AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 6, Number 7. 1990 Mary Ann Liebert, Inc., Publishers
Multiple Antigenic Epitopes Expressed on gag Proteins, p26 and pi5, of a Human Immunodeficiency Virus (HIV) Type 2 as Defined with a Library of Monoclonal Antibodies HIROYOSHI KOMATSU,1 HIDEKI TOZAWA,1 MEIKO KAWAMURA,2 TOSHIAKI KODAMA,2 and MASANORI HAYAMI2
ABSTRACT
Thirty-two hybridoma clones producing monoclonal antibody (MAb) against HIV-2[GH-1] were established from mice immunized with NP-40-disrupted purified whole virus of a Ghanaian isolate of human immunodeficiency virus type 2 (HIV-2), strain HIV-2[GH-1]. Of these 32 MAbs, 20 reacted with p26 and the other MAbs recognized pl5 of the HIV-2[GH-1] isolate. From the results of serological characterization by these MAbs, p26 and pl5 were identified as capsid proteins and matrix protein, respectively, of HIV-2[GH-1] gag products. In addition to two gag proteins, a 55-kD protein corresponding to the primary translational product of gag gene and 39-kD protein corresponding to an intermediate product of cleavage of p55 were recognized by these MAbs in the lysate of HIV-2[GH-l]-infected cells. Moreover, these MAbs were used to analyze the number of antigenic epitopes on p26 and pl5 of HIV-2[GH-1] isolate. The results of cross-reactivity with different HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates and competitive binding assay suggest that there are at least four and five antigenic epitopes in p26 and pl5, respectively, of the HIV-2[GH-1] isolate. The biological activity of MAbs was studied by performing syncytium inhibition assay and infection inhibition assays. However, our MAbs could not inhibit syncytium formation and infection by cell-free virus.
INTRODUCTION
THEciencyimmunodeficiency syndrome (AIDS)
type 1 (HIV-1) is the causative agent of the acquired immunodefiand related disorders.1_3 However, a few cases of AIDS have been linked to a human virus type 2 (HIV-2).4"7 HIV-2 has very similar biological and morphological properties to HIV-1, but it differs in some antigenic components.4 Recently, another human retrovirus was isolated from a seropositive HIV-infected individual in Ghana. The results of Southern blot hybridization, human immunodeficiency virus
'Department of Immunology, School of Hygienic Sciences, Kitasato University, Sagamihara228, Japan,
institute for Virus Research, Kyoto University, Kyoto 606, Japan. 871
KOMATSU ET AL. Western blot analysis (WB), reverse transcriptase activity assay, and morphological examination demonstrated that this isolate belonged to the HIV-2 group, but it differed from HIV-2ROD,47 isolated by Clavel et al. in its restriction patterns, and so was designated as HIV-2[GH-1].8 The proviral gene of HIV-2[GH-1] was cloned, and the complete primary nucleotide sequence was determined.9 The genome of HIV-2[GH-1] is 9,653 nucleotides long in its RNA form, and includes at least six nonstructural genes (vif, vpx, vpr, tat, rev, and nef), in addition to the gag, pol, and env genes. Most of the structural proteins encoded by gag, pol, and env genes are recognized by human antisera from individuals infected with HIV, and the gag gene products are the most detectable of these proteins. The HIV-2 isolate cross-reacted serologically with HIV-1 and simian immunodeficiency virus (SIV) isolates.46 In fact, on Western blot of the serum of the patient from whom HIV-2[GH-1] virus was isolated, the viral proteins of HIV-2[GH-1] cross-reacted with the corresponding proteins of HIV-2ROD, HIV-1, and SIV.8 These findings suggest that it is important to understand the mode of transmission and pathogenicity of HIV-2 and to develop specific reagents that can distinguish HIV-2 from these AIDS-related retroviruses, because epidemiological situations are very complex, particularly in Africa. Here we report the development of MAbs recognizing p26 and pi 5 of the HIV-2[GH-1] isolate and the cross-reactive characteristics of MAbs with various HIV-1, HIV-2, and SIV isolates. In addition, we describe the results of characterization of both p26 and pl5, and the minimal number of epitopes on these proteins.
MATERIALS AND METHODS Virus and cells Various HIV-1 isolates, HTLV-IIIB310 and HIV-lBRU,ln HIV-2 isolates, HIV-2ROD, and HIV-2[GH-1], and SIV isolates, STLV-IIW (SIVMAC),12 and SIVAGM[TYO-l] (SIVAG1V,)1314 used in this study were obtained from the culture fluids of persistently HTLV-IIIB-infected H9 cell line, HIV-lBRU-infected Tall-1 cell line, HIV-2ROD-infected CEM cell line, HIV-2[GH-l]-infected MOLT-4 cell line, SIVMAC-infected HUT-78 cell line, and SIVAGM-infected MOLT-4 cell line, respectively. These virus-infected cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS) and antibiotics. The cells were maintained at 37°C in a humidified atmosphere of 5% C02.
Immunization
of mice
The antigen used for hybridoma cell preparation was the purified HIV-2[GH-1 ] virus from the supernatants of virus-infected MOLT-4 cells by sucrose density gradient centrifugation. The virus (100 p,g) was disrupted with 0.5% (v/v) NP-40 in phosphate-buffered saline (PBS). Two Balb/C mice were immunized by successive intraperitoneal inoculations of solubilized virus emulsified in complete Freund's adjuvant for the first inoculation and in incomplete Freund's adjuvant for following three boosters, given one week apart. Three days following a final intravenous booster with solubilized virus in PBS, the spleen cells were fused with the SP2/0-Agl4 myeloma cells according to the protocol previously described by Galfre et al..15
Enzyme-linked immunosorbent assay (ELISA) Supernatant fluids from the hybridoma cultures were screened by ELISA16 with 1% (v/v) Triton-X 100-solubilized HIV-2[GH-1 ] isolate as antigen. Wells of 96-well plastic plates were coated overnight at 4°C with a solubilized virus at 25 ng of protein per well in 50 p.1 of 0.05 M carbonate buffer (pH 9.6). Each well was washed three times with PBS containing 0.05% (v/v) Tween-20. Next, 50 p.1 of culture fluid was reacted in each well at room temperature for 1 h. After washing, 50 p.1 of peroxidase-conjugated goat anti-mouse IgG (Cappel, West Chester, PA) was added to each well. After the same incubation period, the wells were washed four times with 0.05% Tween-20 in PBS and treated with 100 pJ of the substrate mixture containing 0.1% (w/v) ophenylenediamine and 0.015% (v/v) hydrogen peroxide in 0.5 M citric acid-disodium phosphate buffer (pH 5.0). The reactions were stopped by the addition of 50 p.1 of 2N-H2S04 and the color yield was measured at 492 nm with a Corona microplate photometer, type MTP-22 (Corona, Tokyo, Japan). 872
MABs SPECIFIC FOR HIV-2 gag PROTEINS
Indirect
immunofluorescence assay (IF)
The assay was performed as described previously.14 Virus-infected or uninfected cells were smeared on a glass slide, dried at room temperature for 3 h, and then fixed with acetone at room temperature for 10 min. Cell smears were treated with hybridoma culture fluids at room temperature for 30 min, washed with PBS, and treated with fluorescein-conjugated anti-mouse IgG (Cappel) at room temperature for 30 min.
