|Número de publicación||WO1991000045 A1|
|Tipo de publicación||Solicitud|
|Número de solicitud||PCT/US1990/003591|
|Fecha de publicación||10 Ene 1991|
|Fecha de presentación||29 Jun 1990|
|Fecha de prioridad||3 Jul 1989|
|Número de publicación||PCT/1990/3591, PCT/US/1990/003591, PCT/US/1990/03591, PCT/US/90/003591, PCT/US/90/03591, PCT/US1990/003591, PCT/US1990/03591, PCT/US1990003591, PCT/US199003591, PCT/US90/003591, PCT/US90/03591, PCT/US90003591, PCT/US9003591, WO 1991/000045 A1, WO 1991000045 A1, WO 1991000045A1, WO 9100045 A1, WO 9100045A1, WO-A1-1991000045, WO-A1-9100045, WO1991/000045A1, WO1991000045 A1, WO1991000045A1, WO9100045 A1, WO9100045A1|
|Inventores||Louis E. Henderson, Raymond C. Sowder, Raoul E. Benveniste, Terry D. Copeland, Stephen Oroszlan|
|Solicitante||The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce|
|Exportar cita||BiBTeX, EndNote, RefMan|
|Citas de patentes (1), Otras citas (4), Citada por (1), Clasificaciones (7), Eventos legales (4)|
|Enlaces externos: Patentscope, Espacenet|
REAGENTS FOR DETECTING SIV AND HIV-2 The present invention is related generally to the field of virus diagnostic reagents. More particularly, the present invention is related to providing reagents for detecting the presence of simian immunodeficiency and human immunodeficiency type 2 viruses.
Simian immunodeficiency viruses (SIVs) constitute a group of primate retroviruses that are morphologically and antigenically related to human immunodeficiency viruses (HIVs). The SIV group includes strains isolated from Macaca mulatta (SIVMao). sooty mangabey monkeys (SIVsmm) and a virus isolated from Macaca nemestrina (SIVMno). Macaques infected with the cultured virus develop opportunistic infections and other manifestations of immunodeficiency associated with a loss of CD4+ cells. Some members of the west African human population have been shown to have antibodies that cross- react more extensively with SIV proteins than with proteins from HIV type 1 (HIV-1). These observations led to the suggestion that Viruses more closely related to SIV than to HIV-1 may be a causative factor of the disease in these individual s .
SUMMARY OF THE I NVENTI ON
It is, therefore, an object of the present invention to provide distinguishing markers or entities which distinctively identify SIVs from other viruses, such as HIV1. It is a further object of the present invention to provide kits containing reagents for detecting the presence of SIVs in cells, tissues or biological sample.
Other objects and advantages will become evident from the following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein:
Figure 1 shows immunoblot analysis of antigens present in SIVMno detected with serum from a seropositive pig-tailed macaque infected with SIVMno (Benvenisste et al, 1986, J. Virol 62:2091-2101). Proteins present in purified SIVMno were separated by SDS-PAGE and transferred to nitrocellulose paper as described in Materials and Methods. The nitrocellulose paper strip was incubated with primate plasma (1:50 dilution), and antigen-antibody complexes were detected by incubating .with peroxidase-conjugated anti-human immunoglobulin G antiserum and developing with chloronaphthol. Molecular weights of detected antigens were estimated by reference to the mobility of standard proteins separated on the same gel.
Figure 2 shows the purity of proteins taken for N- terminal Edman degradation and checked by SDS-PAGE visualized by staining with coomassie brilliant blue R250. PANEL A shows a gel lane containing purified gp120env that eluted from the RP-HPLC column in acetonitrile. The positions of molecular marker proteins are indicated by the scale to the left. PANEL B shows a gel containing purified gp32env in lane 1 and purified SIVMno in lane V. The positions of purified gp120env and bands corresponding to viral proteins previously identified as p28gag. p16gag and p14sid are indicated for lane V.
Figure 3 shows the results of tests with polyclonal rabbit antisera prepared against purified gp120env and used for detecting reactive antigens in SIVMno (lanes 3) and cross-reactive antigens in SIVMno (lanes 4), HIV-2 (lanes 2) and HIV-1 (lanes 1). PANEL A shows the results of immunoblot analysis with 1:200 dilution of rabbit sera obtained after three 25 μg injections of purified gp120env. PANEL B shows the results of immunoblot analysis with a 1:200 dilution of rabbit sera obtained after four 25 μg injections.
