Cl). Monoclonal antibodies The six mouse monoclonal antibodies (mAb) used have been characterized previously.3443 The gp 120 used as an immunogen for immunization was prepared from culture fluid of HIV-1 (HTLV-IIIB)-infected H9 cells. All mAb were IgGlk. All six mAb have previously been shown to have a neutralizing property varying between titres of 100-1000 (Table 1 ).43 In addition, one of the mAb showed a capacity to mediate ADCC.34 All mAb react with the same site, amino acids 309318, in the V3 region of gpl2O, but the single amino acid responsible for binding has not yet been identified. HIV neutralization assay Virus neutralization was performed as follows: >50 tissue culture infectious doses of virus (HTLV-IIIB) supernatant (40,000 reverse transcriptase units) were preincubated for 60 min at 37° with serial dilutions (four five-fold steps starting with 1: 20) of sera or mAb. The serum-virus mixture was then added to 5 x 104 peripheral blood mononuclear cells (PBMC) or HUT-78 cells for 60 min at 37°. After washing, the cells were cultured in 96-well plates. Supernatants were collected after 8 days of culture and analysed by HIV antigen capture ELISA.4 Neutralization was defined as a > 80% reduction of p24-viral antigen in the supernatant compared to p24 content when virus was incubated with HIV antibody-negative sera. HIV antibodypositive sera with known neutralization titres were included in each test. ADCC The ADCC assay was performed as described elsewhere.37 Cells of the monocytoid line U937 clone 2, chronically infected with HIV- 1 strain HTLV-IIIB, were used as targets. PBMC obtained from HIV antibody-negative blood donors were used as effector cells. 5'Cr-labelled target cells (I x 104) and effector cells (2 x 105) were mixed with serum or mAb dilutions. Supernatants were harvested after 3 hr and released radioactivity was determined. The spontaneous release never exceeded 10%. HIV antibodypositive sera with known ADCC titres as well as seronegative controls, including non-immunized mice, were included in each test.
Table 1. Clinical stage of patients, and neutralization and ADCC titres of their sera
Serum
Stage Neutralization ADCC
Neutralizing human sera 8 II 180 59 II 180 79 III 180 III 93 1000 167 III 100 III 170 1000 171 III 350 193 III 100 194 III 180 III 204 540 337 III 1000 355 III 125 384 III 300 415 224 363 382 408 22 349 400
III IVA IVA IVA IVA IVC IVC IVC
125 150 150 150 200 1000 350 125
Non-neutralizing human sera III 209 0 297 III 0 414 III 0 416 210 402 418 417
mAb A47/B1 G44/H7 D59/A2 F58/H3 Pl/D12 P4/D1O
III IVA IVA IVA IVC
0 0 0 0 0
100 300 400 1000 500 500
Reactive to peptides*
100 53, 54 0 7000 0 53 2400 51,52,53, 55,56,57,58 0 52, 53, 54, 56 30 53, 54, 55, 56, 57, 58 2000 53, 57, 58 .7000 52,53 0 51,52,53,54, 58 0 53 400 56 52, 53, 7000 51, 52, 53 2000 53, 58 7000 53 0 53, 54 2000 500 53 0 53, 54, 56, 57, 58 0 53, 56 2500
800 >
7000
500 53 250 1000 90 5000 0 51,52,53,
0 0 0 0 0 800
57 58 57 57 57, 58
53, 54 53,54 53,54 53,54 53,54 53, 54
* Residue numbers of peptides: 51, amino acids 294-308; 52, 299313; 53, 304-318; 54, 309-323; 55, 314-328; 56, 319-333; 57, 324-338; 58, 329-343.
Peptides Solid-phase synthesized45 15 amino acid peptides based on the HTLV-IIIB sequence28 were used as antigens in ELISA assays.46 Sequence numbers of the Los Alamos database29 were used. One set of 1 5-mer peptides (C5 1 -C58) with overlapping sequences of 10 amino acids represented the complete putative loop region (amino acids 294-343). Another series of peptides was derived from the sequence SIRIQRGPGRAFVT (306-319) with sequential deletions of one single amino acid.
Peptide ELISA Micro-ELISA plates (Nunc, Odense, Denmark) were coated with 1 pig peptide/well in 100 ,ul 0-05 M sodium carbonate buffer, pH 9-5, held at room temperature overnight and stored at +4°
373
Amino acids in the V3 region ofgp120 U
.0 .
E
a,
I
U
*
cs .
.
to
E.m
I
i-
I.
:
.0 *
U
-
w*
* *
-
W*
m
.
-
* :
* *
U
*
0
£
-
I v
* -t-
0
f -'I-
8
a I
C51 294-308
C52 299-313
C53
C54
C55
C56
C57
C58
304-318
309-323
314-328
319-333
324-338
329-343
Figure 1. Reactivity to overlapping peptides covering the neutralization-including V3 region amino acids 294-343. The individual sera divided into neutralizing (left) and non-neutralizing (right) for each peptide. The median value for each group is shown.
are
until use. The assay was performed as follows.4647 Sera diluted 1:20-1:100 in phosphate-buffered saline (PBS) with 0 5% bovine serum albumin BSA, 0-05% Tween 20 and 5% foetal calf serum (FCS) were added to coated wells. After incubation for 45 min at 370 the plates were washed five times. Horseradish peroxidase-conjugated anti-human IgG (Dakopatts, Copenhagen, Denmark) diluted 1:10,000 was added for 30 min at 37°. The substrate orthophenylendiamine was added for 30 min at room temperature, and the reaction was stopped by adding 2-5 M H2SO4. The absorbance was measured at 490 nm. Values above the mean optical density + 3 SD of negative controls were considered positive. RESULTS
Neutralization and ADCC Of 29 individuals investigated, 21 were found to have neutralizing antibodies (Table 1) with titres between 100 and 1000. Thirteen of them also had an ADCC reactivity. Seven out of eight of the neutralization-negative HIV-seropositive sera had an ADCC activity. The ADCC titres ranged from 30 to more than 7000. No correlation was found between the presence of titres of neutralizing and ADCC-mediating antibodies. Neither was there any significant correlation of appearance of neutralizing or ADCC-mediating antibodies with stage of disease in this limited material.
Antibody reactivity to peptides representing the loop region V3 The human antibody response to the neutralizing-inducing region was analysed by 15-mer peptides with an overlapping sequence of 10 amino acids, representing the sequence of amino acids 294-343 (Fig. 1). The highest frequency of reactivity was seen against peptide C53 (RKSIRIQRGPGRAFV), amino acids 304-318, to which 19/29 (66%) of sera reacted. The conserved region around the C-terminal cystein, represented by C57 and C58, elicited responses in 30% of the sera. When the
divided into two groups depending on their neutralizing capacity, some differences could be noted (Fig. 1). The reactivity towards the central region of the putative loop, C53, was predominant in the neutralization-positive group where 17/ 21 sera were reacting. On the contrary, only 2/8 of the neutralization-negative sera responded to this region. This pattern of reactivity was not seen to any of the other peptides, to which the reactivity was equally distributed between the two groups. Four of the neutralizing sera did not react with any of the peptides used here. sera were
Analysis of human antibody reactivity to sequence 305-319 A set of 14-mer peptides with a sequential deletion of one single amino acid was assayed for reactivity with all sera (Table 2). The OD values against the complete sequence for sera considered to be positive ranged between 0-45 and 1 40. A deletion of an amino acid which decreased the absorbance value by 50% was considered important for antibody binding. In the neutralization-positive group 15/21 sera reacted well with the 14-mer peptides, and it was possible to identify the single amino acids essential for binding to this region (Table 2). The amino acids G, P, G and R (amino acids 312-315) were found to be the most important for antibody binding, but the deletion of the flanking amino acids Q (310), A and F (316-317) also decreased the reactivity in 33-47% of the sera. Only one of eight sera in the neutralization-negative group responded to this region, but its reactivity was decreased when the arginines at positions 311 or 315 were deleted and was not affected by the deletion of G,P,G or R. Three sera were not reactive at all to this set of peptides, even though they showed a weak response to the very similar C53 peptide. Reactivity of neutralizing mAb The mAb were characterized with respect to the binding site between amino acids 306-318. By using a set of omission peptides the single amino acids essential for binding could be identified (Fig. 2). The reactivity to the omission peptides was clearly decreased when any of the amino acids G, P or G were
374
P. A. Broliden et al.
Table 2. Effect of amino acid deletion upon human seroreactivity to a HIV-neutralization inducing site
deleted (Fig. 2). Some reduction, not significant however, was also seen with the deletion of I at position 309 or R at position 315.