Western blot assay
(WB)
HIV-2[GH-l]-infected cells or purified virus were treated with low-salt lysis buffer (10 mM Tris-HCl, (pH 8.0), 0.14 M NaCl, 3 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl-fluoride, 0.5% NP-40] on ice for 30 min. After centrifugation at 14,000 g for 10 min at 4°C, proteins of lysates were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5 % polyacrylamide gel by the '7 procedure of Laemmli, and the fractionated proteins were transferred electrophoretically to a nitrocellulose sheet (ATTO, Tokyo, Japan) using the method of Towbin et al.18 and Burnette.19 The nitrocellulose sheet was treated with Block-Ace (Dainihon Seiyaku, Suita, Japan) overnight at 4°C, and then with culture fluids
containing MAbs. The MAbs bound to blotted proteins were located by enzyme immunoassay.
Competitive binding assay The MAbs obtained from ascites fluids by salting-out with ammonium sulfate were conjugated with peroxidase according to the method described by Wilson and Nakane.20 Wells of 96-well ELISA plates were coated overnight at 4°C with a solubilized virus at 25 ng of protein per well in 50 p.1 of 0.05 M carbonate buffer (pH 9.6). After each well was washed three times with PBS containing 0.05% (v/v) Tween-20, 50 p.1 of a 10-fold dilution of the ascites fluid and 50 p.1 of homologous or heterologous peroxidase-conjugated MAb were added into each well. After incubation for 1 h, the wells were washed and peroxidase activity was detected as described for ELISA. Specific inhibition was calculated as follows. Percent specific inhibition |(ABSmax ABS x) / (ABSmax ABSmin)] x 100, where ABSmax is maximal absorbance in the absence of competitive ascites fluid (competitor), ABSmin is minimal absorbance in the presence of a specific competitor (homologous ascites fluid at 1:10 dilution), and ABSX is absorbance in the presence of the =
-
-
heterologous competitor.
RESULTS
Identification of anti-HIV MAbs In separate fusions, hybridoma cells were generated in 536 wells in hypoxanthine/aminopterin/thymidine (HAT) culture medium. Hybridoma culture fluids were screened for anti-HIV-2[GH-1] activity by ELISA and
by IF using infected and uninfected cells. Sixty-four hybridoma cultures gave positive reactions, and of these, 32 cultures that gave strong positive reactions by ELISA were subcloned twice by limiting dilution. Thirty-one of the MAbs were found to secrete antibodies of the IgG, subclass and one (KB-3) IgG3 by the Ouchterlony method with class- and subclass-specific anti-mouse immunoglobulin typing antisera (Cappel). Characterization
of MAbs
WB was used for identification of viral proteins reacting with the 32 MAbs. The results of WB using HIV-2[GH-l]-infected cell Iysate as antigen are shown in Figure 1. Twenty MAbs reacted with p26, and the other MAbs recognized pl5 of the HIV-2[GH-1] isolate. HIV-2[GH-l]-immunized mouse serum also recognized the p26 and p 15 in the lysates of HIV-2[GH-1 ]-infected cells and of purified virus. However, these proteins were not detected in uninfected cell Iysate. Therefore, both p26 and p 15 were defined as viral-specific
proteins. In addition to these two proteins, a common 55-kD protein was recognized by most of the MAbs and 873
KOMATSU ET AL.
FIG. 1.
of MAbs with HIV-2[GH-1] isolate by WB. Proteins of HIV-2[GH-1] virus Iysate (lane 1), HIV-2[GH-l]-infected cell lysates (lanes 2-10), and uninfected cell Iysate (lane 11) were separated electrophoretically, blotted onto a nitrocellulose sheet and were treated with HIV-2[GH-l]-immunized mouse serum (lanes 1 and 2), KB-4 (lane 3), KB-7 (lane 4), KB-8 (lane 5), KB-9 (lane 6), KT-12 (lane 7), KT-10 (lane 8), KT-6 (lane 9), KT-3 (lane 10), and mixture of these MAbs (lane 11).
Reactivity
mouse antiserum in the infected cell lysates. Thus, the antigenic epitopes of p55 are serologically related to those present on p26 and p 15. A specific band of 39-kD protein also was detected by all anti-p26 MAbs and mouse antiserum, whereas it was not by anti-pl5 MAbs. Therefore, p39 contains an epitope(s) that is present on the p26 molecule and is unrelated to the pi5 molecule.
Cross-reactivity of MAbs with HIV-1, HIV-2, and SIV isolates The cross-reactivities of these anti-p26 and anti-pl5 MAbs with various HIV-1 isolates, HTLV-IIIB and hiv-1bru, HIV-2 isolates, HIV-2ROD, HIV-2[GH-1], and SIV isolates, SIVMAC and SIVAGM were tested by
I Mr
1234561
II
III
234561234561
IV 23
456
66-
4331
p26 2214-
FIG. 2. WB of
anti-p26 MAbs. HTLV-IIIB (lane 1), HIV-2ROD (lane 2), HIV-2[GH-1] (lane 3), SIVMAC (lane 4), SIVAGM (lane 5), and HTLV-I (lane 6) viral proteins were separated electrophoretically, blotted onto a nitrocellulose sheet, and were treated with four MAbs (I, KT-2; II, KT-6; III, KT-1 ; IV, KT-12 MAb). Positions of Mr markers (x 10-3) are
indicated on the left.
874
MABs SPECIFIC FOR HIV-2 gag PROTEINS
II
I Mr
12
III
34561234561234
IV 56
123456
66433322-
-p15
14-
FIG. 3. WB of anti-pl5 MAbs. Lanes are the same as in Fig. 2. Positions of Mr markers (x 10~3) are indicated on the left.
(I), KB-5; (II), KB-9; (III), KB-1; (IV), KB-12 MAb.