Figure 4 shows the relative amounts of viral antigens assayed by the immunoblot analysis shown in Figure 3 and estimated by separating an identical amount of each virus by SDS-PAGE and visualizing proteins by staining with coomassie brilliant blue R250. The order of viral lanes was: lane 1, HIV-1; lane 2, HIV-2; lane 3, SIVMno; lane 4, SIVMno; and is different from the order used in Figure 3. The migration position of purified gp120env is indicated as 120K and the migration positions of molecular weight proteins are indicated by the scale to the left.
DETAILED DESCRIPTION OF THE INVENTION
The above and various other objects and advantages of the present invention are achieved by substantially pure, isolated SIVMno gag proteins p16, p28, p8, p6, p2, p1; env proteins gp120 and gp32 and antibodies having specific binding affinity for one or more of the SIVMno env or gag proteins.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed herein are standard methodologies well known to one of ordinary skill in the art.
The term "substantially pure" as used herein means the protein is as pure as can be obtained by conventional purification techniques known to one of ordinary skill in the art. MATER I AL S AND METHODS
Virus production. A single-cell clone of HuT 78 cells producing SIVMn. was grown, and the virus was purified by sucrose density gradient centrifugation as described by Benveniste et al (1986, J. Virol 60:483-490). SIVMno was purified as described by Daniel et al (1985, Science 228:1201-1204) and supplied by R. Desrosiers and M. Daniel (New England Regional Primate Research Center, Southborough Mass.).
Purification of viral proteins. Viral gag proteins were purified by standard reversed-phase high-pressure liquid chromatography (RP-HPLC). Briefly, 48 ml of sucrose density gradient-purified virus (1,000x concentrated) containing 1.1 mg of protein per ml was made 6 M in guanidine hydrochloride at pH 8 (0.01 M Tris) and 1% (vol/vol) 2-mercaptoethanol The resulting slightly turbid solution was heated to 80ºC for 30 min to allow complete disruption of the virus and reduction of disulfide bonds. After cooling, the solution was adjusted to pH2 with trifluoroacetic acid and injected onto a column of μ-Bondapak C18 (19 X 150 mm, Waters Associates Inc., Milford, Mass.). Proteins were eluted from the column at pH 2 (0.05% trifluoroacetic acid) with gradients of increasing concentration of acetonitrile and detected by UV absorption at 206 nm with a model 2140 rapid spectral detector (LKB Instruments, Inc., Gaithersburg, Md.). Solvents were pumped with model 6000A pumps controlled by a model 660 solvent programmer (Waters). The chromatographic solvent was removed from collected column fractions by lyoph-ilization.
Purification of SIVMno env proteins. 50 ml of 1000x concentrated purified virus was pelleted by centrifugation and resuspended in 5.0 ml of water and made pH 2 with the addition of 50 μl of 20% (v/v) trifluoroacetic acid (TFA). The viral suspension was placed in a sonicating bath at room temperature (22°-24° C) and extracted twice with 4 volumes of water-saturated 1-butanol. The aqueous (a-) phase, which included insoluble protein present at the interface, was separated from the butanol-water (b/w-) phase and the b/w- phase and made 0.1 M in NaCl. Addition of NaCl to water- saturated butanol causes a phase separation and proteins , original ly soluble in the b/w-phase , concentrate in the salt water (s/w-) phase, whereas most of the extracted lipids remain in the butanol phase. Subsequent analysis (SDS-PAGE) revealed that a-phase retained most of the viral proteins including the gp120env and gp32env but the p28gag. p16gag and p14sid were found in the s/w-phase. The a-phase, including the proteins at the interface, was placed under a stream of N2 to evaporate any retained butanol and then adjusted to pH 8 and made 1% (v/v) in 2-mercaptoethanol and placed in a sonicating bath for 30 min. The a-phase was then readjusted to pH 2 (addition of TFA), placed in a sonicating bath for 30 min and then centrifuged to separate insoluble proteins. The pH 2 soluble proteins from the a-phase were separated by reversed phase high pressure liquid chromatography on
u-bondapak C-18. Proteins were eluted from the column at pH 2.0 (0.05% TFA) with a linear gradient of acetonitrile at room temperature followed by a linear gradient of 1-propanol at 50C. Eluted protein was detected by U.V. absorption at 206 and 280 nm and individual column fractions assayed by SDS-PAGE followed by staining with coomassie brilliant blue R250 and/or by immunoblot analysis.
