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Synthetic Peptides in HIV Antibody Screening and Typing" OLIVIERO E. VARNIER,~ALE NARVANEN,' MIRJA KORKOLAINEN,' FLAVIA LILLO,~ SARI KONTIO,~JOSEPH ELM,^ JUKKA SUNI,' ANTTI VAHERI: AND MARJA-LIISA HUHTALACsg bLaboratoiy of Human Retrovirology Institute of Microbiology School of Medicine University of Genoa Genoa, Italy 'Labsystems Research Laboratories Helsinki, Finland dDepartment of Tropical Medicine University of Hawaii at Manoa Honolulu, Hawaii eAurora Hospital Helsinki, Finland fDepartment of Virology University of Helsinki Helsinki, Finland Although the acquired immunodeficiency syndrome (AIDS) was first described in adults, the incidence of human immunodeficiency virus (HIV) infections in children is rising and is currently a major cause of infant and childhood mortality in certain regions of the w0r1d.I~The principal mode of transmission of HIV is through direct contact with infected blood such as by intravenous (i.v.) drug abuse or through sexual transmission.' Pediatric HIV infections are acquired by vertical transmission from mother to fetus or infant and on rare occasions by horizontal tran~mission.~ In the majority of the cases in Europe and the United States the mother has been an i.v. drug abuser or has had sexual contact with i.v. drug abusers,2whereas in developing countries the infection is caused mainly by sexual contact unrelated to drug abuse.4 It has been estimated that 6,000-20,000 new cases of HIV-positive children, infected vertically from women using i.v. drugs, will occur during the next few years in the United state^.^.^ The incubation period of AIDS development for vertically infected children varies, with a median range from 4.1 months to 6.1 years, which gives an overall median of 4.8 years.= Although incubation periods longer than 10 years are rare, symptomless teenagers with HIV infection have been reported.' In the future, more cases with long incubation periods in vertically infected teenagers and adults may be observed especially in the high-risk groups. In the absence of a vaccine or effective therapy the development and use of reliable and sensitive diagnostics is one of the most important preventive measures, with the =This work was supported by grants from the ICSC World Laboratory MCD-2/11. g Address for correspondence: Marja-Liisa Huhtala, Labsystems Research Laboratories, Pultti-
tie 8, SF-00880 Helsinki, Finland. 502
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goal to identify the carriers in an effort to reduce the vertical transmission of the virus. Serology using enzyme immunoassay (EIA) is the most feasible method used for detection of HIV-infected individuals today.8 Many other technologies are also being used for diagnosis of HIV infection, including virus isolation in cell culture, recognition and binding of HIV-specific gene sequences with nucleic acid probes, and the polymerase chain reaction. These hybridization and gene amplification tests, however, can be performed only in specialized centers with costly facilities and well-trained staff. The EIAs currently used for screening of HIV IgG antibodies in blood banks are mostly based on antigens obtained from disrupted whole HIV-1 and HIV-2 virions grown in vitro in human T-cell leukemia lines. The principal method used to confirm positive EIA results has been immunoblotting based on antigens derived from disrupted HIV-1 and HIV-2 virions.8 The use of viral lysates, however, and also proteins derived from recombinant DNA expression products invariably results in some false-positive specimens in the EIA and also in equivocal interpretation in immunoblotting. This is mainly due to the cellular impurities present in these antigens, which are reactive or interfere with serum antibodies present in some sera. Such false-positive results, which are in part based on multiple other microbial infections, represent a serious problem especially in those geographical areas where multiple retroviral infections or distinct HIV type 1 and also type 2 strains are expected to occur. Consequently, it is hard to distinguish cross-reactive antibodies from unspecific reactions and to define the type of the infection in seropositive cases. In addition, there is an increasing incidence of HIV-2 infection in populations previously not thought to be at risk for type 2. Chemically synthesized peptides representing conserved immunodominant epitopes of HIV proteins provide an attractive alternative to virus-derived antigens and also to recombinant proteins in many respects. First of all, they are entirely free from cellular impurities. Second, they may be readily applied to a number of immunoassay types ranging from conventional EIAs to different types of homogenic immunoasThird, synthetic peptides provide the additional advantage over viral lysates and recombinant proteins by offering the possibility to distinguish related retrovirus infections from each other and by being able to define multiple retroviral infections Finally, they are cost effective and safe in use compared to the viral lysates, which are potentially infectious. The mapping of linear epitopes in HIV-1 proteins has demonstrated that there is a uniquely immunogenic region in the HIV- 1 transmembrane protein gp41 ( e m residues 578-613) in eliciting antibody responses during natural infection and that it contains at least two highly immunoreactive native e p i t o p e ~ . ~The - ~ ~corresponding J~ region in the HIV-2 transmembrane protein gp36 (env residues 578-613 ) has been demonstrated to behave similarly.15J6~19 In this paper, we summarize the basic strategy used to define native epitopes from the transmembrane proteins of HIV and demonstrate the function of HIV-derived synthetic peptides in HIV antibody screening and typing of HIV infections. The results of the present study are based on sera collected both in the developed and developing parts of the world.
SYSTEMATIC PROBING OF NATIVE EPITOPES OF HIV PROTEINS USING SYNTHETIC PEPTIDES Systematic epitope scanning technology, developed by Geysen et alLzo was used in order to identify continuous native epitopes from HIV type 1 and 2 structural protein sequences. This fascinating technology is simply based on the procedure of solid-phase
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peptide synthesis as first demonstrated by Merrifield in 19632*and solid phase EIA developed by Engvall and Perlmann in 1971.22 In brief: the technique allows rapid synthesis of hundreds of overlapping peptides simultaneously on a reusable format on polyethylene rods. The format has been designed to fit standard microtitration plates. Once peptides of the desired length have been synthesized on the rods, the immunoreactivity of the synthesized peptides is tested on the rods in microtitration wells using conventional EIA technology. Results obtained from such a systematic approach are independent of the accuracy of any predictive algorithm based on computer-processing of the primary sequence, and provide a comprehensive data base from which accurate relationships can be formulated.20An additional advantage of this systematic approach is that the number of negative controls incorporated into any experiment is very large, thus providing a statistical basis to set criteria for a significant positive interaction.