WB using purified virus as antigen and by IF using infected and uninfected cells. The results of cross-reactivities by WB are shown in Figures 2 and 3. For example, KT-12 (anti-p26) cross-reacted with p24 of HTLV-IIIB, p25 of HIV-2ROD, p28 of SIVMAC, and p26 of SIVAGM (Fig. 2). KB-12 (anti-pl5) also cross-reacted with pl6 of HIV-2ROD and pl5 of SIVMAC (Fig.3). From the cross-reactivities of these MAbs, anti-p26 and anti-pl5 MAbs were both divided into four groups. The anti-p26-I group reacted only with the
Table 1. Reactivity of
ANTi-p26
MAbs
with
HIV-1, HIV-2,
and
SIV Isolates Examined
by WB and
IF
Reactivity with virus isolate3 HIV-2 MAb
Group I II III
IV
Clone KT-2 KT-19 KT-6 KT-20 KT-1 KT-4 KT-5 KT-7 KT-8 KT-9 KT-11 KT-13 KT-14 KT-15 KT-16 KT-17 KT-18 KT-3 KT-12 KT-10
HTLV-Mb
HIV-1 bru
WB
IF
WB
+ ++
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
+ + ++
IF
—
+ ++
HIV-2rod WB
IF
w
w
+ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
w
+ w
+ + w w w
w
+ w
+ w w
[GH-1] WB
IF
++ ++ + + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
w
++ ++ ++
SIVMAC WB
IF
+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
+
SIVagm WB
IF
++ ++
+ +
++ ++ ++ ++ + + + + + + +
++ ++ + + + ++ ++ ++ + +
+ ++
+
w w w
w w w
+ + + w w w
+ w
++ ++ ++ + + ++ ++ ++ ++ + ++ ++ ++ ++ ++ ++
aHTLV-IIIB-infected H9 cells, HIV-lBRu-infected Tall-1 cells, HIV-2R0D-infected CEM cells, HIV-2[GH-1]infected MOLT-4 cells, SIVniAC-infected HUT-78 cells, andSIVAGM-infected MOLT-4 cells were used. Uninfected cell lines were used as negative controls. Key: +-1-, strong positive reaction; +, positive reaction; w, weak reaction; —, no reaction; ND, not determined. 875
KOMATSU ET AL. Table 2. Reactivity of
MAbs with HIV-1, HIV-2, Examined by WB and IF
ANTi-pl5
Reactivity with
and
SIV Isolates
virus isolate3
HIV-2 MAb
Group
HIV-1 bru
HIV-2 rod
WB
WB
WB
IF
KB-2 KB-5 KB-11 KB-6 KB-9
II III
IV 3
Clone
HTLV-Mb
ND ND ND ND ND ND ND ND ND ND ND ND
KB-1 KB-3 KB-4 KB-7 KB-8 KB-12 KB-13
Explanations as for Table
IF
+ ++
w
++
IF
w
+
[GH-I]
SIVMAC WB
IF
w
++ ++ + ++ ++ ++
++ ++ ++ ++ ++ ++
w
w
WB
IF
++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +
+ + + + + + + + + +
SIVagm WB
IF
1.
Key: Explanations as for Table
1.
HIV-2[GH-1] isolate, while the anti-p26-ll group cross-reacted with HIV-2ROD and SIVMAC isolates, the
anti-p26-HI group cross-reacted with SIVAGM in addition to HIV-2ROD and SIVMAC, and the anti-p26-IV group reacted with HTLV-IIIB, HIV-2s and all SIVs except KT-3, which shows no reactivity with SIVAGM (Table 1 and Fig. 2). This anti-p26-IV group containing KT-12 detected double bands in lanes 2-4. The bands that were observed below the major band may correspond to degenerative capsid proteins of respective virus
FIG. 4. IF reactivity of MAbs with virus-infected cells. (A) HIV-2[GH-l]-infected MOLT-4 cells were treated with culture fluid of P3-X63-Ag8 as a negative control. (B) HIV-2[GH-1 ]-infected MOLT-4 cells were treated with KB-1. (C) HIV-2ROD-infected CEM cells were treated with KT-1 and (D) SIVMAC-infected cells were treated with KB-1.
876
MABs SPECIFIC FOR HIV-2 gag PROTEINS
isolates, and these proteins express the antigenic epitope that is recognized only by anti-p26-IV group. On the other hand, the anti-pl5-I group reacted only with the HIV-2[GH-1] isolate, whereas the anti-pl5-II group cross-reacted with the HIV-2ROD isolate, the anti-pl5-III group cross-reacted with SIVMAC, and the anti-pl5-IV group reacted with HIV-2s and SIVMAC. Interestingly, none of these anti-pl5 MAbs crossreacted with HTLV-IIIB or SIVAGM (Table 2 and Fig. 3). By IF, the anti-p26 and anti-pl5 MAbs exhibited very weak positivity with HIV-2ROD- and HIV-2[GH-1 ]-infected cells. Examples of the IF reactivity of KB-1 and KT-1 are shown in Figure 4. These two MAbs gave strong positive reactions with SIVMAC-infected HUT-78 cells. The data from an extensive survey of IF reactivities with various isolates are summarized in Tables 1 and 2, and correlate well with the reactivities found by WB.