Deletion-induced loss of antibody binding Serum
S
8 22 93 167 170 171 193 194 204 224 337 . 363 384 408 415 "Y,
I
R
I
Q
R G P G R A F V T + + + + + + + + + + + + +
--+++ ++ -
---
+ +
-
_ -
0
mAb A47/B I G44/H7 D59/A2 F58/H3 Pl/D12 P4/DI0
-
-
-
-
_
_
++
-
+ + + + + + + +
+ + + + + + + + +
+ + + +
+ + + +
+ + + + + - - . + + - _ _ + + + + + + + - - - - - + + + 0 13 13 33 7 739393
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+ + + + + +
+ + + + + +
+ + + + + +
+ + + _ + + + +
+ + + 87 33 47 13 7
-
-
-
-
-
._ _ _
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
The individual amino acids determining IgG binding of each serum shown. A set of peptides where the amino acids were deleted one by one was used. Amino acid residue not affecting antibody binding when deleted. + Decreased absorbance value .500% when deleted. The OD values for positive sera against the complete sequence ranged between 0 45 and 1-40.
are
-
2E0 E N 0)
a)
1hO
(a)
co\
//
P4/D1O0
(a) F58/H3
0
co 0.5
I
S
R
Q R G P G R
I
A
F V T
-
Figure 2. Effect of single amino acid deletions on mAb reactivity to the neutralization-inducing site. The seroreactivity of two representative human sera and two mAb are shown.
DISCUSSION Sera from HIV-1-infected individuals in different stages of disease were analysed for site-directed antibody reactivity to the V3 region of gpl2O, virus neutralization and ADCC. The V3 region forms a loop between two cysteins,27 and the conserved sequence GPGR is predicted to form a fl-turn which may give a good exposure to antibodies. An interesting question is whether a site important for neutralization could be identified in HIV-1-infected humans. A correlation between reactivity to the entire V3 region, amino acids 296-331, and neutralization has been found recently.'4 It has also been shown that sera from HIV-infected individuals bound to a peptide (aino acids 308-322) which elicited neutralizing antibodies in animals.48 Our analysis showed that the highest frequency of reactivity of human sera was directed against the middle (C53-54, amino acids 304-323) and the C-terminal part of the V3 region (C57-58 amino acids 328-343). Sera that had a neutralizing capacity were predominantly reactive with the C53 (amino acids 304323) peptide, which contains the conserved sequence GPGR. Seventeen out of 21 sera reacted with C53 in this group, compared with 2/8 among sera negative in neutralization. Experiments with omission peptides showed that the amino acids G, P. G and R were the most important for antibody binding to this region in most of the sera with a neutralizing capacity. The flanking amino acids Q, A and F seemed to be of importance in some of the sera. Six monoclonal antibodies with a neutralizing property all reacted with this region, and where the sequence GPG were the most important for binding. This is a conserved sequence within a variable region, shared by almost all sequenced HIV- I isolates from Europe and the U.S.A. published to date.29'30 These mAb have shown reactivity against a broad range of HIV- I strains by using peptides representing the same region of eight different isolates.43 Furthermore, a set of peptides covering amino acids 306-319 where the amino acids were sequentially deleted one by one showed that amino acids GPG were essential for binding, and also proved the feasibility of using omission peptides for the identification of important single amino acids. The same conclusion could be made when looking at a set of replacement peptides (data not shown). None of the amino acids GPG could be fully substituted by any other amino acid, even though a partial substitution was seen when proline was replaced with alanine or valine. Although the mAb were raised against HTLVITIB, which contain the rarely present residues Q and R (amino acids 310-311) flanking the GPG sequence, the deletion of any of these two amino acids did, not decrease the reactivity of the mAb, while the residues I at position 309 and R at position 315 which flank GPG in most isolates30 might be of some importance for the mAb reactivity (Fig. 2). The data strongly suggest that the conserved sequence GPGR is essential for binding of neutralizing antibodies, not only murine monoclonal but also human antibodies. While human HIV-1 seropositive sera are capable of neutralizing a wide range of different HIV-I isolates,'8 sera raised in animals to proteins or peptides tend only to neutralize the strain from
Amino acids in the V3 region ofgp120 which the antigen was derived.5 ' The similarity in epitopic reactivity between the panel of mAb and the human sera indicate a broad neutralizing capacity of these mAb. Neutralization-resistant strains have been generated in vitro in the presence of neutralizing mAb,49 and amino-acid substitutions within the V3 region in some of these variants. The use of neutralizing monoclonal antibodies and the importance of such variants in vivo are of great interest. Further studies of neutralizing antibodies in vivo are of interest. It is interesting to note that four sera with a neutralizing capacity did not react at all with peptides derived from the V3 region. Other regions may thus also play an important role for neutralization.57"0 The seroreactivity against C57 (amino acids 324-338) could not be associated with neutralization or ADCC reactivity. However, serological reactivity to peptide C57 is related to good prognostic possibilities to escape from HIV-1 infection in newborns of infected mothers. It is not known whether this is a reflection of a generally high reactivity towards the whole neutralizing region, in which case the neutralizing antibodies might have been adsorbed by antigen, or whether there is a direct functional activity of antibodies directed to the C-terminal cystein of the V3 region. The lack of correlation between ADCC activity and serological response to a region known to induce ADCC,M indicates that there are several epitopes for induction of ADCC-mediating antibodies. Other authors have described such determinants on the C-terminal end of gpI2039 and on gp41,50 but not on any other HIV protein. An HIV vaccine should contain determinants for both Tand B-cell immunity. The V3 neutralization-inducing region is a strong candidate to be included as one of the subcomponents. Even though the essential sequence is known, further knowledge about type specificity and variability is necessary, as well as the structure of the protein or peptide that will elicit the most efficient and broadly reactive neutralizing response.
immunodeficiency virus-infected cells bind a 24-amino acid sequence of the viral envelope, gpl2O. Proc. nati. Acad. Sci. U.S.A. 85, 3198. 8. DALGLEISH A., CHANH T., KENNEDY R., KANDA P., CLAPHAM P. & WEISS R. (1988) Neutralization of diverse HIV-1 strains by monoclonal antibodies raised against a gp4I synthetic peptide. Virology, 165, 209. 9. EVANS D.J., MCKEATING J., MEREDITH J.M., BURKE K., KATRAH K., JOHN A., FERGUSON M., MINOR P., WEISS R. & ALMOND J. (1989) An engineered polioviruschimaera elicits broadly reactive HIV- I neutralizing antibodies. Nature, 339, 385. 10. PAPSIDERO L.D., SHEU M. & RUSCETTI F.W. (1989) Human Immunodeficiency Virus type- 1 neutralizing monoclonal antibodies which react with p17 core protein: characterization and epitope mapping. J. Virol. 63, 267. 11. ALBERT J., ABRAHAMSSON B., NAGY K., AURELIUS E., GAINES H., KROOK A., NYSTROM G. & FENYO E.M. (1990) Rapid development of isolate-specific neutralizing antibodies after primary HIV-1 infection and consequent emergence of virus variants which resist neutralization by autologous sera. AIDS, 4, 107. 12. WEBER J., WEISS R., ROBERTS C., WELLER I., TEDDER R., CLAPHAM P. et al. (1987) Human immunodeficiency virus infection in two cohorts of homosexual men: neutralising sera and association of anti-gag antibody with prognosis. Lancet, i, 119. 13. RANKI A.M., WEISS S.H., VALLE S.L., ANTONEN J., KROHN K.J.E. (1987) Neutralizing antibodies in HIV (HTLV-III) infection: correlation with clinical outcome and antibody response against different viral proteins. Clin. exp. Immunol 69, 23 1. 14. BOTTIGER B., KARLSSON A., ANDREASSO P., NAUCLtR A., MENDES COSTA C., NORRBY E. & BIBERFIELD G. (1990) Envelope crossreactivity between human immunodeficiency virus types 1 and 2 detected by different serological methods: correlation between cross-neutralization and reactivity against the main neutralizing site. J. Virol. 64, 3492. 15. ROBERT-GUROFF M., OLESKE J.M., CONNOR E.M., EPSTEIN L.G., MINNEFOR A.B. & GALLO R.C. (1987) Relationship between HTLV-III neutralising antibodies and clinical status of pediatric
16.