Epitope Scanning Profiles of HIV-1 and HIV-2 Transmembrane Proteins In FIGURE 1 is shown a typical antibody-binding profile of one HIV type 1 antibody positive serum with overlapping peptides covering the whole transmembrane protein of HIV type 1. The protein sequence has been subdivided into 14 amino acid windows, and the window has been moved along the sequence in steps of three amino acids. The peptide scan begins from the amino-terminal end of the protein and goes to the COOH-terminal end. There are two highly immunoreactive continuous native epitope regions designated as 1 and 2 in this protein. The first region is a uniquely immunogenic region being highly reactive with almost all HIV-1 antibody positive sera. FIGURE 2 summarizes five scans from the first region with five different sera derived from five different individuals in different stages of HIV- 1 infection. These scans clearly show that the highly immunoreactive region 1 containing 41 amino acids is likely to be composed of several different native epitope sites that are strongly immunoreactive with HIV antibody positive sera and that different individuals may respond differently to antigenic sites. In addition, there may be a correlation to the stage of the infection. A typical immunoreactive profile of one HIV-2 positive serum with overlapping 3. peptides covering the whole transmembrane protein of HIV-2 is shown in FIGURE There is a clear difference in the content of sequential epitope regions between the transmembrane proteins of HIV- 1 and HIV-2. The second highly immunoreactive region of HIV-2 gp34 in FIGURE3 corresponds to the uniquely immunogenic first region located also in the HIV-1 transmembrane protein (FIG. 1). In addition, to the uniquely immunogenic first region, HIV-2 contains nine additional highly immunoreactive regions.
The Highly Immunoreactive Regions of HIV-1 and HZV-2 For HIV antibody screening, after preliminary experimentation, we have found it optimal to use a test combination of overlapping pentadecapeptides covering the first region of HIV type 1 transmembrane protein and the fourth region of HIV type 2 (FIGURES 1 and 3). In HIV typing tests these immunoreactive regions are applied as single pentadecapeptides in separate tests. These sequence regions show considerable
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1
2
1
10
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FIGURE 1. Antibody-binding profile of gp41 (HIV-I, isolate HXB2). The results are shown as vertical lines proportional to the absorbance value obtained in the antibody binding EIA, plotted above the number giving the location within the gp41 sequence of the amino terminal sequence of each peptide.
A405
seroconu.
ASX
ASX
A IDS
ASX
I
44 1
10
1
10
1
10
1
10
1
10
FIGURE 2. Antibody-binding profiles of the first immunoreactive region derived from FIG. 1 with 5 different sera (ASX represents asymptomatic phase of the infection). The first vertical line in each scan in this FIGURE represents peptide number 25 in FIG.1.
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mx
2.50s 2
I
1
3
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1
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10 20 30 40 50 60 70 80 90 100 110 FIGURE 3. Antibody-binding profile of gp36 (HIV-2, isolate ROD). The results are shown as vertical lines proportional to the absorbance value obtained in the antibody-bindingEIA, plotted above the number giving location within the gp36 sequence of the amino terminal sequence of each peptide.
conservation between different isolates within the HIV type but minimal homology 4A and between the types, thus permitting the distinction between the types (FIGURES B). It should be noted that we have not been able to define any sequential highly immunoreactive epitopes from structural or regulatory HIV proteins that would be superior over the epitopes located in the transmembrane proteins.
ACCURACY OF SYNTHETIC PEPTIDE-BASED TESTS IN SIMULTANEOUS DETECTION OF HIV TYPE 1 AND 2 INFECTIONS AND TYPING OF THE INFECTIONS Three different types of assays were developed, designated as HIV-1, HIV-2, and HIV 1 +2 env peptide EIAs, using pentadecapeptides as antigens and derived from the 4A and B. transmembrane protein regions indicated in FIGURES
+
The HIV 1 2 env Peptide EIA The sensitivity of HIV 1 + 2 env peptide EIA was tested with 1245 individuals at various stages of HIV-1 infection from Finland (n = 43), Italy (n = 1076), and the Pacific area (n = 126), being seropositive in immunoblotting, and with 191 HIV-2 sera from West Africa. The specificity of the assay was determined with HIV antibody negative blood donor sera from Finland (n = 3579).
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The screening results shown in FIGURE 5 indicate 100% sensitivity for simultaneous detection of both HIV infections. The specificity with this material is 99.6 percent. It should be noted that the overall distinction between HIV antibody positive and negative sera is clear. Ninety-nine percent of the HIV-1 positive sera gave values over the measuring range, and 73% of the studied HIV-2 positive sera gave values over the measuring range. The sensitivity of the HIV 1 + 2 env peptide EIA to detect HIV infections was further studied with sera derived from donors undergoing seroconversion to HIV- 1 (seroconversion panels C and G, Boston Biomedica Inc., West Bridgewater, MA) and
HIV-1
HXBZ
QLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWS
BRU
MN
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N
26
T
2321
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sl I
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IT
I
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VT I K
Q GAR NS
AFRQV H T
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GAQ NS
AFRQV H S
VNDTL
MM142
N
T VS I K
GAQ NA
AFRQV H S
PNATL
K6W
M
T VS I K
E GAQ NA
AFRQV H S
PNATL
FIGURE 4A. The alignment of env residues 575-615 (HIV-1 isolate HXBZ) derived from the first immunoreactive region of HIV-1 gp41 with the different HIV-1 isolates and with the corresponding HIV-2 isolates.
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HIV-2
HIV-1
ROD
EQAQIQQEKNMYELQKLNSWDIFGNWFDLTSWVKYIQYGVL
NIHZ
E
V T
MM142
E
X6W
E
MAL
ES I
EX
LE DK ASLW
SISK LW
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ES T
EX
LE DK ASLW
SI Q LW
KIFIM
26
ES T
EQ
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NI Q LW
KIFIM
2321
ES T
I ERD LA DK ANLW
DISN LW
KIFIM
JY1
NS I
I
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ES N
LE DK ASLW
NI N LW
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ES N
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NI N LW
KIFIM
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KS T
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DI N LW
KIFIM
sc
ES N
LE DK ASLW
NI N LW
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ES N
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CDC4
ES N
LQ DK ASLWT
DI K LW
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DI N LW
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EQ
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FIGURE 4B. T h e alignment of e m residues 642-682 (HIV-2 isolate ROD) derived from the fourth immunoreactive region of HIV-2 gp36 with different HIV-2 isolates. Only amino acid substitutions with respect to isolates HXB2 in HIV-1 and ROD in HIV-2 are noted.
from donors having low titers of antibodies to HIV-1 (low titer serum panel B01, Boston Biomedica Inc.). The test results were compared to recombinant and other peptide-based tests (Wellcozyme HIV Recombinant, Wellcome Diagnostics, Dartford, England; HIV- W-ZELISA, Biochrom KG, Berlin; Abbott Recombinant HIV- 1/ HIV-2 EIA, Abbott Laboratories, Chicago, USA; HIV l&2 Combi IgG EIA, Pharmacia, Uppsala, Sweden) (FIGURES 6A and B). Comparison of these assays indicates that the HIV 1 + 2 env peptide EIA test is the best of the three for early detection of HIV seroconversion. For the detection of low levels of serum HIV antibodies, the HIV 1+ 2 env peptide EIA and the Wellcozyme recombinant gene product test are equal, and the results also indicate that these different test technologies have different sensitivities for individual serum specimens.