Competitive binding assay anti-p26 and anti-pl5 MAbs were divided into four groups, respectively. We attempted competitive binding assay to reveal whether the different groups of MAbs are directed to distinct or related epitopes. From the results, 20 anti-p26 MAbs were clearly divided into four groups corresponding to the results of cross-reactivity, except KT-19 belonging to anti-p26-I group, which inhibited the binding characteristics of peroxidase-conjugated anti-p26-III group (KT-11 and KT-15) (Table 3). The anti-pl5 MAbs were classified into five groups by competitive binding assay, but some MAbs belonging to I, II, and III were in competition against one another (Table 4). For example, KB-6 inhibited both KB-2 and KB-5, and KB-4 blocked KB-6 and KB-9 in this analysis. Moreover, the anti-pl5-IV group could be further subdivided into two groups, as KB-12 and KB-13 did not compete against each other. These findings indicate that the antigenic epitopes of pl5 are closely associated. As stated above, the
DISCUSSION We describe the generation and characterization of 32 MAbs to HIV-2[GH-1] isolate. On WB with lysates of HIV-2[GH-l]-infected cell and purified virus as antigen, 20 of 32 MAbs reacted with p26 and the other MAbs detected p 15 of the HIV-2[GH-1 ] isolate. Antiserum derived from an HIV-2[GH-1 ]-immunized mouse also recognized both p26 and p 15 in the lysates of infected cells and of purified virus. Our MAbs could not detect these species in uninfected cell lysates (Fig.l), suggesting that both anti-p26 and anti-pl5 MAbs specifically reacted with HIV-2[GH-l]-infected cells. Moreover, anti-p26 and anti-pl5 MAbs cross-reacted with viral components corresponding to the capsid proteins and matrix proteins, respectively, of AIDS-related retroviruses (Figs. 2 and 3).4~7'10"14 Therefore, we identified the p26 and pl5 as capsid protein and matrix protein, respectively, which are encoded by the gag gene of HIV-2[GH-1]. From the result of nucleotide sequence analysis of HIV-2[GH-1], the capsid protein size and matrix protein size have been defined as 26and 16-kD, respectively.9 Although the HIV-2[GH-1] capsid protein recognized by WB is the same size as that defined by the genetic investigation, the molecular weight ( 15-kD) of matrix protein which was identified by WB is slightly smaller than that (16-kD) of the product identified by the nucleotide sequence analysis. These MAbs also detected a 55-kD protein in the infected cell lysates (Fig. 1). Thus the antigenic epitopes recognized on p26 and pl5 are present on the p55 molecule, and the cellular p55 was identified as the precursor of gag protein of HI V-2[GH-1 ] isolate. In addition to p55, a 39-kD protein was detected by anti-p26 MAbs and mouse antiserum in the infected cell Iysate, but not by anti-p 15 MAbs. It is known that intermediate precursor proteins are derived from the precursor (p53-p55) oí gag protein.21 These findings identified p39 as the intermediate precursor protein containing sequence from both p26 and pi2 which are encoded by carboxy terminus of HIV-2[GH-1] gag gene.9 The cross-reactivities of MAbs against gag proteins with different HIV-1, HIV-2, and SIV isolates have been reported by several authors.22"27 Niedrig et al.24 found that five MAbs against capsid protein, p24, of the HTLV-IIIB isolate reacted with the corresponding antigens of HIV-2ROD and SIVMAC. Moreover, two anti-p24 MAbs raised against HIV-1CBU cross-reacted with HIV-2ROD and HIV-2CBL20 isolates, whereas MAbs against matrix protein, pi8, failed to cross-react.27 Minassian et al.23 also reported that two MAbs against capsid protein, p24, of the HIV-2NIH.Z isolate cross-reacted with various HIV-1, HIV-2, and SIV
877
KOMATSU ET AL.
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++ + +
+ + + + ++ + +
+ + + I + +
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MABs SPECIFIC FOR HIV-2 gag PROTEINS Table 4. Analysis of Epitpopes of
the
pl5
in
Competitive Binding Assay BY
ANTi-pl5
MAbs
Competitors Peroxidase-
I
II
IV
III
-
-
conjugated
KB-
KB-
2
5
KB-2 KB-5 KB-6 KB-9 KB-8 KB-1 KB-12 KB-13
+++++ +++++++-------++++++++++++++++++++ ++ ++ ++ ++ + -+++++++ + --______++----++
MAb
KBII
KB6
KB-
-
9
KB4
KB7
KB1
KB8
KB-
3
KB12
KB13
-
--------
-
-
-
-
-
-
-
-
-
-
-
-
---
--
The serial 10-fold dilutions (L10-L105) of ascites fluid Key: Explanation as for Table 3.
were
used
as
competitors.
isolates, but that MAbs against matrix protein, pi6, did not cross-react with HIV-1 and SIV isolates. Thus, these previous studies could not demonstrate that the MAbs against the matrix proteins cross-reacted with the different subgroups of AIDS-related viruses. However, three of our MAbs to the matrix protein of HIV-2[GH-1] isolate, anti-pl5-III group, KB-12, and KB-13, cross-reacted with the matrix protein of SIVMAC. This finding demonstrates that there is at least three common epitopes in the matrix proteins of HIV-2[GH-1] and SIVMAC isolate. The anti-p26-IV group also cross-reacted with the corresponding capsid proteins of HIV-1 and SIV isolates, in addition to the HIV-2ROD isolate. This result suggests that the capsid proteins, p24-p28, of HIV-1, HIV-2, and SIV isolates used in this study express at least one conserved immunogenic epitope that is serologically recognizable. The MAbs against the conserved epitope may be effective for detecting HIV or SIV isolates in body fluids, tissues, and cultured cells. Finally, to study the biological activity of the MAbs, two neutralization assays, syncytium inhibition assay and infection inhibition assay, were performed as described previously.28 Regrettably, the MAbs described in this study failed to neutralize the HIV-2[GH-1 ] isolate. Papsidero et al.29 reported that two MAbs to the matrix protein of HTLV-IIIB neutralized infection by the cell-free virus, and that there are two regions identified by the MAbs on the matrix protein of HTLV-IIIB isolate. This result indicates that epitopes which are available for antibody-mediated virus inhibition exist within the matrix protein of HTLV-IIIB. The epitopes may be present in matrix proteins of other AIDS-related retroviruses, because one (Glu-Leu-Asp-Arg-Trp-GluLys-Ile) of the two regions identified by these MAbs are highly conserved in those of HIVs and SIVs. ACKNOWLEDGMENTS We are grateful to Dr. Y. Tanaka for helpful and constructive discussion. This work was partly supported by a Grant-in-Aid for Research from the Ministry of Education, Science and Culture of Japan.
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proteins during
the
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of the head of
bacteriophage
T4. Nature
18. Towbin H, Staehelin T. and Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheet: Procedure and some applications. Proc Nati Acad Sei (USA) 1979;76:4350-4354. 19. Burnette WN: Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographie detection with antibody and radioiodinated protein A. Anal Biochem
1981;112:195-203. 20. Wilson BM and Nakane PK: Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In: Immunofluorescence and Related Staining Techniques. W. Knapp, K. Holubar and G. Wick (eds.). Elsevier, Amsterdam, 1978, pp. 215-224. 21. Veronese FD, Copeland TD, Oroszlan S, Gallo RC, and Sarngadharan MG: Biochemical and immunological analysis of human immunodeficiency virus gag gene products pi7 and p24. J. Virol 1988;62:795-801. 22. Koito A, Hattori T, Matsushita S, Maeda Y, Nozaki C, SagawaK, and Takatsuki K: Conserved immunogenic region of a major core protein (p24) of human and simian immunodeficiency viruses. AIDS Res Human Retroviruses
1988;4:409-417.
880
MABs SPECIFIC FOR HIV-2 gag PROTEINS 23. Minassian AA, Kalyanaraman VS. Gallo RC, and Popovic M: Monoclonal antibodies against human immunodeficiency virus (HIV) type 2 core proteins: cross-reactivity with HIV type 1 and simian immunodeficiency virus. Proc Nati Acad Sei (USA) 1988;85:6939-6943. 24.