REFERENCES I. ROBERT-GUROFF M., BROWN M. & GALLO R.C. (1985) HTLV-III neutralising antibodies in patients with AIDS and AIDS related complex. Nature, 316, 72. 2. WEiSS R.A., CLAPHAM P., WEBER J., CHEINSONG-POPOv R., DALGLEISH A., CARNE A, WELLER I. & TEDDER R.S. (1985) Neutralization of human T-lymphotropic virus type III sera of AIDS and AIDS-risk patients. Nature, 316, 69. 3. LASKY L.A., GROOPMAN J.E., FENNIE C.W., BENZ P.N., NUNES W.M., RENZ M.E. & BERMAN P.W. (1986) Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein. Science, 233, 209. 4. PUTNEY S.D, MATrHEWs T.J., ROBEY W.G., LYNN D.L., ROBERTGUROFF M., MUELLER W.T. et al. (1986) HTLVIII/LAV-neutralizing antibodies to an E. coli-produced fragment of the virus envelope. Science, 234, 1392. 5. Ho D.D., KAPLAN J.C., RACKAUSKAS I.E. & GURNEY M.E. (1988) Second conserved domain of gp 120 is important for HIV infectivity and antibody neutralization. Science, 239, 1021. 6. PALKER T.J., CLARK M.E., LANGLOIS A., MATTHEWS T.J., WEINHOLD K.J., RANDALL R.R., BOLOGNEsI D.I. & HAYNES B.F. (1988) Type-specific neutralization of the human immunodeficiency virus with antibodies toenv-encoded synthetic peptides. Proc. nati. Acad. Sci. U.S.A. 85, 1932. 7. RuscE J.A., JAVAHERIAN K., McDANAL C., PETRO J., LYNN D.L., GRIMAILA R. et al. (1988) Antibodies that inhibit fusion of human
375
17. 18.
19.
20.
acquired imunodeficiency syndrome (AIDS) and AIDS-related complex cases. Pediatric Res. 21, 547. LJUNGGREN K., MOSCHESE V., BROLIDEN P.-A., FENYO E.-M., WAHREN B., JONDAL M. & Rossi P. (1990) Antibodies mediating cellular cytotoxicity and neutralization correlate with a better clinical stage in children born to HIV- 1 infected mothers. J. infect. Dis. 161, 198. FULTZ B.N., SRINIVASAN A., GREENE C., BUTLER D., SWENSON R. & MCCLURE H. (1987) Superinfection of a chimpanzee with a second strain of human immunodeficiency virus. J. Virol. 61, 4026. PRINCE A.M., HOROWITZ B., BAKER L., SHULMAN R., RALPH H., VALIKSKY J. et al. (1988) Failure of a human immunodeficiency virus (HIV) immunoglobulin to protect chimpanzees against experimental challenge with HIV. Proc. natl. Acad. Sci. U.S.A. 85, 6944. BERMAN P., GREGORY T., RIDDLE L., NAKAMURA G., CHAMPE M., PORTER J., WURM F., HERSCHBERG R., COBBS E. & EICHBERG J. (1990) Protection of chimpanzees from infection by HIV-1 after vaccination with recombinant glycoprotein gpl20 but not gpl60. Nature, 345, 622. DESROSIERS R., WYAND M., KODAMA T., RINGLER D., ARTHUR L.,
SEHGAL P., LETVIN N., KING N. & DANIEL M. (1989) Vaccine protection against simian immunodeficiency virus infection. Proc. nat!. Acad. Sci U.S.A. 86, 6353. 21. MURPHEY-CORB M., MARTIN L., DAVISON-FAIRBURN B., MONTELARO R., MILLER M., WEST M., OHKAWA S., BASKIN G., ZHANG J., PUTNEY S., ALLISON A. & EPPSTEIN D. (1989) A formalininactivated whole SIV vaccine confers protection in macaques. Science, 246, 1293. 22. PUTKONEN P., THORSTENSSON R., ALBERT J., NORRBY E., BIBERFELD P. & BIBERFELD G. (1991) Infection of cynomolgus monkeys with
376
23.
24.
25.
26.
27.
28. 29.
30. 31.
32.
33.
34.
35.
P. A. Broliden et al.
HIV type 2 protects against pathogenic consequences of a subsequent SIV infection. AIDS (in press). JACKSON G.G., RUBENIS M., KNIGGE M., PERKINS J.T., PAUL D.A., DESPOTEs J.C. & SPENCER P. (1988) Passive immunoneutralisation of human immunodeficiency virus in patients with advanced AIDS. Lancet, ii, 647. MODROW S., HAHN B.H., SHAW G.M., GALLO R.C., WONG-STAAL F. & WOLF H. (1987) Computer-assisted analysis of envelope protein sequences of seven human immunodeficiency virus isolates: prediction of antigenic epitopes in conserved and variable regions. J. Virol. 61, 570. GoUDSMIT J., DEBOUCK C., MELOEN R., SMIT L., BAKKER M., ASHER D.M., WOLFF A.V., GIBBS C.J. & GAJDUSEK D.C. (1988) Human immunodeficiency virus type I neutralization epitope with conserved architecture elicits early type-specific antibodies in experimentally infected chimpanzees. Proc. nat!. Acad. Sci. U.S.A. 85, 4478. MELOEN R., LISKAMP R.M. & GOUDSMIT J. (1989) Specificity and function of the individual amino acids of an important determinant of human immunodeficiency virus type i that induces neutralizing activity. J. Gen. Virol. 70, 1505. GREGORY T., LEONARD C., RIDDLE L., THOMAS J., HARRIS R. & SPELLMAN M. (1990) Disulfide bond assignment and characterization of N-linked glycosylation sites in recombinant HIV- I type IIIB gpl2O produced in CHO cells. Abstract UCLA symposia on Molecular & Cellular biology. J. Cell. Biochem., suppl. 14D, L418. RATNER L., HASELTINE W., PATARCA R., LIVAK K., STARCICH B. & JOSEPHS S. (1985) Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature, 313, 277. MYERS G., JOSEPHS S., RABSON A., SMITH T. & WONG-STAAL F. (1988) Database Human Retroviruses and AIDS. Los Alamos Natl Lab, Los Alamos. LAROSA G., DAVIDE J., WEINHOLD K., WATERBURY J., PROFY A. & LEWIS J. (1990) Conserved sequence and structural elements in the HIV-1 principal neutralizing determinant. Science, 249, 932. SKINNER M.A., TING R., LANGLOIS A., WEINHOLD K.J., LYERLY K., JAVAHERIAN K. & MATTHEWS T.J. (1988) Characteristics of a neutralizing monoclonal antibody to the HIV envelope glycoprotein. AIDS Res. Hum. Retroviruses, 4, 187. LINSLEY P., LEDBETrER J., KINNEY-THOMAS E. & Hu S.-L. (1988) Effects of anti-gpl20 monoclonal antibodies on CD4 receptor binding by the env protein of human immunodeficiency virus type 1. J. Virol. 62, 3695. MATSUSHITA S., ROBERT-GUROFF M., RUSCHE J., KoITo A., HATTORI T., HOSHINo H., JAVVAHERIAN K., TAKATSUKI K. & PUTNEY S. (1988) Characterization of a human immunodeficiency virus neutralizing monoclonal antibody and mapping of the neutralizing epitope. J. Virol. 62, 2107. BROLIDEN P.-A., LJUNGGREN K., HINKULA J., NORRBY E., AKERBLOM L. & WAHREN B. (1990) A monoclonal antibody to HIV-1 mediating cellular cytotoxicity (ADCC) and neutralization. J. Virol. 64, 936. WAHREN B., MORFELDT-MANSSON L., BIBERFELD G., MOBERG L., SONNERBORG A., LJUNGMAN P., WERNER A., KURTH R., GALLO R. & BOLOGNESI D. (1987) Characteristics of the specific cell-mediated immune response in human immunodeficiency virus infection. J. Virol. 61, 2017.