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Type Specificity of HZV-I and HZV-2 env Peptide EZAs The capacity of the HIV-1 and HIV-2 env peptide EIAs to distinguish the infections is shown in FIGURES 7A and B. Type specificity of the HIV-1 peptide EIA was tested with 1194 HIV-1 positive sera and with 187 HIV-2 positive sera (FIG.7A). The type specificity of the studied sera is based on immunoblotting data. There was a slight 99% OF HIV- I POSITIVE SAMPLES ARE OVER T H E MEASURING RANGE
737. OF HIV-2 POSITIVE SAMPLES ARE OVER T H E MEASURING RANGE
EIU
200
150
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.
-
-
.
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*.
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-
0.
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HIV - NEGATIVE
HIV-1 POSITIVE SERA
BLOOD DONORS
HIV-2 POSITIVE SERA
n = 3579 n = 1245 n = 191 FIGURE 5. Reactivityof the HIV 1 2 env peptide EIA with HIV- 1 and HIV-2 antibody-positive sera and HIV antibody-negativesera. The results are expressed as enzyme immunoassay units (EIU). The EIUs were calculated using the following formula: EIU = (S-B)/(C-B) X 100, where S is the absorbanceof the sample, B is the absorbanceof the reagent blank, and C is the absorbance of positive control.
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MYLTIPLES OF CUT-OFF
3.6
r
SEROCONVERSION PANEL C
7
2.6 2 1.6
1
0.6 0 0
7
9
14
16
21
23
28
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MULTIPLES OF CUT-OFF SEROCONVERSION PANEL G
3
2.6 2 1.5
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0.6 0
0
3
7
11
14
DAY
FIGURE 6A. Comparison of the sensitivity of the HIV 1 +2 env peptide EIA (Labsystems: HIV 1+2 env peptide EIA, *) with the recombinant-based EIAs (Abbott: HIV-1/HIV-2 EIA, 0, Wellcozyme: HIV Recombinant, U) and other synthetic peptide-based EIAs (Phannacia: HIV 1&2 Combi IgG EIA, X; Biochrom: HIV-l/-2-ELISA, +), with seroconversion panel C (upper panel) and seroconversion panel G (lower panel) derived from Boston Biomedica.
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cross-reaction with some HIV-2 positive sera. When the cutoff level was adjusted to 60 EIU, however, the cross-reactive rate of HIV-2 positive sera was only 1.6 percent. The overall distinction between type 1 and 2 sera is clear: 97% of the type 1 cases gave values over the measuring range, and 96% of the type 2 cases gave more than twice below the cutoff level. The sensitivity of the test still remains at 100 percent. The type specificity of HIV-2 env peptide EIA was tested with 378 HIV-2 positive sera from West Africa and with 658 HIV-1 positive sera from Italy (FIG.7B). Again, there was some cross-reaction with some (6.8%) HIV-1 positive sera. It should be noted, however, that these sera had over-scale absorbance values in their corresponding type 1 specific assays. As a whole, the distinction between type 2 and type 1 values is clear: the figure for type 2 specificity with this material and this test constellation is
Multiples of cut-off
1
nu
nnnnn 0
/r
0
0
+0 +
0
: j o+ g
1
2
+ + o+
+ +
3
6
4
5
7
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+
8
9
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0
0
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FIGURE 6B. Comparison of the sensitivity of the HIV 1 + 2 env peptide EIA (Labsystems: HIV 1 + 2 env peptide EIA, 0)with the recombinant-based EIA (Wellcozyme:HIV Recombinant, +) and another peptide-based EIA (Biochrom: HIV-1/-2-ELISA, 0 ), with the low-titer serum panel derived from Boston Biomedica (BO, 1-15).
93% and the sensitivity 100 percent. It should be noted that the rate of cross-reactivity in virion-based tests with this serum material ranges from 30 to 50% depending on the manufacturer of the test kit.
SUMMARY We have defined continuous native epitopes of HIV proteins by using a systematic epitope-scanning technology. We have demonstrated that there is a highly immunoreactive continuous native epitope region in the transmembrane protein gp41 of HIV-1 that
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EIU
250
9 7 % OF HIV-1 SAMPLES ARE OVER T H E MEASURING RANGE
1
0 0
0 0 0
200
0
150 0.0 0 0.0 0
... 0.0
100
0
60
HIV-NEGATIVE BLOOD DONORS n = 2808
HIV- 1 POSITIVE SERA
HIV-2 POSITIVE SERA
n = 1194
n = 187
FIGURE 7A. The reactivity of HIV-1 env peptide EIA (Labsystems: HIV-1 env peptide EIA) with HIV-1 and HIV-2 antibody-positive sera and with HIV antibody-negative sera.
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83%OF HIV-2 SAMPLES ARE OVER THE MEASURING RANGE
EIU
200
-
. 150
-
100
-
60
-
........ ...... ....... CUL
HIV-NEGATIVE BLOOD DONORS n = 2412
oil
HIV- 1 POSITIVE SERA
HIV-2 POSITIVE SERA
n = 658
n = 378
FIGURE 7B. The reactivity of HIV-2 env peptide EIA (Labsystems: HIV-2 env peptide EIA) with HIV-2 and HIV-1 antibody-positive sera and with HIV antibody-negative sera.
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is immunoreactive with all studied HIV- 1 antibody-positive sera. The corresponding region in HIV-2 gp34 behaves similarly. There is a clear difference, however, between HIV type 1 and type 2 transmembrane proteins in the number of highly immunoreactive continuous epitope regions. W e have further demonstrated that these highly immunoreactive regions, when presented properly as synthetic antigens in solid-phase EIA, can provide tests unusually suitable for early and reliable diagnosis of HIV-1 and HIV-2 infections and for type-specific distinction of the two types of HIV infections.