Niedrig M, Rabanus JP, L'Age StehrJ, GelderblomHR, and Pauli G: Monoclonal antibodies directed against human immunodeficiency virus (HIV) gag proteins with specificity for conserved epitopes in HIV-1, HIV-2 and simian immunodeficiency virus. J Gen Virol 1988;69:2109-2114. 25. Ferns RB, Tedder RS. and Weiss RA: Characterization of monoclonal antibodies against the human immunodeficiency virus (HIV) gag products and their use in monitoring HIV isolate variation. J Gen Virol 1987:68:1543-1551. 26. Tersmette M, Winkel IN, Groenink M, Gruters RA, Spence RP, Saman E, Van der Groen G, Miedema F, and Huisman JG: Detection and subtyping of HIV-1 isolates with a panel of characterized monoclonal antibodies to HIV p24iw. Virology 1989;171:149-155. 27. Ferns RB, Partridge JC, Spence RP, Hunt N, and Tedder RS: Epitope location of 13 anti-gag HIV-1 monoclonal antibodies using oligopeptides and their cross reactivity with HIV-2. AIDS Res Human Retroviruses 1989; 3:829-834.
Weber JN, McClure J, Clapham PR, Singhai MC, Shriver K, and Weiss RA: monoclonal antibodies to the AIDS virus. AIDS 1988;2:25-29.
28.
Kinney Thomas E,
29.
Papsidero LD, Sheu M, andRuscetti FW: Human immunodeficiency virus type 1-neutralizing monoclonal antibodies which react with pl7 core protein: Characterization and epitope mapping. J Virol 1989;63:267-272.
Neutralizing
reprint requests to: Hiroyoshi Komatsu Department of Immunology School of Hygienic Sciences Kitasato University 1-15-1 Kitasato, Sagamihara Kanagawa 228, Japan Address
881
Multiple Antigenic Epitopes Expressed on gag Proteins, p26 and pi5, of a Human Immunodeficiency Virus (HIV) Type 2 as Defined with a Library of Monoclonal Antibodies HIROYOSHI KOMATSU,1 HIDEKI TOZAWA,1 MEIKO KAWAMURA,2 TOSHIAKI KODAMA,2 and MASANORI HAYAMI2
ABSTRACT
Thirty-two hybridoma clones producing monoclonal antibody (MAb) against HIV-2[GH-1] were established from mice immunized with NP-40-disrupted purified whole virus of a Ghanaian isolate of human immunodeficiency virus type 2 (HIV-2), strain HIV-2[GH-1]. Of these 32 MAbs, 20 reacted with p26 and the other MAbs recognized pl5 of the HIV-2[GH-1] isolate. From the results of serological characterization by these MAbs, p26 and pl5 were identified as capsid proteins and matrix protein, respectively, of HIV-2[GH-1] gag products. In addition to two gag proteins, a 55-kD protein corresponding to the primary translational product of gag gene and 39-kD protein corresponding to an intermediate product of cleavage of p55 were recognized by these MAbs in the lysate of HIV-2[GH-l]-infected cells. Moreover, these MAbs were used to analyze the number of antigenic epitopes on p26 and pl5 of HIV-2[GH-1] isolate. The results of cross-reactivity with different HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates and competitive binding assay suggest that there are at least four and five antigenic epitopes in p26 and pl5, respectively, of the HIV-2[GH-1] isolate. The biological activity of MAbs was studied by performing syncytium inhibition assay and infection inhibition assays. However, our MAbs could not inhibit syncytium formation and infection by cell-free virus.
INTRODUCTION
THEciencyimmunodeficiency syndrome (AIDS)
type 1 (HIV-1) is the causative agent of the acquired immunodefiand related disorders.1_3 However, a few cases of AIDS have been linked to a human virus type 2 (HIV-2).4"7 HIV-2 has very similar biological and morphological properties to HIV-1, but it differs in some antigenic components.4 Recently, another human retrovirus was isolated from a seropositive HIV-infected individual in Ghana. The results of Southern blot hybridization, human immunodeficiency virus
'Department of Immunology, School of Hygienic Sciences, Kitasato University, Sagamihara228, Japan,
institute for Virus Research, Kyoto University, Kyoto 606, Japan. 871
KOMATSU ET AL. Western blot analysis (WB), reverse transcriptase activity assay, and morphological examination demonstrated that this isolate belonged to the HIV-2 group, but it differed from HIV-2ROD,47 isolated by Clavel et al. in its restriction patterns, and so was designated as HIV-2[GH-1].8 The proviral gene of HIV-2[GH-1] was cloned, and the complete primary nucleotide sequence was determined.9 The genome of HIV-2[GH-1] is 9,653 nucleotides long in its RNA form, and includes at least six nonstructural genes (vif, vpx, vpr, tat, rev, and nef), in addition to the gag, pol, and env genes. Most of the structural proteins encoded by gag, pol, and env genes are recognized by human antisera from individuals infected with HIV, and the gag gene products are the most detectable of these proteins. The HIV-2 isolate cross-reacted serologically with HIV-1 and simian immunodeficiency virus (SIV) isolates.46 In fact, on Western blot of the serum of the patient from whom HIV-2[GH-1] virus was isolated, the viral proteins of HIV-2[GH-1] cross-reacted with the corresponding proteins of HIV-2ROD, HIV-1, and SIV.8 These findings suggest that it is important to understand the mode of transmission and pathogenicity of HIV-2 and to develop specific reagents that can distinguish HIV-2 from these AIDS-related retroviruses, because epidemiological situations are very complex, particularly in Africa. Here we report the development of MAbs recognizing p26 and pi 5 of the HIV-2[GH-1] isolate and the cross-reactive characteristics of MAbs with various HIV-1, HIV-2, and SIV isolates. In addition, we describe the results of characterization of both p26 and pl5, and the minimal number of epitopes on these proteins.
MATERIALS AND METHODS Virus and cells Various HIV-1 isolates, HTLV-IIIB310 and HIV-lBRU,ln HIV-2 isolates, HIV-2ROD, and HIV-2[GH-1], and SIV isolates, STLV-IIW (SIVMAC),12 and SIVAGM[TYO-l] (SIVAG1V,)1314 used in this study were obtained from the culture fluids of persistently HTLV-IIIB-infected H9 cell line, HIV-lBRU-infected Tall-1 cell line, HIV-2ROD-infected CEM cell line, HIV-2[GH-l]-infected MOLT-4 cell line, SIVMAC-infected HUT-78 cell line, and SIVAGM-infected MOLT-4 cell line, respectively. These virus-infected cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS) and antibiotics. The cells were maintained at 37°C in a humidified atmosphere of 5% C02.