36. Rossi P., MOSCHESE V., BROLIDEN P.-A., FUNDARO C., QUINTI I., PLEBANI A. et al. (1989) Antibodies to HIV-l gpl20 env epitopes correlate with uninfected status of children born to seropositive mothers. Proc. nati. Acad. Sci. U.S.A. 86, 8055. 37. LJUNGGREN K., BOTTIGER B., BIBERFELD G., KARLSSON A., FENYO E.M. & JONDAL M. (1987) Antibody dependent cellular cytotoxicity-inducing antibodies against human immunodeficiency virus: Presence at different clinical stages. J. Immunol. 139, 2263. 38. LJUNGGREN K., BROLIDEN P.A., MORFELDT-MANSSON L., JONDAL M. & WAHREN B. (1988) IgG subclass response to HIV in relation to antibody-dependent cellular cytotoxicity at different clinical stages. Clin. exp. Immunol. 73, 343. 39. BOLOGNESi D.P. (1988) Natural immunity to HIV and its possible relationship to vaccine strategies. Microbiol. Sci. 5, 236. 40. LYERLY H.K., REED D., MATTHEWS T.J. LANGLOiS A.J., AHEARNE P.A., PETTEWAY S.R. & WEINHOLD K.J. (1987) Anti-gpI20 antibodies from HIV seropositive individuals mediate broadly reactive anti-HIV ADCC. AIDS Res. Hum. Retroviruses 3, 409. 41. B6TTIGER B., LJUNGGREN K., KARLSSON A., KROHN K., FENY6 E.M., JONDAL M. & BIBERFELD G. (1988) Neutralizing antibodies in relation to antibody-dependent cellular cytotoxicity-inducing antibodies against human immunodeficiency virus type 1. Clin. exp. Immunol. 73, 339. 42. CENTERS FOR DISEASE CONTROL (1986) Classification system for HTLV-III/LAV infections. Morbidity and Mortality Weekly Report, 34, 373. 43. AKERBLOM L., HINKULA J., BROLIDEN P.A., FRIDBERGER T., ROSEN J., ERICSSON-DE-LA CiUZ M., MOREIN B. & WAHREN B. (1990) Neutralizing cross-reactive and non-neutralizing monoclonal antibodies to HIV-l gp 120. AIDS, 4, 953. 44. SUNDQVIST V.-A., ALBERT J., OHLSSON E., HINHULA J., FENYO E.M. & WAHREN B. (1990). HIV-1 p24 antigen variation in tissue culture of isolates with defined growth characteristics. J. Med. Virol. 27, 170. 45. HOUGHTEN R.A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigenantibody interaction at the level of individual amino acids. Proc. natl. Acad. Sci. U.S.A. 82, 5131. 46. MATHIESEN T., BROLIDEN P.-A., ROSEN J. & WAHREN B. (1989) Mapping of IgG subclass- and T-cell epitopes on HIV-proteins by synthetic peptides. Immunology, 67, 453. 47. BROLIDEN P.-A., MORFELDT-MANSSON L., ROSEN J., JONDAL M. & WAHREN B. (1989) Fine specificity of IgG subclass response in HIV infected patients. Clin. exp. Immunol. 76, 216. 48. KENEALY W., MATTHEWS T., GANFIELD M.C., LANGLOIS A., WASELEFSKY D. & PETTEWAY S. (1989) Antibodies from human immunodeficiency virus-infected individuals bind to a short amino acid sequence that elicits neutralizing antibodies in animals. AIDS Res. Hum. Retroviruses 5: 173. 49. MCKEATING J.A., Gow J., GoUDSMIT J., PEARL L., MULDER C. & WEISS R. (1989) Characterization of HIV-1 neutralization escape mutants. AIDS, 3, 777. 50. KOWALSKI M., POTZ J., BASIRIPOUR L., DORFMAN T., GOH W., TERWILLIGEN E., DAYTON A., ROSEN C., HASELTINE W. & SODROSHI J. (1987) Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science, 237, 1351.
ADONIS 0019280591001678
Identification of amino acids in the V3 region of gpl20 critical for virus neutralization by human HIV-1-specific antibodies P. A. BROLIDEN,*t$ B. MAKITALO,* L. AKERBLOM,§ J. ROSEN,¶ K. BROLIDEN,*t G. UTTERt M. JONDAL,$ E. NORRBYt & B. WAHREN* *Department of Virology, National Bacteriological Laboratory, tDepartment of Virology, Karolinska Institute, $Department of Immunology, Karolinska Institute, Stockholm, §Department of Veterinary Microbiology, Biomedical Center, Uppsala, Sweden and ¶Johnson & Johnson Biotechnology, La Jolla, California, U.S.A.
Acceptedfor publication 24 April 1991
SUMMARY The importance of the dependence on single amino acids in the V3 region of HIV-1 gpl20 was evaluated for virus neutralization and antibody-dependent cellular cytotoxicity (ADCC). Synthetic overlapping 15-mer peptides and a set of omission peptides covering amino acids 301-317 were used. Sera from 29 HIV-I-infected individuals at different stages of disease were tested for neutralization, ADCC and specific IgG reactivity. Six HIV-1 neutralizing monoclonal antibodies (mAb) acted as controls. All mAb reacted with a region (amino acids 304-318) of gp 120, previously shown to induce neutralizing antibodies. The amino acids essential for reactivity were identified to be within the sequence GPGR (amino acids 312-315). The importance of this region for occurrence of neutralizing antibodies in infected humans was investigated using the same set of peptides. Out of 29 individuals, 21 were found to have neutralizing antibodies in titres between 100 and 1000. Among the neutralization-positive sera, 17/21 (81 %) reacted with amino acids 304-318, compared with only one of eight sera ( 13%) negative in neutralization. When any of the four amino acids G, P. G or R were deleted, the seroreactivity decreased considerably. The conserved sequence GPGR was therefore considered to be the most important for neutralization in this region in human sera as well. Thus, the conserved sequence GPGR in the V3 region of gpl2O is critical for virus neutralization by human HIV-i-specific antibodies. INTRODUCTION The immune response to human immunodeficiency virus (HIV) consists of both cellular and humoral responses. Antibodies which are able to neutralize HIV-l 1.2 have been identified to various regions of gp 120,3-7 gp418 9 and p 17. '0 The importance of neutralizing antibodes in vivo is not yet fully understood. It was recently shown that HIV-1 -infected individuals produce neutralizing antibodies that recognize only the virus that initiated the infection, and are unable to neutralize newly emerging variants." No correlation between the presence of neutralizing antibodies and the clinical stage of disease in HIV-I-infected adults has been found,'2-14 but in paediatric HIV-infection it was correlated with a better clinical status.'5 6 Although immunoglobulins are capable of inhibiting HIV-1 infection in vitro, vaccines and passive immunization have not been able to induce protection in animals,'7"8 until recently when protection in chimpanzees vaccinated with HIV-l gpl20 was reported.'9 Moreoever, protection against simian immunodeficiency virus Correspondence: Dr P. A. Broliden, Dept. of Virology, National Bacteriological Laboratory, S-105 21 Stockholm, Sweden.