REFERENCES MOSCATO,F. VEBER,M-J. MAYAUX, C. 1. BLANCHE,S., C. R o u z ~ o u x ,M-L. GUIHARD JACOMET, J. TRICOIRE, A. DEVILLE,M. VIAL,G. FIRTON,A. DE CREPY,D. DOUARD, M. ROBIN,C. COURPOTIN, N. CIRARU-VIGNERON, F. LE DEIST& C. GRISCELLI.1989. A prospective study of infants born to women seropositive for human immunodeficiency virus type 1. N. Engl. J. Med. 320 1643-1648. 2. PIZZO,P. A. 1990. Pediatric AIDS: Problems within problems. J. Infect. Dis. 161: 316-325. 3. SCOTT,G.B., C. HUYTO, R. W. MAKUCH,M. T. MASTRUCCI, T. OCONNOR,C. D. MITCHELL,E. J. TRAPIDO& W. P. PARKS.1989. Survival in children with perinatally acquired human immunodeficiency virus type 1 infection. N. Engl. J. Med. 321: 1791- 1796. 4. RYDER,R. W., W. NSA,S. E. HASSING, F. BEHETS,M. RAYFIELD, B. EKUNGOLA, A. M. NELSON,U.MULENDA, H. FRANCIS, K. MWANDAGALIRWA, F. DAVACHI, M. ROGERS, N. NZILAMBI,A. GREENBERG, J. MA”, T. C. QUINN,P. PIT & J. W. CURRAN. 1989. Perinatal transmission of the human immunodeficiency virus type 1 to infants of seropositive women in Zaire. N. Engl. J. Med. 320 1637-1642. E. E., D. HARTEL,P. A. SELWYN, R. S. KLEIN,K. DAVENNY, M. ROGERS, 5. SCHOENBAUM, C. FEINER & G. FRIENDLAND. 1989. Risk factors for human immunodeficiency virus infection in intravenous drug users. N. Engl. J. Med. 321: 874-879. 6. AUGER,I., P. THOMAS,V. DE GRUTTOLA,D. MORSE,D. MOORE,R. WILLIAMS,B. TRUMAN & C. E. LAWRENCE. 1988. Incubation periods for pediatric AIDS patients. Nature 336 575-577. R. GRIMSON, A. KAELL,K. FLATHERTY, J. GULLA,R. A. 7. BURGER,H., A.-L. BELLMAN, GIBBS,P.-N. NGUYUN & B. WEISER.1990. Long HIV-1 incubation periods and dynamics of transmission within a family. Lancet 21: 134-136. 8. JACKSON, J. & H. H. BALFOUR, JR. 1988. Practical diagnostic testing for human immunodeficiency virus. Clin. Microbiol. Rev. 1: 124-138. 9. WANG,J. J. G., S.STEEL,R. WISNIEWOLKSKI & C. Y.WANG.1986. Detection of antibodies to human T-lymphotrophic virus type 111 by using a synthetic peptide of 21 amino acid residues correspondingto a highly antigenic segment of gp41 envelope protein. Proc. Natl. Acad. Sci. USA 83: 6159-6163. 10. ROSEN,J., Y-L. HOM,A. WHALLEY, R. SMITH& R. B. NASO.1987. Detection of antibodies to HIV using synthetic peptides derived from the gp41 envelope protein. In Vaccines. 87: 188-193. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York. 11. GNANN,J. W., P. L. SCHWIMMBECK JR, J. A. NELSON,A. B. TRUAX& B. A. OLDSTONE. 1987. Diagnostic of AIDS by using a 12-aminoacid peptide representing an immunodominant epitope of the human immunodeficiency virus. J. Infec. Dis. 156: 261-267. J. ALBERT,E. M. FENYO,H. GAINES,M. VON SYDOW, 12. CHIODI,F., A. VON GEGERFELDT, G. BIBERFELD,E. PARKS& E. NORRBY.1987. Site-directed ELISA with synthetic peptides representing the HIV transmembrane glycoprotein. J. Med. Virol. 23: 1-9. 13. NARVANEN,A,, M. KORKOLAINEN, J. SUNI,J. KORPELA,S. KONTIO,P. PARTANEN, A. VAHERI & M.-L. HUHTALA.1988. Synthetic env gp41 peptide as a sensitive and specific diagnostic reagent in different stages of human immunodeficiency virus type 1 infection. J. Med. Virol. 2 6 111-118.
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R. R. DOHERTY,D. A. MCPHEE,D. 14. KEMP,B. E., D. B. RYLATT,P. G. BUNDESEN, STAPLETON, L. E. C O ~ I SK., WILSON,M. A. JOHN,J. M. WAN,D. P. DIHN,S. MILES & C. J. HILLYARD. 1988. Autologous red cell agglutination assay for HIV-1 antibodies: amplified test with whole blood. Science 241: 1352-1354. S. MITCHELL, J. A. NELSON& M. B. A. OLDSTONE. 15. GNANN,JR., J. W., J. B. MCGORMICK, 1987. Synthetic peptide immunoassay distinguishes HIV type 1 and HIV type 2 infections. Science 237: 1346-1349. F. CHIODI,A. VON GEGERFELD, A. NAUCLER, E. PARKS& 16. NORRBY,E., G. BIBERFELD, R. LERNER.1987. Discrimination between antibodies to HIV and to related retroviruses using site-directed serology. Nature : 329-350. M., A. NARVANEN, 0. VARNIER, F. LILLO,S. KONTIO,R. RESCALDANI, 17. KORKOLAINEN, A. VISCONTI& M.-L. HUHTALA.1990. Epitope mapping of the transmembrane proteins of human retroviruses by using solid phase synthesis on the rods. In Innovation and Perspectives in Solid Phase Synthesis-Peptides, Polypeptides and Oliconucleotides: Macro-organic Reagent and Catalyst. R. Epton, Ed.: 511-518. SPCC (UK) Ltd. West Midland, U.K. A., M. KORKOLAINEN, F. LILLO,0.VARNIER, R. RESCALDANI, A. VISCONTI, 18. NARVANEN, E. DE GOURVILLE, A. VAHERI& M.-L. HUHTALA.1990. Synthetic peptides derived from HIV-1, HIV-2 and HTLV-I envelope proteins in human retrovirus serology. In Rapid Methods and Automation in Microbiology and Immunology. A. Balows, R. Tilton & A. Vaheri, MS. Springer Verlag, Berlin. In press. E., E. PARKS,G. UTTER,R. HOUGHTEN & R. LERNER.1989. Immunochemistry 19. NORRBY, of the dominating antigenic region Ala(582) to Cys(604) in the transmembranous protein of simian and human immunodeficiency virus. J. Immunol. 143: 3602-3608. 1984. Use of peptide synthesis to probe 20. GEYSEN,H. M., R. H. MELOEN& S.J. BARTELING. viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA 81: 3998-4002. R. B. 1963. Solid phase peptide synthesis I. The synthesis of a tetrapeptide. 21. MERRIFIELD, J. Am. Chem. SOC.85: 2149-2154. E. & P. PERLMANN. 1971. Enzyme-linked immunosorbent assay (ELSA). Quan22. ENGVALL, titative assay of immunoglobulin G. Immunochemistry 8: 874-879.