Immunization
of mice
The antigen used for hybridoma cell preparation was the purified HIV-2[GH-1 ] virus from the supernatants of virus-infected MOLT-4 cells by sucrose density gradient centrifugation. The virus (100 p,g) was disrupted with 0.5% (v/v) NP-40 in phosphate-buffered saline (PBS). Two Balb/C mice were immunized by successive intraperitoneal inoculations of solubilized virus emulsified in complete Freund's adjuvant for the first inoculation and in incomplete Freund's adjuvant for following three boosters, given one week apart. Three days following a final intravenous booster with solubilized virus in PBS, the spleen cells were fused with the SP2/0-Agl4 myeloma cells according to the protocol previously described by Galfre et al..15
Enzyme-linked immunosorbent assay (ELISA) Supernatant fluids from the hybridoma cultures were screened by ELISA16 with 1% (v/v) Triton-X 100-solubilized HIV-2[GH-1 ] isolate as antigen. Wells of 96-well plastic plates were coated overnight at 4°C with a solubilized virus at 25 ng of protein per well in 50 p.1 of 0.05 M carbonate buffer (pH 9.6). Each well was washed three times with PBS containing 0.05% (v/v) Tween-20. Next, 50 p.1 of culture fluid was reacted in each well at room temperature for 1 h. After washing, 50 p.1 of peroxidase-conjugated goat anti-mouse IgG (Cappel, West Chester, PA) was added to each well. After the same incubation period, the wells were washed four times with 0.05% Tween-20 in PBS and treated with 100 pJ of the substrate mixture containing 0.1% (w/v) ophenylenediamine and 0.015% (v/v) hydrogen peroxide in 0.5 M citric acid-disodium phosphate buffer (pH 5.0). The reactions were stopped by the addition of 50 p.1 of 2N-H2S04 and the color yield was measured at 492 nm with a Corona microplate photometer, type MTP-22 (Corona, Tokyo, Japan). 872
MABs SPECIFIC FOR HIV-2 gag PROTEINS
Indirect
immunofluorescence assay (IF)
The assay was performed as described previously.14 Virus-infected or uninfected cells were smeared on a glass slide, dried at room temperature for 3 h, and then fixed with acetone at room temperature for 10 min. Cell smears were treated with hybridoma culture fluids at room temperature for 30 min, washed with PBS, and treated with fluorescein-conjugated anti-mouse IgG (Cappel) at room temperature for 30 min.
Western blot assay
(WB)
HIV-2[GH-l]-infected cells or purified virus were treated with low-salt lysis buffer (10 mM Tris-HCl, (pH 8.0), 0.14 M NaCl, 3 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl-fluoride, 0.5% NP-40] on ice for 30 min. After centrifugation at 14,000 g for 10 min at 4°C, proteins of lysates were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5 % polyacrylamide gel by the '7 procedure of Laemmli, and the fractionated proteins were transferred electrophoretically to a nitrocellulose sheet (ATTO, Tokyo, Japan) using the method of Towbin et al.18 and Burnette.19 The nitrocellulose sheet was treated with Block-Ace (Dainihon Seiyaku, Suita, Japan) overnight at 4°C, and then with culture fluids
containing MAbs. The MAbs bound to blotted proteins were located by enzyme immunoassay.
Competitive binding assay The MAbs obtained from ascites fluids by salting-out with ammonium sulfate were conjugated with peroxidase according to the method described by Wilson and Nakane.20 Wells of 96-well ELISA plates were coated overnight at 4°C with a solubilized virus at 25 ng of protein per well in 50 p.1 of 0.05 M carbonate buffer (pH 9.6). After each well was washed three times with PBS containing 0.05% (v/v) Tween-20, 50 p.1 of a 10-fold dilution of the ascites fluid and 50 p.1 of homologous or heterologous peroxidase-conjugated MAb were added into each well. After incubation for 1 h, the wells were washed and peroxidase activity was detected as described for ELISA. Specific inhibition was calculated as follows. Percent specific inhibition |(ABSmax ABS x) / (ABSmax ABSmin)] x 100, where ABSmax is maximal absorbance in the absence of competitive ascites fluid (competitor), ABSmin is minimal absorbance in the presence of a specific competitor (homologous ascites fluid at 1:10 dilution), and ABSX is absorbance in the presence of the =
-
-
heterologous competitor.
RESULTS
Identification of anti-HIV MAbs In separate fusions, hybridoma cells were generated in 536 wells in hypoxanthine/aminopterin/thymidine (HAT) culture medium. Hybridoma culture fluids were screened for anti-HIV-2[GH-1] activity by ELISA and
by IF using infected and uninfected cells. Sixty-four hybridoma cultures gave positive reactions, and of these, 32 cultures that gave strong positive reactions by ELISA were subcloned twice by limiting dilution. Thirty-one of the MAbs were found to secrete antibodies of the IgG, subclass and one (KB-3) IgG3 by the Ouchterlony method with class- and subclass-specific anti-mouse immunoglobulin typing antisera (Cappel). Characterization
of MAbs
WB was used for identification of viral proteins reacting with the 32 MAbs. The results of WB using HIV-2[GH-l]-infected cell Iysate as antigen are shown in Figure 1. Twenty MAbs reacted with p26, and the other MAbs recognized pl5 of the HIV-2[GH-1] isolate. HIV-2[GH-l]-immunized mouse serum also recognized the p26 and p 15 in the lysates of HIV-2[GH-1 ]-infected cells and of purified virus. However, these proteins were not detected in uninfected cell Iysate. Therefore, both p26 and p 15 were defined as viral-specific
proteins. In addition to these two proteins, a common 55-kD protein was recognized by most of the MAbs and 873
KOMATSU ET AL.
FIG. 1.
of MAbs with HIV-2[GH-1] isolate by WB. Proteins of HIV-2[GH-1] virus Iysate (lane 1), HIV-2[GH-l]-infected cell lysates (lanes 2-10), and uninfected cell Iysate (lane 11) were separated electrophoretically, blotted onto a nitrocellulose sheet and were treated with HIV-2[GH-l]-immunized mouse serum (lanes 1 and 2), KB-4 (lane 3), KB-7 (lane 4), KB-8 (lane 5), KB-9 (lane 6), KT-12 (lane 7), KT-10 (lane 8), KT-6 (lane 9), KT-3 (lane 10), and mixture of these MAbs (lane 11).
Reactivity
mouse antiserum in the infected cell lysates. Thus, the antigenic epitopes of p55 are serologically related to those present on p26 and p 15. A specific band of 39-kD protein also was detected by all anti-p26 MAbs and mouse antiserum, whereas it was not by anti-pl5 MAbs. Therefore, p39 contains an epitope(s) that is present on the p26 molecule and is unrelated to the pi5 molecule.