371
(SIV) infection in monkeys by immunization with non-replicating SIV202' or modification of disease by pretreatment with infectious HIV-222 has recently been shown. In humans promising results have been reported with passive immunization with high-titred HIV-specific neutralizing plasma in patients with acquired immunodeficiency syndrome (AIDS).23 The principal neutralizing determinant is located within the V3 region of gpI2024 25 including 34-36 amino acids depending upon the HIV strain, and has been shown to form a loop between two cysteins.2627 In the strain HTLV-IIIB, these cysteins are located at positions 296 and 331.2829 Conserved sequences of this region occur around both cysteins and halfway between, while the remainder is variable. The site in the middle between the cysteins has a sequence of GPGR, which is highly conserved between isolates.30 Neutralizing monoclonal antibodies have been found to bind to these four conserved amino acids.63' 34 The region is also one of the few immunodominant parts of gpl2O, with both T- and B-cell activating sites.35 Antibodies against the N- and C-terminal cystein regions appear to be of particular interest as they are possibly protective against transmission of HIV- I to newborns from infected mothers.36
372
P. A. Broliden et al.
Another potentially important mechanism for protection against viral spread is antibody-dependent cellular cytotoxicity (ADCC).37 In this reaction HIV-specific antibodies bind to HIV antigens on the surface of infected cells, which are then killed by Fc-receptor positive effector cells. It has been shown that ADCC to HIV-infected cells is mediated by IgG138 and that the HIV envelope glycoprotein constitutes a target for this reaction.37'39'4' A correlation between the presence of neutralizing and ADCCmediating antibodies in human sera has also been suggested.4' In this study we have analysed the serological response of human sera with or without neutralizing and/or ADCCmediating capacity to the V3 region of gpl2O. MATERIAL AND METHODS
Study population Serum samples from 29 HIV-1 seropositive males at different stages of disease were used. The study included two asymptomatic patients, 16 patients in CDC Group 111,42 seven patients in Group IV:A and four patients with AIDS (Group IV:Cl). Monoclonal antibodies The six mouse monoclonal antibodies (mAb) used have been characterized previously.3443 The gp 120 used as an immunogen for immunization was prepared from culture fluid of HIV-1 (HTLV-IIIB)-infected H9 cells. All mAb were IgGlk. All six mAb have previously been shown to have a neutralizing property varying between titres of 100-1000 (Table 1 ).43 In addition, one of the mAb showed a capacity to mediate ADCC.34 All mAb react with the same site, amino acids 309318, in the V3 region of gpl2O, but the single amino acid responsible for binding has not yet been identified. HIV neutralization assay Virus neutralization was performed as follows: >50 tissue culture infectious doses of virus (HTLV-IIIB) supernatant (40,000 reverse transcriptase units) were preincubated for 60 min at 37° with serial dilutions (four five-fold steps starting with 1: 20) of sera or mAb. The serum-virus mixture was then added to 5 x 104 peripheral blood mononuclear cells (PBMC) or HUT-78 cells for 60 min at 37°. After washing, the cells were cultured in 96-well plates. Supernatants were collected after 8 days of culture and analysed by HIV antigen capture ELISA.4 Neutralization was defined as a > 80% reduction of p24-viral antigen in the supernatant compared to p24 content when virus was incubated with HIV antibody-negative sera. HIV antibodypositive sera with known neutralization titres were included in each test. ADCC The ADCC assay was performed as described elsewhere.37 Cells of the monocytoid line U937 clone 2, chronically infected with HIV- 1 strain HTLV-IIIB, were used as targets. PBMC obtained from HIV antibody-negative blood donors were used as effector cells. 5'Cr-labelled target cells (I x 104) and effector cells (2 x 105) were mixed with serum or mAb dilutions. Supernatants were harvested after 3 hr and released radioactivity was determined. The spontaneous release never exceeded 10%. HIV antibodypositive sera with known ADCC titres as well as seronegative controls, including non-immunized mice, were included in each test.
Table 1. Clinical stage of patients, and neutralization and ADCC titres of their sera
Serum
Stage Neutralization ADCC
Neutralizing human sera 8 II 180 59 II 180 79 III 180 III 93 1000 167 III 100 III 170 1000 171 III 350 193 III 100 194 III 180 III 204 540 337 III 1000 355 III 125 384 III 300 415 224 363 382 408 22 349 400
III IVA IVA IVA IVA IVC IVC IVC
125 150 150 150 200 1000 350 125
Non-neutralizing human sera III 209 0 297 III 0 414 III 0 416 210 402 418 417
mAb A47/B1 G44/H7 D59/A2 F58/H3 Pl/D12 P4/D1O
III IVA IVA IVA IVC
0 0 0 0 0
100 300 400 1000 500 500
Reactive to peptides*
100 53, 54 0 7000 0 53 2400 51,52,53, 55,56,57,58 0 52, 53, 54, 56 30 53, 54, 55, 56, 57, 58 2000 53, 57, 58 .7000 52,53 0 51,52,53,54, 58 0 53 400 56 52, 53, 7000 51, 52, 53 2000 53, 58 7000 53 0 53, 54 2000 500 53 0 53, 54, 56, 57, 58 0 53, 56 2500
800 >
7000
500 53 250 1000 90 5000 0 51,52,53,
0 0 0 0 0 800
57 58 57 57 57, 58
53, 54 53,54 53,54 53,54 53,54 53, 54
* Residue numbers of peptides: 51, amino acids 294-308; 52, 299313; 53, 304-318; 54, 309-323; 55, 314-328; 56, 319-333; 57, 324-338; 58, 329-343.
Peptides Solid-phase synthesized45 15 amino acid peptides based on the HTLV-IIIB sequence28 were used as antigens in ELISA assays.46 Sequence numbers of the Los Alamos database29 were used. One set of 1 5-mer peptides (C5 1 -C58) with overlapping sequences of 10 amino acids represented the complete putative loop region (amino acids 294-343). Another series of peptides was derived from the sequence SIRIQRGPGRAFVT (306-319) with sequential deletions of one single amino acid.
Peptide ELISA Micro-ELISA plates (Nunc, Odense, Denmark) were coated with 1 pig peptide/well in 100 ,ul 0-05 M sodium carbonate buffer, pH 9-5, held at room temperature overnight and stored at +4°
373
Amino acids in the V3 region ofgp120 U
.0 .
E
a,
I
U
*
cs .
.
to
E.m
I
i-
I.
:
.0 *
U
-
w*
* *
-
W*
m
.