Synthetic Peptides in HIV Antibody Screening and Typing" OLIVIERO E. VARNIER,~ALE NARVANEN,' MIRJA KORKOLAINEN,' FLAVIA LILLO,~ SARI KONTIO,~JOSEPH ELM,^ JUKKA SUNI,' ANTTI VAHERI: AND MARJA-LIISA HUHTALACsg bLaboratoiy of Human Retrovirology Institute of Microbiology School of Medicine University of Genoa Genoa, Italy 'Labsystems Research Laboratories Helsinki, Finland dDepartment of Tropical Medicine University of Hawaii at Manoa Honolulu, Hawaii eAurora Hospital Helsinki, Finland fDepartment of Virology University of Helsinki Helsinki, Finland Although the acquired immunodeficiency syndrome (AIDS) was first described in adults, the incidence of human immunodeficiency virus (HIV) infections in children is rising and is currently a major cause of infant and childhood mortality in certain regions of the w0r1d.I~The principal mode of transmission of HIV is through direct contact with infected blood such as by intravenous (i.v.) drug abuse or through sexual transmission.' Pediatric HIV infections are acquired by vertical transmission from mother to fetus or infant and on rare occasions by horizontal tran~mission.~ In the majority of the cases in Europe and the United States the mother has been an i.v. drug abuser or has had sexual contact with i.v. drug abusers,2whereas in developing countries the infection is caused mainly by sexual contact unrelated to drug abuse.4 It has been estimated that 6,000-20,000 new cases of HIV-positive children, infected vertically from women using i.v. drugs, will occur during the next few years in the United state^.^.^ The incubation period of AIDS development for vertically infected children varies, with a median range from 4.1 months to 6.1 years, which gives an overall median of 4.8 years.= Although incubation periods longer than 10 years are rare, symptomless teenagers with HIV infection have been reported.' In the future, more cases with long incubation periods in vertically infected teenagers and adults may be observed especially in the high-risk groups. In the absence of a vaccine or effective therapy the development and use of reliable and sensitive diagnostics is one of the most important preventive measures, with the =This work was supported by grants from the ICSC World Laboratory MCD-2/11. g Address for correspondence: Marja-Liisa Huhtala, Labsystems Research Laboratories, Pultti-
tie 8, SF-00880 Helsinki, Finland. 502
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goal to identify the carriers in an effort to reduce the vertical transmission of the virus. Serology using enzyme immunoassay (EIA) is the most feasible method used for detection of HIV-infected individuals today.8 Many other technologies are also being used for diagnosis of HIV infection, including virus isolation in cell culture, recognition and binding of HIV-specific gene sequences with nucleic acid probes, and the polymerase chain reaction. These hybridization and gene amplification tests, however, can be performed only in specialized centers with costly facilities and well-trained staff. The EIAs currently used for screening of HIV IgG antibodies in blood banks are mostly based on antigens obtained from disrupted whole HIV-1 and HIV-2 virions grown in vitro in human T-cell leukemia lines. The principal method used to confirm positive EIA results has been immunoblotting based on antigens derived from disrupted HIV-1 and HIV-2 virions.8 The use of viral lysates, however, and also proteins derived from recombinant DNA expression products invariably results in some false-positive specimens in the EIA and also in equivocal interpretation in immunoblotting. This is mainly due to the cellular impurities present in these antigens, which are reactive or interfere with serum antibodies present in some sera. Such false-positive results, which are in part based on multiple other microbial infections, represent a serious problem especially in those geographical areas where multiple retroviral infections or distinct HIV type 1 and also type 2 strains are expected to occur. Consequently, it is hard to distinguish cross-reactive antibodies from unspecific reactions and to define the type of the infection in seropositive cases. In addition, there is an increasing incidence of HIV-2 infection in populations previously not thought to be at risk for type 2. Chemically synthesized peptides representing conserved immunodominant epitopes of HIV proteins provide an attractive alternative to virus-derived antigens and also to recombinant proteins in many respects. First of all, they are entirely free from cellular impurities. Second, they may be readily applied to a number of immunoassay types ranging from conventional EIAs to different types of homogenic immunoasThird, synthetic peptides provide the additional advantage over viral lysates and recombinant proteins by offering the possibility to distinguish related retrovirus infections from each other and by being able to define multiple retroviral infections Finally, they are cost effective and safe in use compared to the viral lysates, which are potentially infectious. The mapping of linear epitopes in HIV-1 proteins has demonstrated that there is a uniquely immunogenic region in the HIV- 1 transmembrane protein gp41 ( e m residues 578-613) in eliciting antibody responses during natural infection and that it contains at least two highly immunoreactive native e p i t o p e ~ . ~The - ~ ~corresponding J~ region in the HIV-2 transmembrane protein gp36 (env residues 578-613 ) has been demonstrated to behave similarly.15J6~19 In this paper, we summarize the basic strategy used to define native epitopes from the transmembrane proteins of HIV and demonstrate the function of HIV-derived synthetic peptides in HIV antibody screening and typing of HIV infections. The results of the present study are based on sera collected both in the developed and developing parts of the world.
SYSTEMATIC PROBING OF NATIVE EPITOPES OF HIV PROTEINS USING SYNTHETIC PEPTIDES Systematic epitope scanning technology, developed by Geysen et alLzo was used in order to identify continuous native epitopes from HIV type 1 and 2 structural protein sequences. This fascinating technology is simply based on the procedure of solid-phase
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peptide synthesis as first demonstrated by Merrifield in 19632*and solid phase EIA developed by Engvall and Perlmann in 1971.22 In brief: the technique allows rapid synthesis of hundreds of overlapping peptides simultaneously on a reusable format on polyethylene rods. The format has been designed to fit standard microtitration plates. Once peptides of the desired length have been synthesized on the rods, the immunoreactivity of the synthesized peptides is tested on the rods in microtitration wells using conventional EIA technology. Results obtained from such a systematic approach are independent of the accuracy of any predictive algorithm based on computer-processing of the primary sequence, and provide a comprehensive data base from which accurate relationships can be formulated.20An additional advantage of this systematic approach is that the number of negative controls incorporated into any experiment is very large, thus providing a statistical basis to set criteria for a significant positive interaction.
Epitope Scanning Profiles of HIV-1 and HIV-2 Transmembrane Proteins In FIGURE 1 is shown a typical antibody-binding profile of one HIV type 1 antibody positive serum with overlapping peptides covering the whole transmembrane protein of HIV type 1. The protein sequence has been subdivided into 14 amino acid windows, and the window has been moved along the sequence in steps of three amino acids. The peptide scan begins from the amino-terminal end of the protein and goes to the COOH-terminal end. There are two highly immunoreactive continuous native epitope regions designated as 1 and 2 in this protein. The first region is a uniquely immunogenic region being highly reactive with almost all HIV-1 antibody positive sera. FIGURE 2 summarizes five scans from the first region with five different sera derived from five different individuals in different stages of HIV- 1 infection. These scans clearly show that the highly immunoreactive region 1 containing 41 amino acids is likely to be composed of several different native epitope sites that are strongly immunoreactive with HIV antibody positive sera and that different individuals may respond differently to antigenic sites. In addition, there may be a correlation to the stage of the infection. A typical immunoreactive profile of one HIV-2 positive serum with overlapping 3. peptides covering the whole transmembrane protein of HIV-2 is shown in FIGURE There is a clear difference in the content of sequential epitope regions between the transmembrane proteins of HIV- 1 and HIV-2. The second highly immunoreactive region of HIV-2 gp34 in FIGURE3 corresponds to the uniquely immunogenic first region located also in the HIV-1 transmembrane protein (FIG. 1). In addition, to the uniquely immunogenic first region, HIV-2 contains nine additional highly immunoreactive regions.