Column fractions containing the viral transmembrane protein (gp32env) and the envelope glycoprotein (gp120env) were identified by immunoblot analysis using antisera from experimentally infected macaques.
Partly purified gp120env was detected in fractions eluted from the column with about 40% acetonitrile at room temperature and also in later fractions eluted with about 26% 1-propanol at 50ºC. Partly purified gp32env was detected in column fractions eluted with 39% 1-propanol.
Fractions containing partly purified gp120env were pooled, lyophilized and redissolved in 8M guanidine hydrochloride at pH 8.0 containing 1% (v/v) 2-mercaptoethanol and kept under N2 for 3H to reduce disulfide bonds Following reduction, the pH was adjusted to 2.0 and the sample rechromatographed on u-bondapak C-18. Column fractions were analyzed as before and most of the detected gp120env (approximately 75%) was found in fractions eluting from the column in about 40% racetonitrile at room temperature and the remainder of the gp120env eluted with 1-propanol (26%) at 50°C. The gp120env eluting in 1-propanol was rechromatographed a second time and eluted in about 40% acetonitrile.
Fig. 1 shows the results of immunoblot analysis and Fig. 2 (panel A) shows the results of SDS-PAGE analysis of the rechromatographed gp120env that eluted in about 40% acetonitrile. The gel photographed for Fig. 2 was stained with coomassie brilliant blue (R250) and purified gp120env gave a rose-pink color that was easily distinguished by the eye from the blue-purple color obtained with most other proteins.
Fractions containing partly purified gp32env were pooled and 1.0 ml of 8M guanidine hydrochloride added before lyophilizing to dryness. The sample was redissolved in 1.0 ml of 0.05% TFA and applied to a u-bondapak C-18 column equilibrated with 15% 1-propanol (0.05% TFA) and eluted with a linear gradient of 1-propanol at 50ºC. Eluted protein was detected as before and the results of SDS-PAGE analysis of rechromatographed gp32env is shown in Fig. 2, panel B, lane 1.
Puri f ied gp120env and gp32env (Fig. 2) were each analyzed by Edman degradation in an automatic gas-phase sequencer to determine their N-terminal amino acid sequences and the results are summarized in Table 1. The results for gp120env gave positive assignments for the first 19 residues except that the tryptophan tentatively iden.t-ified in step 13 gave a very low yield and no residue was identified for step 15. The determined N-terrainal amino acid sequence (Table 1) is identical to a sequence predicted by the DNA sequence of S IVM n o beginni ng at codon 23 i n t he ENV open reading f rame 90RF). The DNA sequence predicts an asparagine residue at position 15 in gp120env in the sequence -Asn-Ala-Thr-. The asparagine (Asn) in this sequence is a putative s i te f or N-l inked glycosylati on .
The determined N-terminal amino acid sequence of gp32env (Table 1) is identical to a predicted sequence in SIVMno beginning with codon 529 of the env ORF except that arginine was indicated by step 16 whereas the nucleotide sequence predicted a serine residue. Steps 1 and 17 of the Edman degradation each gave two residues (glycine and glutamine in step 1, and alanine and leucine in step 17).
Of the 39 residues determined by sequence analysis of purified proteins from SIVMno (18 for gpl20, and 21 for gp32) 38 were identical to residues predicted by the DNA sequence of the SIVMno env gene. Thus, in the compared portion of the env gene, SIVMno and SIVMno are about 98% identical.