-
* :
* *
U
*
0
£
-
I v
* -t-
0
f -'I-
8
a I
C51 294-308
C52 299-313
C53
C54
C55
C56
C57
C58
304-318
309-323
314-328
319-333
324-338
329-343
Figure 1. Reactivity to overlapping peptides covering the neutralization-including V3 region amino acids 294-343. The individual sera divided into neutralizing (left) and non-neutralizing (right) for each peptide. The median value for each group is shown.
are
until use. The assay was performed as follows.4647 Sera diluted 1:20-1:100 in phosphate-buffered saline (PBS) with 0 5% bovine serum albumin BSA, 0-05% Tween 20 and 5% foetal calf serum (FCS) were added to coated wells. After incubation for 45 min at 370 the plates were washed five times. Horseradish peroxidase-conjugated anti-human IgG (Dakopatts, Copenhagen, Denmark) diluted 1:10,000 was added for 30 min at 37°. The substrate orthophenylendiamine was added for 30 min at room temperature, and the reaction was stopped by adding 2-5 M H2SO4. The absorbance was measured at 490 nm. Values above the mean optical density + 3 SD of negative controls were considered positive. RESULTS
Neutralization and ADCC Of 29 individuals investigated, 21 were found to have neutralizing antibodies (Table 1) with titres between 100 and 1000. Thirteen of them also had an ADCC reactivity. Seven out of eight of the neutralization-negative HIV-seropositive sera had an ADCC activity. The ADCC titres ranged from 30 to more than 7000. No correlation was found between the presence of titres of neutralizing and ADCC-mediating antibodies. Neither was there any significant correlation of appearance of neutralizing or ADCC-mediating antibodies with stage of disease in this limited material.
Antibody reactivity to peptides representing the loop region V3 The human antibody response to the neutralizing-inducing region was analysed by 15-mer peptides with an overlapping sequence of 10 amino acids, representing the sequence of amino acids 294-343 (Fig. 1). The highest frequency of reactivity was seen against peptide C53 (RKSIRIQRGPGRAFV), amino acids 304-318, to which 19/29 (66%) of sera reacted. The conserved region around the C-terminal cystein, represented by C57 and C58, elicited responses in 30% of the sera. When the
divided into two groups depending on their neutralizing capacity, some differences could be noted (Fig. 1). The reactivity towards the central region of the putative loop, C53, was predominant in the neutralization-positive group where 17/ 21 sera were reacting. On the contrary, only 2/8 of the neutralization-negative sera responded to this region. This pattern of reactivity was not seen to any of the other peptides, to which the reactivity was equally distributed between the two groups. Four of the neutralizing sera did not react with any of the peptides used here. sera were
Analysis of human antibody reactivity to sequence 305-319 A set of 14-mer peptides with a sequential deletion of one single amino acid was assayed for reactivity with all sera (Table 2). The OD values against the complete sequence for sera considered to be positive ranged between 0-45 and 1 40. A deletion of an amino acid which decreased the absorbance value by 50% was considered important for antibody binding. In the neutralization-positive group 15/21 sera reacted well with the 14-mer peptides, and it was possible to identify the single amino acids essential for binding to this region (Table 2). The amino acids G, P, G and R (amino acids 312-315) were found to be the most important for antibody binding, but the deletion of the flanking amino acids Q (310), A and F (316-317) also decreased the reactivity in 33-47% of the sera. Only one of eight sera in the neutralization-negative group responded to this region, but its reactivity was decreased when the arginines at positions 311 or 315 were deleted and was not affected by the deletion of G,P,G or R. Three sera were not reactive at all to this set of peptides, even though they showed a weak response to the very similar C53 peptide. Reactivity of neutralizing mAb The mAb were characterized with respect to the binding site between amino acids 306-318. By using a set of omission peptides the single amino acids essential for binding could be identified (Fig. 2). The reactivity to the omission peptides was clearly decreased when any of the amino acids G, P or G were
374
P. A. Broliden et al.
Table 2. Effect of amino acid deletion upon human seroreactivity to a HIV-neutralization inducing site
deleted (Fig. 2). Some reduction, not significant however, was also seen with the deletion of I at position 309 or R at position 315.
Deletion-induced loss of antibody binding Serum
S
8 22 93 167 170 171 193 194 204 224 337 . 363 384 408 415 "Y,
I
R
I
Q
R G P G R A F V T + + + + + + + + + + + + +
--+++ ++ -
---
+ +
-
_ -
0
mAb A47/B I G44/H7 D59/A2 F58/H3 Pl/D12 P4/DI0
-
-
-
-
_
_
++
-
+ + + + + + + +
+ + + + + + + + +
+ + + +
+ + + +
+ + + + + - - . + + - _ _ + + + + + + + - - - - - + + + 0 13 13 33 7 739393
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+ + + + + +
+ + + + + +
+ + + + + +
+ + + _ + + + +
+ + + 87 33 47 13 7
-
-
-
-
-
._ _ _
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
The individual amino acids determining IgG binding of each serum shown. A set of peptides where the amino acids were deleted one by one was used. Amino acid residue not affecting antibody binding when deleted. + Decreased absorbance value .500% when deleted. The OD values for positive sera against the complete sequence ranged between 0 45 and 1-40.
are
-
2E0 E N 0)
a)
1hO
(a)
co\
//
P4/D1O0
(a) F58/H3
0
co 0.5
I
S
R
Q R G P G R
I
A
F V T
-
Figure 2. Effect of single amino acid deletions on mAb reactivity to the neutralization-inducing site. The seroreactivity of two representative human sera and two mAb are shown.
DISCUSSION Sera from HIV-1-infected individuals in different stages of disease were analysed for site-directed antibody reactivity to the V3 region of gpl2O, virus neutralization and ADCC. The V3 region forms a loop between two cysteins,27 and the conserved sequence GPGR is predicted to form a fl-turn which may give a good exposure to antibodies. An interesting question is whether a site important for neutralization could be identified in HIV-1-infected humans. A correlation between reactivity to the entire V3 region, amino acids 296-331, and neutralization has been found recently.'4 It has also been shown that sera from HIV-infected individuals bound to a peptide (aino acids 308-322) which elicited neutralizing antibodies in animals.48 Our analysis showed that the highest frequency of reactivity of human sera was directed against the middle (C53-54, amino acids 304-323) and the C-terminal part of the V3 region (C57-58 amino acids 328-343). Sera that had a neutralizing capacity were predominantly reactive with the C53 (amino acids 304323) peptide, which contains the conserved sequence GPGR. Seventeen out of 21 sera reacted with C53 in this group, compared with 2/8 among sera negative in neutralization. Experiments with omission peptides showed that the amino acids G, P. G and R were the most important for antibody binding to this region in most of the sera with a neutralizing capacity. The flanking amino acids Q, A and F seemed to be of importance in some of the sera. Six monoclonal antibodies with a neutralizing property all reacted with this region, and where the sequence GPG were the most important for binding. This is a conserved sequence within a variable region, shared by almost all sequenced HIV- I isolates from Europe and the U.S.A. published to date.29'30 These mAb have shown reactivity against a broad range of HIV- I strains by using peptides representing the same region of eight different isolates.43 Furthermore, a set of peptides covering amino acids 306-319 where the amino acids were sequentially deleted one by one showed that amino acids GPG were essential for binding, and also proved the feasibility of using omission peptides for the identification of important single amino acids. The same conclusion could be made when looking at a set of replacement peptides (data not shown). None of the amino acids GPG could be fully substituted by any other amino acid, even though a partial substitution was seen when proline was replaced with alanine or valine. Although the mAb were raised against HTLVITIB, which contain the rarely present residues Q and R (amino acids 310-311) flanking the GPG sequence, the deletion of any of these two amino acids did, not decrease the reactivity of the mAb, while the residues I at position 309 and R at position 315 which flank GPG in most isolates30 might be of some importance for the mAb reactivity (Fig. 2). The data strongly suggest that the conserved sequence GPGR is essential for binding of neutralizing antibodies, not only murine monoclonal but also human antibodies. While human HIV-1 seropositive sera are capable of neutralizing a wide range of different HIV-I isolates,'8 sera raised in animals to proteins or peptides tend only to neutralize the strain from
Amino acids in the V3 region ofgp120 which the antigen was derived.5 ' The similarity in epitopic reactivity between the panel of mAb and the human sera indicate a broad neutralizing capacity of these mAb. Neutralization-resistant strains have been generated in vitro in the presence of neutralizing mAb,49 and amino-acid substitutions within the V3 region in some of these variants. The use of neutralizing monoclonal antibodies and the importance of such variants in vivo are of great interest. Further studies of neutralizing antibodies in vivo are of interest. It is interesting to note that four sera with a neutralizing capacity did not react at all with peptides derived from the V3 region. Other regions may thus also play an important role for neutralization.57"0 The seroreactivity against C57 (amino acids 324-338) could not be associated with neutralization or ADCC reactivity. However, serological reactivity to peptide C57 is related to good prognostic possibilities to escape from HIV-1 infection in newborns of infected mothers. It is not known whether this is a reflection of a generally high reactivity towards the whole neutralizing region, in which case the neutralizing antibodies might have been adsorbed by antigen, or whether there is a direct functional activity of antibodies directed to the C-terminal cystein of the V3 region. The lack of correlation between ADCC activity and serological response to a region known to induce ADCC,M indicates that there are several epitopes for induction of ADCC-mediating antibodies. Other authors have described such determinants on the C-terminal end of gpI2039 and on gp41,50 but not on any other HIV protein. An HIV vaccine should contain determinants for both Tand B-cell immunity. The V3 neutralization-inducing region is a strong candidate to be included as one of the subcomponents. Even though the essential sequence is known, further knowledge about type specificity and variability is necessary, as well as the structure of the protein or peptide that will elicit the most efficient and broadly reactive neutralizing response.