Cross-reactivity of MAbs with HIV-1, HIV-2, and SIV isolates The cross-reactivities of these anti-p26 and anti-pl5 MAbs with various HIV-1 isolates, HTLV-IIIB and hiv-1bru, HIV-2 isolates, HIV-2ROD, HIV-2[GH-1], and SIV isolates, SIVMAC and SIVAGM were tested by
I Mr
1234561
II
III
234561234561
IV 23
456
66-
4331
p26 2214-
FIG. 2. WB of
anti-p26 MAbs. HTLV-IIIB (lane 1), HIV-2ROD (lane 2), HIV-2[GH-1] (lane 3), SIVMAC (lane 4), SIVAGM (lane 5), and HTLV-I (lane 6) viral proteins were separated electrophoretically, blotted onto a nitrocellulose sheet, and were treated with four MAbs (I, KT-2; II, KT-6; III, KT-1 ; IV, KT-12 MAb). Positions of Mr markers (x 10-3) are
indicated on the left.
874
MABs SPECIFIC FOR HIV-2 gag PROTEINS
II
I Mr
12
III
34561234561234
IV 56
123456
66433322-
-p15
14-
FIG. 3. WB of anti-pl5 MAbs. Lanes are the same as in Fig. 2. Positions of Mr markers (x 10~3) are indicated on the left.
(I), KB-5; (II), KB-9; (III), KB-1; (IV), KB-12 MAb.
WB using purified virus as antigen and by IF using infected and uninfected cells. The results of cross-reactivities by WB are shown in Figures 2 and 3. For example, KT-12 (anti-p26) cross-reacted with p24 of HTLV-IIIB, p25 of HIV-2ROD, p28 of SIVMAC, and p26 of SIVAGM (Fig. 2). KB-12 (anti-pl5) also cross-reacted with pl6 of HIV-2ROD and pl5 of SIVMAC (Fig.3). From the cross-reactivities of these MAbs, anti-p26 and anti-pl5 MAbs were both divided into four groups. The anti-p26-I group reacted only with the
Table 1. Reactivity of
ANTi-p26
MAbs
with
HIV-1, HIV-2,
and
SIV Isolates Examined
by WB and
IF
Reactivity with virus isolate3 HIV-2 MAb
Group I II III
IV
Clone KT-2 KT-19 KT-6 KT-20 KT-1 KT-4 KT-5 KT-7 KT-8 KT-9 KT-11 KT-13 KT-14 KT-15 KT-16 KT-17 KT-18 KT-3 KT-12 KT-10
HTLV-Mb
HIV-1 bru
WB
IF
WB
+ ++
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
+ + ++
IF
—
+ ++
HIV-2rod WB
IF
w
w
+ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
w
+ w
+ + w w w
w
+ w
+ w w
[GH-1] WB
IF
++ ++ + + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
w
++ ++ ++
SIVMAC WB
IF
+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
+
SIVagm WB
IF
++ ++
+ +
++ ++ ++ ++ + + + + + + +
++ ++ + + + ++ ++ ++ + +
+ ++
+
w w w
w w w
+ + + w w w
+ w
++ ++ ++ + + ++ ++ ++ ++ + ++ ++ ++ ++ ++ ++
aHTLV-IIIB-infected H9 cells, HIV-lBRu-infected Tall-1 cells, HIV-2R0D-infected CEM cells, HIV-2[GH-1]infected MOLT-4 cells, SIVniAC-infected HUT-78 cells, andSIVAGM-infected MOLT-4 cells were used. Uninfected cell lines were used as negative controls. Key: +-1-, strong positive reaction; +, positive reaction; w, weak reaction; —, no reaction; ND, not determined. 875
KOMATSU ET AL. Table 2. Reactivity of
MAbs with HIV-1, HIV-2, Examined by WB and IF
ANTi-pl5
Reactivity with
and
SIV Isolates
virus isolate3
HIV-2 MAb
Group
HIV-1 bru
HIV-2 rod
WB
WB
WB
IF
KB-2 KB-5 KB-11 KB-6 KB-9
II III
IV 3
Clone
HTLV-Mb
ND ND ND ND ND ND ND ND ND ND ND ND
KB-1 KB-3 KB-4 KB-7 KB-8 KB-12 KB-13
Explanations as for Table
IF
+ ++
w
++
IF
w
+
[GH-I]
SIVMAC WB
IF
w
++ ++ + ++ ++ ++
++ ++ ++ ++ ++ ++
w
w
WB
IF
++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +
+ + + + + + + + + +
SIVagm WB
IF
1.
Key: Explanations as for Table
1.
HIV-2[GH-1] isolate, while the anti-p26-ll group cross-reacted with HIV-2ROD and SIVMAC isolates, the
anti-p26-HI group cross-reacted with SIVAGM in addition to HIV-2ROD and SIVMAC, and the anti-p26-IV group reacted with HTLV-IIIB, HIV-2s and all SIVs except KT-3, which shows no reactivity with SIVAGM (Table 1 and Fig. 2). This anti-p26-IV group containing KT-12 detected double bands in lanes 2-4. The bands that were observed below the major band may correspond to degenerative capsid proteins of respective virus
FIG. 4. IF reactivity of MAbs with virus-infected cells. (A) HIV-2[GH-l]-infected MOLT-4 cells were treated with culture fluid of P3-X63-Ag8 as a negative control. (B) HIV-2[GH-1 ]-infected MOLT-4 cells were treated with KB-1. (C) HIV-2ROD-infected CEM cells were treated with KT-1 and (D) SIVMAC-infected cells were treated with KB-1.
876
MABs SPECIFIC FOR HIV-2 gag PROTEINS
isolates, and these proteins express the antigenic epitope that is recognized only by anti-p26-IV group. On the other hand, the anti-pl5-I group reacted only with the HIV-2[GH-1] isolate, whereas the anti-pl5-II group cross-reacted with the HIV-2ROD isolate, the anti-pl5-III group cross-reacted with SIVMAC, and the anti-pl5-IV group reacted with HIV-2s and SIVMAC. Interestingly, none of these anti-pl5 MAbs crossreacted with HTLV-IIIB or SIVAGM (Table 2 and Fig. 3). By IF, the anti-p26 and anti-pl5 MAbs exhibited very weak positivity with HIV-2ROD- and HIV-2[GH-1 ]-infected cells. Examples of the IF reactivity of KB-1 and KT-1 are shown in Figure 4. These two MAbs gave strong positive reactions with SIVMAC-infected HUT-78 cells. The data from an extensive survey of IF reactivities with various isolates are summarized in Tables 1 and 2, and correlate well with the reactivities found by WB.