The Highly Immunoreactive Regions of HIV-1 and HZV-2 For HIV antibody screening, after preliminary experimentation, we have found it optimal to use a test combination of overlapping pentadecapeptides covering the first region of HIV type 1 transmembrane protein and the fourth region of HIV type 2 (FIGURES 1 and 3). In HIV typing tests these immunoreactive regions are applied as single pentadecapeptides in separate tests. These sequence regions show considerable
VARNIER ef al.: SYNTHETIC PEPTIDES
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1
2
1
10
20
30
50
40
60
70
80
100
90
110
FIGURE 1. Antibody-binding profile of gp41 (HIV-I, isolate HXB2). The results are shown as vertical lines proportional to the absorbance value obtained in the antibody binding EIA, plotted above the number giving the location within the gp41 sequence of the amino terminal sequence of each peptide.
A405
seroconu.
ASX
ASX
A IDS
ASX
I
44 1
10
1
10
1
10
1
10
1
10
FIGURE 2. Antibody-binding profiles of the first immunoreactive region derived from FIG. 1 with 5 different sera (ASX represents asymptomatic phase of the infection). The first vertical line in each scan in this FIGURE represents peptide number 25 in FIG.1.
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mx
2.50s 2
I
1
3
4
6
1
1
1
1
1
1
1
7
1
8
1
1
1
1
1
10 20 30 40 50 60 70 80 90 100 110 FIGURE 3. Antibody-binding profile of gp36 (HIV-2, isolate ROD). The results are shown as vertical lines proportional to the absorbance value obtained in the antibody-bindingEIA, plotted above the number giving location within the gp36 sequence of the amino terminal sequence of each peptide.
conservation between different isolates within the HIV type but minimal homology 4A and between the types, thus permitting the distinction between the types (FIGURES B). It should be noted that we have not been able to define any sequential highly immunoreactive epitopes from structural or regulatory HIV proteins that would be superior over the epitopes located in the transmembrane proteins.
ACCURACY OF SYNTHETIC PEPTIDE-BASED TESTS IN SIMULTANEOUS DETECTION OF HIV TYPE 1 AND 2 INFECTIONS AND TYPING OF THE INFECTIONS Three different types of assays were developed, designated as HIV-1, HIV-2, and HIV 1 +2 env peptide EIAs, using pentadecapeptides as antigens and derived from the 4A and B. transmembrane protein regions indicated in FIGURES
+
The HIV 1 2 env Peptide EIA The sensitivity of HIV 1 + 2 env peptide EIA was tested with 1245 individuals at various stages of HIV-1 infection from Finland (n = 43), Italy (n = 1076), and the Pacific area (n = 126), being seropositive in immunoblotting, and with 191 HIV-2 sera from West Africa. The specificity of the assay was determined with HIV antibody negative blood donor sera from Finland (n = 3579).
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The screening results shown in FIGURE 5 indicate 100% sensitivity for simultaneous detection of both HIV infections. The specificity with this material is 99.6 percent. It should be noted that the overall distinction between HIV antibody positive and negative sera is clear. Ninety-nine percent of the HIV-1 positive sera gave values over the measuring range, and 73% of the studied HIV-2 positive sera gave values over the measuring range. The sensitivity of the HIV 1 + 2 env peptide EIA to detect HIV infections was further studied with sera derived from donors undergoing seroconversion to HIV- 1 (seroconversion panels C and G, Boston Biomedica Inc., West Bridgewater, MA) and
HIV-1
HXBZ
QLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWS
BRU
MN
sc
T
SF2
CDC4 WMJ2 RF
PIAL
F
ELI
N
26
T
2321
P N
sl I
JY1
IT
I
HIV-2
1
ROD
VT I K
Q GAR NS
AFRQV H T
VNDSL
NIHZ
VT I K
GAQ NS
AFRQV H S
VNDTL
MM142
N
T VS I K
GAQ NA
AFRQV H S
PNATL
K6W
M
T VS I K
E GAQ NA
AFRQV H S
PNATL
FIGURE 4A. The alignment of env residues 575-615 (HIV-1 isolate HXBZ) derived from the first immunoreactive region of HIV-1 gp41 with the different HIV-1 isolates and with the corresponding HIV-2 isolates.
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HIV-2
HIV-1
ROD
EQAQIQQEKNMYELQKLNSWDIFGNWFDLTSWVKYIQYGVL
NIHZ
E
V T
MM142
E
X6W
E
MAL
ES I
EX
LE DK ASLW
SISK LW
RIFIM
EL I
ES T
EX
LE DK ASLW
SI Q LW
KIFIM
26
ES T
EQ
LE DK ASLW
NI Q LW
KIFIM
2321
ES T
I ERD LA DK ANLW
DISN LW
KIFIM
JY1
NS I
I
EQD LQ DK ASLW
S I / K LW
KIFIM
HXB 2
ES N
LE DK ASLW
NI N LW
KLFIM
BZU
ES N
LE DK ASLW
NI N LW
KIFIM
M: I
KS T
LE DK ASLW
DI N LW
KIFIM
sc
ES N
LE DK ASLW
NI N LW
KIFIM
SF 2
ES N
LE DK ASLW
SI N LW
XIFIM
CDC4
ES N
LQ DK ASLWT
DI K LW
KIFIM
EQ
LE DK ASLW
DI N LW
KIFIM
EQ
LE DK ASLW
DI Q LW
RIFIM
F
IR
IY
V
I
IY
V
I
IY
FIGURE 4B. T h e alignment of e m residues 642-682 (HIV-2 isolate ROD) derived from the fourth immunoreactive region of HIV-2 gp36 with different HIV-2 isolates. Only amino acid substitutions with respect to isolates HXB2 in HIV-1 and ROD in HIV-2 are noted.
from donors having low titers of antibodies to HIV-1 (low titer serum panel B01, Boston Biomedica Inc.). The test results were compared to recombinant and other peptide-based tests (Wellcozyme HIV Recombinant, Wellcome Diagnostics, Dartford, England; HIV- W-ZELISA, Biochrom KG, Berlin; Abbott Recombinant HIV- 1/ HIV-2 EIA, Abbott Laboratories, Chicago, USA; HIV l&2 Combi IgG EIA, Pharmacia, Uppsala, Sweden) (FIGURES 6A and B). Comparison of these assays indicates that the HIV 1 + 2 env peptide EIA test is the best of the three for early detection of HIV seroconversion. For the detection of low levels of serum HIV antibodies, the HIV 1+ 2 env peptide EIA and the Wellcozyme recombinant gene product test are equal, and the results also indicate that these different test technologies have different sensitivities for individual serum specimens.