immunodeficiency virus-infected cells bind a 24-amino acid sequence of the viral envelope, gpl2O. Proc. nati. Acad. Sci. U.S.A. 85, 3198. 8. DALGLEISH A., CHANH T., KENNEDY R., KANDA P., CLAPHAM P. & WEISS R. (1988) Neutralization of diverse HIV-1 strains by monoclonal antibodies raised against a gp4I synthetic peptide. Virology, 165, 209. 9. EVANS D.J., MCKEATING J., MEREDITH J.M., BURKE K., KATRAH K., JOHN A., FERGUSON M., MINOR P., WEISS R. & ALMOND J. (1989) An engineered polioviruschimaera elicits broadly reactive HIV- I neutralizing antibodies. Nature, 339, 385. 10. PAPSIDERO L.D., SHEU M. & RUSCETTI F.W. (1989) Human Immunodeficiency Virus type- 1 neutralizing monoclonal antibodies which react with p17 core protein: characterization and epitope mapping. J. Virol. 63, 267. 11. ALBERT J., ABRAHAMSSON B., NAGY K., AURELIUS E., GAINES H., KROOK A., NYSTROM G. & FENYO E.M. (1990) Rapid development of isolate-specific neutralizing antibodies after primary HIV-1 infection and consequent emergence of virus variants which resist neutralization by autologous sera. AIDS, 4, 107. 12. WEBER J., WEISS R., ROBERTS C., WELLER I., TEDDER R., CLAPHAM P. et al. (1987) Human immunodeficiency virus infection in two cohorts of homosexual men: neutralising sera and association of anti-gag antibody with prognosis. Lancet, i, 119. 13. RANKI A.M., WEISS S.H., VALLE S.L., ANTONEN J., KROHN K.J.E. (1987) Neutralizing antibodies in HIV (HTLV-III) infection: correlation with clinical outcome and antibody response against different viral proteins. Clin. exp. Immunol 69, 23 1. 14. BOTTIGER B., KARLSSON A., ANDREASSO P., NAUCLtR A., MENDES COSTA C., NORRBY E. & BIBERFIELD G. (1990) Envelope crossreactivity between human immunodeficiency virus types 1 and 2 detected by different serological methods: correlation between cross-neutralization and reactivity against the main neutralizing site. J. Virol. 64, 3492. 15. ROBERT-GUROFF M., OLESKE J.M., CONNOR E.M., EPSTEIN L.G., MINNEFOR A.B. & GALLO R.C. (1987) Relationship between HTLV-III neutralising antibodies and clinical status of pediatric
16.
REFERENCES I. ROBERT-GUROFF M., BROWN M. & GALLO R.C. (1985) HTLV-III neutralising antibodies in patients with AIDS and AIDS related complex. Nature, 316, 72. 2. WEiSS R.A., CLAPHAM P., WEBER J., CHEINSONG-POPOv R., DALGLEISH A., CARNE A, WELLER I. & TEDDER R.S. (1985) Neutralization of human T-lymphotropic virus type III sera of AIDS and AIDS-risk patients. Nature, 316, 69. 3. LASKY L.A., GROOPMAN J.E., FENNIE C.W., BENZ P.N., NUNES W.M., RENZ M.E. & BERMAN P.W. (1986) Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein. Science, 233, 209. 4. PUTNEY S.D, MATrHEWs T.J., ROBEY W.G., LYNN D.L., ROBERTGUROFF M., MUELLER W.T. et al. (1986) HTLVIII/LAV-neutralizing antibodies to an E. coli-produced fragment of the virus envelope. Science, 234, 1392. 5. Ho D.D., KAPLAN J.C., RACKAUSKAS I.E. & GURNEY M.E. (1988) Second conserved domain of gp 120 is important for HIV infectivity and antibody neutralization. Science, 239, 1021. 6. PALKER T.J., CLARK M.E., LANGLOIS A., MATTHEWS T.J., WEINHOLD K.J., RANDALL R.R., BOLOGNEsI D.I. & HAYNES B.F. (1988) Type-specific neutralization of the human immunodeficiency virus with antibodies toenv-encoded synthetic peptides. Proc. nati. Acad. Sci. U.S.A. 85, 1932. 7. RuscE J.A., JAVAHERIAN K., McDANAL C., PETRO J., LYNN D.L., GRIMAILA R. et al. (1988) Antibodies that inhibit fusion of human
375
17. 18.
19.
20.
acquired imunodeficiency syndrome (AIDS) and AIDS-related complex cases. Pediatric Res. 21, 547. LJUNGGREN K., MOSCHESE V., BROLIDEN P.-A., FENYO E.-M., WAHREN B., JONDAL M. & Rossi P. (1990) Antibodies mediating cellular cytotoxicity and neutralization correlate with a better clinical stage in children born to HIV- 1 infected mothers. J. infect. Dis. 161, 198. FULTZ B.N., SRINIVASAN A., GREENE C., BUTLER D., SWENSON R. & MCCLURE H. (1987) Superinfection of a chimpanzee with a second strain of human immunodeficiency virus. J. Virol. 61, 4026. PRINCE A.M., HOROWITZ B., BAKER L., SHULMAN R., RALPH H., VALIKSKY J. et al. (1988) Failure of a human immunodeficiency virus (HIV) immunoglobulin to protect chimpanzees against experimental challenge with HIV. Proc. natl. Acad. Sci. U.S.A. 85, 6944. BERMAN P., GREGORY T., RIDDLE L., NAKAMURA G., CHAMPE M., PORTER J., WURM F., HERSCHBERG R., COBBS E. & EICHBERG J. (1990) Protection of chimpanzees from infection by HIV-1 after vaccination with recombinant glycoprotein gpl20 but not gpl60. Nature, 345, 622. DESROSIERS R., WYAND M., KODAMA T., RINGLER D., ARTHUR L.,
SEHGAL P., LETVIN N., KING N. & DANIEL M. (1989) Vaccine protection against simian immunodeficiency virus infection. Proc. nat!. Acad. Sci U.S.A. 86, 6353. 21. MURPHEY-CORB M., MARTIN L., DAVISON-FAIRBURN B., MONTELARO R., MILLER M., WEST M., OHKAWA S., BASKIN G., ZHANG J., PUTNEY S., ALLISON A. & EPPSTEIN D. (1989) A formalininactivated whole SIV vaccine confers protection in macaques. Science, 246, 1293. 22. PUTKONEN P., THORSTENSSON R., ALBERT J., NORRBY E., BIBERFELD P. & BIBERFELD G. (1991) Infection of cynomolgus monkeys with
376
23.
24.
25.