Competitive binding assay anti-p26 and anti-pl5 MAbs were divided into four groups, respectively. We attempted competitive binding assay to reveal whether the different groups of MAbs are directed to distinct or related epitopes. From the results, 20 anti-p26 MAbs were clearly divided into four groups corresponding to the results of cross-reactivity, except KT-19 belonging to anti-p26-I group, which inhibited the binding characteristics of peroxidase-conjugated anti-p26-III group (KT-11 and KT-15) (Table 3). The anti-pl5 MAbs were classified into five groups by competitive binding assay, but some MAbs belonging to I, II, and III were in competition against one another (Table 4). For example, KB-6 inhibited both KB-2 and KB-5, and KB-4 blocked KB-6 and KB-9 in this analysis. Moreover, the anti-pl5-IV group could be further subdivided into two groups, as KB-12 and KB-13 did not compete against each other. These findings indicate that the antigenic epitopes of pl5 are closely associated. As stated above, the
DISCUSSION We describe the generation and characterization of 32 MAbs to HIV-2[GH-1] isolate. On WB with lysates of HIV-2[GH-l]-infected cell and purified virus as antigen, 20 of 32 MAbs reacted with p26 and the other MAbs detected p 15 of the HIV-2[GH-1 ] isolate. Antiserum derived from an HIV-2[GH-1 ]-immunized mouse also recognized both p26 and p 15 in the lysates of infected cells and of purified virus. Our MAbs could not detect these species in uninfected cell lysates (Fig.l), suggesting that both anti-p26 and anti-pl5 MAbs specifically reacted with HIV-2[GH-l]-infected cells. Moreover, anti-p26 and anti-pl5 MAbs cross-reacted with viral components corresponding to the capsid proteins and matrix proteins, respectively, of AIDS-related retroviruses (Figs. 2 and 3).4~7'10"14 Therefore, we identified the p26 and pl5 as capsid protein and matrix protein, respectively, which are encoded by the gag gene of HIV-2[GH-1]. From the result of nucleotide sequence analysis of HIV-2[GH-1], the capsid protein size and matrix protein size have been defined as 26and 16-kD, respectively.9 Although the HIV-2[GH-1] capsid protein recognized by WB is the same size as that defined by the genetic investigation, the molecular weight ( 15-kD) of matrix protein which was identified by WB is slightly smaller than that (16-kD) of the product identified by the nucleotide sequence analysis. These MAbs also detected a 55-kD protein in the infected cell lysates (Fig. 1). Thus the antigenic epitopes recognized on p26 and pl5 are present on the p55 molecule, and the cellular p55 was identified as the precursor of gag protein of HI V-2[GH-1 ] isolate. In addition to p55, a 39-kD protein was detected by anti-p26 MAbs and mouse antiserum in the infected cell Iysate, but not by anti-p 15 MAbs. It is known that intermediate precursor proteins are derived from the precursor (p53-p55) oí gag protein.21 These findings identified p39 as the intermediate precursor protein containing sequence from both p26 and pi2 which are encoded by carboxy terminus of HIV-2[GH-1] gag gene.9 The cross-reactivities of MAbs against gag proteins with different HIV-1, HIV-2, and SIV isolates have been reported by several authors.22"27 Niedrig et al.24 found that five MAbs against capsid protein, p24, of the HTLV-IIIB isolate reacted with the corresponding antigens of HIV-2ROD and SIVMAC. Moreover, two anti-p24 MAbs raised against HIV-1CBU cross-reacted with HIV-2ROD and HIV-2CBL20 isolates, whereas MAbs against matrix protein, pi8, failed to cross-react.27 Minassian et al.23 also reported that two MAbs against capsid protein, p24, of the HIV-2NIH.Z isolate cross-reacted with various HIV-1, HIV-2, and SIV
877
KOMATSU ET AL.
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I
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I
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++ ++
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++ ++ + + + + ++ + +
65
+ + + +
+ + + +
++ + +
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++ + + ++ ++
666>
++ ++
6-
++ + +
+ + + + ++ + +
+ + + I + +
I
+ + I ++
I
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MABs SPECIFIC FOR HIV-2 gag PROTEINS Table 4. Analysis of Epitpopes of
the
pl5
in
Competitive Binding Assay BY
ANTi-pl5
MAbs
Competitors Peroxidase-
I
II
IV
III
-
-
conjugated
KB-
KB-
2
5
KB-2 KB-5 KB-6 KB-9 KB-8 KB-1 KB-12 KB-13
+++++ +++++++-------++++++++++++++++++++ ++ ++ ++ ++ + -+++++++ + --______++----++
MAb
KBII
KB6
KB-
-
9
KB4
KB7
KB1
KB8
KB-
3
KB12
KB13
-
--------
-
-
-
-
-
-
-
-
-
-
-
-
---
--
The serial 10-fold dilutions (L10-L105) of ascites fluid Key: Explanation as for Table 3.
were
used
as
competitors.
isolates, but that MAbs against matrix protein, pi6, did not cross-react with HIV-1 and SIV isolates. Thus, these previous studies could not demonstrate that the MAbs against the matrix proteins cross-reacted with the different subgroups of AIDS-related viruses. However, three of our MAbs to the matrix protein of HIV-2[GH-1] isolate, anti-pl5-III group, KB-12, and KB-13, cross-reacted with the matrix protein of SIVMAC. This finding demonstrates that there is at least three common epitopes in the matrix proteins of HIV-2[GH-1] and SIVMAC isolate. The anti-p26-IV group also cross-reacted with the corresponding capsid proteins of HIV-1 and SIV isolates, in addition to the HIV-2ROD isolate. This result suggests that the capsid proteins, p24-p28, of HIV-1, HIV-2, and SIV isolates used in this study express at least one conserved immunogenic epitope that is serologically recognizable. The MAbs against the conserved epitope may be effective for detecting HIV or SIV isolates in body fluids, tissues, and cultured cells. Finally, to study the biological activity of the MAbs, two neutralization assays, syncytium inhibition assay and infection inhibition assay, were performed as described previously.28 Regrettably, the MAbs described in this study failed to neutralize the HIV-2[GH-1 ] isolate. Papsidero et al.29 reported that two MAbs to the matrix protein of HTLV-IIIB neutralized infection by the cell-free virus, and that there are two regions identified by the MAbs on the matrix protein of HTLV-IIIB isolate. This result indicates that epitopes which are available for antibody-mediated virus inhibition exist within the matrix protein of HTLV-IIIB. The epitopes may be present in matrix proteins of other AIDS-related retroviruses, because one (Glu-Leu-Asp-Arg-Trp-GluLys-Ile) of the two regions identified by these MAbs are highly conserved in those of HIVs and SIVs. ACKNOWLEDGMENTS We are grateful to Dr. Y. Tanaka for helpful and constructive discussion. This work was partly supported by a Grant-in-Aid for Research from the Ministry of Education, Science and Culture of Japan.
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Levy JA. Hoffman AD. Kramer SM, Shimabukuro JM. and Oshiro LS: Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS. Science 1984;225:840-842. 879
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reprint requests to: Hiroyoshi Komatsu Department of Immunology School of Hygienic Sciences Kitasato University 1-15-1 Kitasato, Sagamihara Kanagawa 228, Japan Address
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