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Type Specificity of HZV-I and HZV-2 env Peptide EZAs The capacity of the HIV-1 and HIV-2 env peptide EIAs to distinguish the infections is shown in FIGURES 7A and B. Type specificity of the HIV-1 peptide EIA was tested with 1194 HIV-1 positive sera and with 187 HIV-2 positive sera (FIG.7A). The type specificity of the studied sera is based on immunoblotting data. There was a slight 99% OF HIV- I POSITIVE SAMPLES ARE OVER T H E MEASURING RANGE
737. OF HIV-2 POSITIVE SAMPLES ARE OVER T H E MEASURING RANGE
EIU
200
150
I00
.
-
-
.
me.
mm
*.
.. . me
-
0.
..
0
HIV - NEGATIVE
HIV-1 POSITIVE SERA
BLOOD DONORS
HIV-2 POSITIVE SERA
n = 3579 n = 1245 n = 191 FIGURE 5. Reactivityof the HIV 1 2 env peptide EIA with HIV- 1 and HIV-2 antibody-positive sera and HIV antibody-negativesera. The results are expressed as enzyme immunoassay units (EIU). The EIUs were calculated using the following formula: EIU = (S-B)/(C-B) X 100, where S is the absorbanceof the sample, B is the absorbanceof the reagent blank, and C is the absorbance of positive control.
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510
MYLTIPLES OF CUT-OFF
3.6
r
SEROCONVERSION PANEL C
7
2.6 2 1.6
1
0.6 0 0
7
9
14
16
21
23
28
18
24
28
DAY 3.5
MULTIPLES OF CUT-OFF SEROCONVERSION PANEL G
3
2.6 2 1.5
1
0.6 0
0
3
7
11
14
DAY
FIGURE 6A. Comparison of the sensitivity of the HIV 1 +2 env peptide EIA (Labsystems: HIV 1+2 env peptide EIA, *) with the recombinant-based EIAs (Abbott: HIV-1/HIV-2 EIA, 0, Wellcozyme: HIV Recombinant, U) and other synthetic peptide-based EIAs (Phannacia: HIV 1&2 Combi IgG EIA, X; Biochrom: HIV-l/-2-ELISA, +), with seroconversion panel C (upper panel) and seroconversion panel G (lower panel) derived from Boston Biomedica.
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SYNTHETIC PEPTIDES
cross-reaction with some HIV-2 positive sera. When the cutoff level was adjusted to 60 EIU, however, the cross-reactive rate of HIV-2 positive sera was only 1.6 percent. The overall distinction between type 1 and 2 sera is clear: 97% of the type 1 cases gave values over the measuring range, and 96% of the type 2 cases gave more than twice below the cutoff level. The sensitivity of the test still remains at 100 percent. The type specificity of HIV-2 env peptide EIA was tested with 378 HIV-2 positive sera from West Africa and with 658 HIV-1 positive sera from Italy (FIG.7B). Again, there was some cross-reaction with some (6.8%) HIV-1 positive sera. It should be noted, however, that these sera had over-scale absorbance values in their corresponding type 1 specific assays. As a whole, the distinction between type 2 and type 1 values is clear: the figure for type 2 specificity with this material and this test constellation is
Multiples of cut-off
1
nu
nnnnn 0
/r
0
0
+0 +
0
: j o+ g
1
2
+ + o+
+ +
3
6
4
5
7
*
+
8
9
o
0
0
++++
10 11 1 2 1 3 1 4 1 5
FIGURE 6B. Comparison of the sensitivity of the HIV 1 + 2 env peptide EIA (Labsystems: HIV 1 + 2 env peptide EIA, 0)with the recombinant-based EIA (Wellcozyme:HIV Recombinant, +) and another peptide-based EIA (Biochrom: HIV-1/-2-ELISA, 0 ), with the low-titer serum panel derived from Boston Biomedica (BO, 1-15).
93% and the sensitivity 100 percent. It should be noted that the rate of cross-reactivity in virion-based tests with this serum material ranges from 30 to 50% depending on the manufacturer of the test kit.
SUMMARY We have defined continuous native epitopes of HIV proteins by using a systematic epitope-scanning technology. We have demonstrated that there is a highly immunoreactive continuous native epitope region in the transmembrane protein gp41 of HIV-1 that
ANNALS NEW YORK ACADEMY OF SCIENCES
512
EIU
250
9 7 % OF HIV-1 SAMPLES ARE OVER T H E MEASURING RANGE
1
0 0
0 0 0
200
0
150 0.0 0 0.0 0
... 0.0
100
0
60
HIV-NEGATIVE BLOOD DONORS n = 2808
HIV- 1 POSITIVE SERA
HIV-2 POSITIVE SERA
n = 1194
n = 187
FIGURE 7A. The reactivity of HIV-1 env peptide EIA (Labsystems: HIV-1 env peptide EIA) with HIV-1 and HIV-2 antibody-positive sera and with HIV antibody-negative sera.
513
VARNIER el al.: SYNTHETIC PEPTIDES
83%OF HIV-2 SAMPLES ARE OVER THE MEASURING RANGE
EIU
200
-
. 150
-
100
-
60
-
........ ...... ....... CUL
HIV-NEGATIVE BLOOD DONORS n = 2412
oil
HIV- 1 POSITIVE SERA
HIV-2 POSITIVE SERA
n = 658
n = 378
FIGURE 7B. The reactivity of HIV-2 env peptide EIA (Labsystems: HIV-2 env peptide EIA) with HIV-2 and HIV-1 antibody-positive sera and with HIV antibody-negative sera.
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ANNALS NEW YORK ACADEMY OF SCIENCES
is immunoreactive with all studied HIV- 1 antibody-positive sera. The corresponding region in HIV-2 gp34 behaves similarly. There is a clear difference, however, between HIV type 1 and type 2 transmembrane proteins in the number of highly immunoreactive continuous epitope regions. W e have further demonstrated that these highly immunoreactive regions, when presented properly as synthetic antigens in solid-phase EIA, can provide tests unusually suitable for early and reliable diagnosis of HIV-1 and HIV-2 infections and for type-specific distinction of the two types of HIV infections.