26.
27.
28. 29.
30. 31.
32.
33.
34.
35.
P. A. Broliden et al.
HIV type 2 protects against pathogenic consequences of a subsequent SIV infection. AIDS (in press). JACKSON G.G., RUBENIS M., KNIGGE M., PERKINS J.T., PAUL D.A., DESPOTEs J.C. & SPENCER P. (1988) Passive immunoneutralisation of human immunodeficiency virus in patients with advanced AIDS. Lancet, ii, 647. MODROW S., HAHN B.H., SHAW G.M., GALLO R.C., WONG-STAAL F. & WOLF H. (1987) Computer-assisted analysis of envelope protein sequences of seven human immunodeficiency virus isolates: prediction of antigenic epitopes in conserved and variable regions. J. Virol. 61, 570. GoUDSMIT J., DEBOUCK C., MELOEN R., SMIT L., BAKKER M., ASHER D.M., WOLFF A.V., GIBBS C.J. & GAJDUSEK D.C. (1988) Human immunodeficiency virus type I neutralization epitope with conserved architecture elicits early type-specific antibodies in experimentally infected chimpanzees. Proc. nat!. Acad. Sci. U.S.A. 85, 4478. MELOEN R., LISKAMP R.M. & GOUDSMIT J. (1989) Specificity and function of the individual amino acids of an important determinant of human immunodeficiency virus type i that induces neutralizing activity. J. Gen. Virol. 70, 1505. GREGORY T., LEONARD C., RIDDLE L., THOMAS J., HARRIS R. & SPELLMAN M. (1990) Disulfide bond assignment and characterization of N-linked glycosylation sites in recombinant HIV- I type IIIB gpl2O produced in CHO cells. Abstract UCLA symposia on Molecular & Cellular biology. J. Cell. Biochem., suppl. 14D, L418. RATNER L., HASELTINE W., PATARCA R., LIVAK K., STARCICH B. & JOSEPHS S. (1985) Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature, 313, 277. MYERS G., JOSEPHS S., RABSON A., SMITH T. & WONG-STAAL F. (1988) Database Human Retroviruses and AIDS. Los Alamos Natl Lab, Los Alamos. LAROSA G., DAVIDE J., WEINHOLD K., WATERBURY J., PROFY A. & LEWIS J. (1990) Conserved sequence and structural elements in the HIV-1 principal neutralizing determinant. Science, 249, 932. SKINNER M.A., TING R., LANGLOIS A., WEINHOLD K.J., LYERLY K., JAVAHERIAN K. & MATTHEWS T.J. (1988) Characteristics of a neutralizing monoclonal antibody to the HIV envelope glycoprotein. AIDS Res. Hum. Retroviruses, 4, 187. LINSLEY P., LEDBETrER J., KINNEY-THOMAS E. & Hu S.-L. (1988) Effects of anti-gpl20 monoclonal antibodies on CD4 receptor binding by the env protein of human immunodeficiency virus type 1. J. Virol. 62, 3695. MATSUSHITA S., ROBERT-GUROFF M., RUSCHE J., KoITo A., HATTORI T., HOSHINo H., JAVVAHERIAN K., TAKATSUKI K. & PUTNEY S. (1988) Characterization of a human immunodeficiency virus neutralizing monoclonal antibody and mapping of the neutralizing epitope. J. Virol. 62, 2107. BROLIDEN P.-A., LJUNGGREN K., HINKULA J., NORRBY E., AKERBLOM L. & WAHREN B. (1990) A monoclonal antibody to HIV-1 mediating cellular cytotoxicity (ADCC) and neutralization. J. Virol. 64, 936. WAHREN B., MORFELDT-MANSSON L., BIBERFELD G., MOBERG L., SONNERBORG A., LJUNGMAN P., WERNER A., KURTH R., GALLO R. & BOLOGNESI D. (1987) Characteristics of the specific cell-mediated immune response in human immunodeficiency virus infection. J. Virol. 61, 2017.
36. Rossi P., MOSCHESE V., BROLIDEN P.-A., FUNDARO C., QUINTI I., PLEBANI A. et al. (1989) Antibodies to HIV-l gpl20 env epitopes correlate with uninfected status of children born to seropositive mothers. Proc. nati. Acad. Sci. U.S.A. 86, 8055. 37. LJUNGGREN K., BOTTIGER B., BIBERFELD G., KARLSSON A., FENYO E.M. & JONDAL M. (1987) Antibody dependent cellular cytotoxicity-inducing antibodies against human immunodeficiency virus: Presence at different clinical stages. J. Immunol. 139, 2263. 38. LJUNGGREN K., BROLIDEN P.A., MORFELDT-MANSSON L., JONDAL M. & WAHREN B. (1988) IgG subclass response to HIV in relation to antibody-dependent cellular cytotoxicity at different clinical stages. Clin. exp. Immunol. 73, 343. 39. BOLOGNESi D.P. (1988) Natural immunity to HIV and its possible relationship to vaccine strategies. Microbiol. Sci. 5, 236. 40. LYERLY H.K., REED D., MATTHEWS T.J. LANGLOiS A.J., AHEARNE P.A., PETTEWAY S.R. & WEINHOLD K.J. (1987) Anti-gpI20 antibodies from HIV seropositive individuals mediate broadly reactive anti-HIV ADCC. AIDS Res. Hum. Retroviruses 3, 409. 41. B6TTIGER B., LJUNGGREN K., KARLSSON A., KROHN K., FENY6 E.M., JONDAL M. & BIBERFELD G. (1988) Neutralizing antibodies in relation to antibody-dependent cellular cytotoxicity-inducing antibodies against human immunodeficiency virus type 1. Clin. exp. Immunol. 73, 339. 42. CENTERS FOR DISEASE CONTROL (1986) Classification system for HTLV-III/LAV infections. Morbidity and Mortality Weekly Report, 34, 373. 43. AKERBLOM L., HINKULA J., BROLIDEN P.A., FRIDBERGER T., ROSEN J., ERICSSON-DE-LA CiUZ M., MOREIN B. & WAHREN B. (1990) Neutralizing cross-reactive and non-neutralizing monoclonal antibodies to HIV-l gp 120. AIDS, 4, 953. 44. SUNDQVIST V.-A., ALBERT J., OHLSSON E., HINHULA J., FENYO E.M. & WAHREN B. (1990). HIV-1 p24 antigen variation in tissue culture of isolates with defined growth characteristics. J. Med. Virol. 27, 170. 45. HOUGHTEN R.A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigenantibody interaction at the level of individual amino acids. Proc. natl. Acad. Sci. U.S.A. 82, 5131. 46. MATHIESEN T., BROLIDEN P.-A., ROSEN J. & WAHREN B. (1989) Mapping of IgG subclass- and T-cell epitopes on HIV-proteins by synthetic peptides. Immunology, 67, 453. 47. BROLIDEN P.-A., MORFELDT-MANSSON L., ROSEN J., JONDAL M. & WAHREN B. (1989) Fine specificity of IgG subclass response in HIV infected patients. Clin. exp. Immunol. 76, 216. 48. KENEALY W., MATTHEWS T., GANFIELD M.C., LANGLOIS A., WASELEFSKY D. & PETTEWAY S. (1989) Antibodies from human immunodeficiency virus-infected individuals bind to a short amino acid sequence that elicits neutralizing antibodies in animals. AIDS Res. Hum. Retroviruses 5: 173. 49. MCKEATING J.A., Gow J., GoUDSMIT J., PEARL L., MULDER C. & WEISS R. (1989) Characterization of HIV-1 neutralization escape mutants. AIDS, 3, 777. 50. KOWALSKI M., POTZ J., BASIRIPOUR L., DORFMAN T., GOH W., TERWILLIGEN E., DAYTON A., ROSEN C., HASELTINE W. & SODROSHI J. (1987) Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science, 237, 1351.