REFERENCES MOSCATO,F. VEBER,M-J. MAYAUX, C. 1. BLANCHE,S., C. R o u z ~ o u x ,M-L. GUIHARD JACOMET, J. TRICOIRE, A. DEVILLE,M. VIAL,G. FIRTON,A. DE CREPY,D. DOUARD, M. ROBIN,C. COURPOTIN, N. CIRARU-VIGNERON, F. LE DEIST& C. GRISCELLI.1989. A prospective study of infants born to women seropositive for human immunodeficiency virus type 1. N. Engl. J. Med. 320 1643-1648. 2. PIZZO,P. A. 1990. Pediatric AIDS: Problems within problems. J. Infect. Dis. 161: 316-325. 3. SCOTT,G.B., C. HUYTO, R. W. MAKUCH,M. T. MASTRUCCI, T. OCONNOR,C. D. MITCHELL,E. J. TRAPIDO& W. P. PARKS.1989. Survival in children with perinatally acquired human immunodeficiency virus type 1 infection. N. Engl. J. Med. 321: 1791- 1796. 4. RYDER,R. W., W. NSA,S. E. HASSING, F. BEHETS,M. RAYFIELD, B. EKUNGOLA, A. M. NELSON,U.MULENDA, H. FRANCIS, K. MWANDAGALIRWA, F. DAVACHI, M. ROGERS, N. NZILAMBI,A. GREENBERG, J. MA”, T. C. QUINN,P. PIT & J. W. CURRAN. 1989. Perinatal transmission of the human immunodeficiency virus type 1 to infants of seropositive women in Zaire. N. Engl. J. Med. 320 1637-1642. E. E., D. HARTEL,P. A. SELWYN, R. S. KLEIN,K. DAVENNY, M. ROGERS, 5. SCHOENBAUM, C. FEINER & G. FRIENDLAND. 1989. Risk factors for human immunodeficiency virus infection in intravenous drug users. N. Engl. J. Med. 321: 874-879. 6. AUGER,I., P. THOMAS,V. DE GRUTTOLA,D. MORSE,D. MOORE,R. WILLIAMS,B. TRUMAN & C. E. LAWRENCE. 1988. Incubation periods for pediatric AIDS patients. Nature 336 575-577. R. GRIMSON, A. KAELL,K. FLATHERTY, J. GULLA,R. A. 7. BURGER,H., A.-L. BELLMAN, GIBBS,P.-N. NGUYUN & B. WEISER.1990. Long HIV-1 incubation periods and dynamics of transmission within a family. Lancet 21: 134-136. 8. JACKSON, J. & H. H. BALFOUR, JR. 1988. Practical diagnostic testing for human immunodeficiency virus. Clin. Microbiol. Rev. 1: 124-138. 9. WANG,J. J. G., S.STEEL,R. WISNIEWOLKSKI & C. Y.WANG.1986. Detection of antibodies to human T-lymphotrophic virus type 111 by using a synthetic peptide of 21 amino acid residues correspondingto a highly antigenic segment of gp41 envelope protein. Proc. Natl. Acad. Sci. USA 83: 6159-6163. 10. ROSEN,J., Y-L. HOM,A. WHALLEY, R. SMITH& R. B. NASO.1987. Detection of antibodies to HIV using synthetic peptides derived from the gp41 envelope protein. In Vaccines. 87: 188-193. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York. 11. GNANN,J. W., P. L. SCHWIMMBECK JR, J. A. NELSON,A. B. TRUAX& B. A. OLDSTONE. 1987. Diagnostic of AIDS by using a 12-aminoacid peptide representing an immunodominant epitope of the human immunodeficiency virus. J. Infec. Dis. 156: 261-267. J. ALBERT,E. M. FENYO,H. GAINES,M. VON SYDOW, 12. CHIODI,F., A. VON GEGERFELDT, G. BIBERFELD,E. PARKS& E. NORRBY.1987. Site-directed ELISA with synthetic peptides representing the HIV transmembrane glycoprotein. J. Med. Virol. 23: 1-9. 13. NARVANEN,A,, M. KORKOLAINEN, J. SUNI,J. KORPELA,S. KONTIO,P. PARTANEN, A. VAHERI & M.-L. HUHTALA.1988. Synthetic env gp41 peptide as a sensitive and specific diagnostic reagent in different stages of human immunodeficiency virus type 1 infection. J. Med. Virol. 2 6 111-118.
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R. R. DOHERTY,D. A. MCPHEE,D. 14. KEMP,B. E., D. B. RYLATT,P. G. BUNDESEN, STAPLETON, L. E. C O ~ I SK., WILSON,M. A. JOHN,J. M. WAN,D. P. DIHN,S. MILES & C. J. HILLYARD. 1988. Autologous red cell agglutination assay for HIV-1 antibodies: amplified test with whole blood. Science 241: 1352-1354. S. MITCHELL, J. A. NELSON& M. B. A. OLDSTONE. 15. GNANN,JR., J. W., J. B. MCGORMICK, 1987. Synthetic peptide immunoassay distinguishes HIV type 1 and HIV type 2 infections. Science 237: 1346-1349. F. CHIODI,A. VON GEGERFELD, A. NAUCLER, E. PARKS& 16. NORRBY,E., G. BIBERFELD, R. LERNER.1987. Discrimination between antibodies to HIV and to related retroviruses using site-directed serology. Nature : 329-350. M., A. NARVANEN, 0. VARNIER, F. LILLO,S. KONTIO,R. RESCALDANI, 17. KORKOLAINEN, A. VISCONTI& M.-L. HUHTALA.1990. Epitope mapping of the transmembrane proteins of human retroviruses by using solid phase synthesis on the rods. In Innovation and Perspectives in Solid Phase Synthesis-Peptides, Polypeptides and Oliconucleotides: Macro-organic Reagent and Catalyst. R. Epton, Ed.: 511-518. SPCC (UK) Ltd. West Midland, U.K. A., M. KORKOLAINEN, F. LILLO,0.VARNIER, R. RESCALDANI, A. VISCONTI, 18. NARVANEN, E. DE GOURVILLE, A. VAHERI& M.-L. HUHTALA.1990. Synthetic peptides derived from HIV-1, HIV-2 and HTLV-I envelope proteins in human retrovirus serology. In Rapid Methods and Automation in Microbiology and Immunology. A. Balows, R. Tilton & A. Vaheri, MS. Springer Verlag, Berlin. In press. E., E. PARKS,G. UTTER,R. HOUGHTEN & R. LERNER.1989. Immunochemistry 19. NORRBY, of the dominating antigenic region Ala(582) to Cys(604) in the transmembranous protein of simian and human immunodeficiency virus. J. Immunol. 143: 3602-3608. 1984. Use of peptide synthesis to probe 20. GEYSEN,H. M., R. H. MELOEN& S.J. BARTELING. viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA 81: 3998-4002. R. B. 1963. Solid phase peptide synthesis I. The synthesis of a tetrapeptide. 21. MERRIFIELD, J. Am. Chem. SOC.85: 2149-2154. E. & P. PERLMANN. 1971. Enzyme-linked immunosorbent assay (ELSA). Quan22. ENGVALL, titative assay of immunoglobulin G. Immunochemistry 8: 874-879.