The env ORF of SIVMno contains 736 codons and presumably directs the synthesis of a 736 residue polyprotein which begins with a 'leader peptide" (lp) and is ultimat ly glycosylated [at Asn-X-Thr or Ser sites iand; cleaved (tol remove the 1p and generate the viral surface glycoprotein (gp120 env) and transmembrane protein (gp32env) 1. The data are entirely consistent with this view. Proteolytic cleavage to remove the putative 22 residue "leader peptide" apparently takes place at a Cys-Ile bond and the "leader peptide" and cleavage site is similar to the peptide and cleavage site of human k chains. Proteolytic cleavage to separate gpl20 from gp32 apparently takes place at an Arg-Gly bond and this cleavage site is similar to functionally homologous cleavage sites identified in other retroviral env polyproteins. The calculated molecular weights for gp120env and gp32env [based on the proteolyti c cl eavages , predicted amino acid sequences and glycosylation sites (taking 2,800 as an average value for the MW of the carbohydrate at each site)] are in agreement with their observed mobilities in SDS-PAGE.
Purified gp120env (as in Fig. 2) was cut from an SDS- PAGE gel and crushed gel containing approximately 100 yg of protein was mixed with Freund's incomplete adjuvant and used to immunize a rabbit (25 μg initial inoculation and 25 μg booster shots every two weeks). Test bleeds (10 ml of whole blood) were taken after the first and second boost and the animal bleed out two weeks after the last boost. These antisera were used in immunoblot analysis to detect gp120env in SIVMno and cross-reactive antigens in HIV-1, HIV-2 and SIVMno (Fig. 3). Antisera from the second test bleed (Fig. 3, panel A) detected a 120K antigen in SIVMno and SIVMno but did not detect cross-reactive antigens in HIV-2 and HIV-1. Antisera from the bleed out (Fig. 3, panel B) detected a 120K antigen in SIVMno, SIVMno. and HIV-2 and also reacted with.lower molecular weight antigens in the 24 to 28K range in SIVMno, SIVMno, HIV-2 and HIV-1. In separate experiments (results not shown) the bleed out antisera was shown to reac t with purified p28gag of SIVMno. Thus the lower molecular weight antigen detected by the bleed out antisera (Fig. 3, panel B) appears to be the major gag antigen (p28 for SIV. p26 for HIV-2 and p24 for HIV-1) present in each viral lane. The reasons for the cross-reactivity with the gag protein is unknown, but it is possible that the purified gp120env used for the immunizations was contaminated with enough gag protein to elicit an antibody response after the fourth injection.
In any event, the data presented in Fig. 3 shows that antisera to SIVMno gp120env cross-reacts with the homologous proteins in SIVMno and HIV-2, but not HIV-1.
The available sequence information shows that, at the amino acid sequence level, SIVMno is greater than 90% identical to SIVMno and greater than 80% identical to HIV-2. Thus, polyclonal antisera to a purified viral protein would be expected to show extensive cross-reactivity with homologous proteins in the other viruses. The results of immunoblotting with antisera to SIVMno gp120env (Fig. 3) show that the 120K antigen reacts much more strongly in the SIVMno lane than in the SIVMmo and HIV-2 lanes. This result suggests a high level of strain specificity for the polyclonal anti sera but seems at least partly related to the amount of detected antigen present in each viral lane. The relative amounts of 120K protein present in each viral lane can be estimated by inspection of the SDS-PAGE gel stained with coomassie brilliant blue (R250) shown in Fig. 4. In the 24 to 28K region of the gel, there is one prominent band in each viral lane which is determined to be the major gag antigen. These bands are of about equal intensity, indicating that each lane contains about the same amount of virus. In the 120K region of the gel, a diffuse band of protein is clearly visible in SIVMno (lane 3). The other viral lanes also show some staining in this region of the gel but the staining in the SIVMno lane is more intense. The 120K diffuse band in the SIVMno lane is reddish-purple in color and has a rose-pink hue at the outer edges of the lane. These subtle shades of color are visible to the eye and distinguish the 120K band in the SIVMno lane from most other proteins bands in the gel. Purified SIVMno gp120env stains rose-pink with coomassie brilliant blue R250 and it is believed that the distinguishing color of the 120K diffuse band in the SIVMno lane is due in large part to the presence of this protein. This observation also indicates that the biologically cloned strain of SIVMno retains substantially more gp120env after purification of the virus than i s normal ly f ound with other purified viruses of this class (HTV-1, HIV-2 and SIVmac). of course, the protein can be purified directly from virus by the methods described herein. The availability of the substantially pure, isolated env and gag proteins of the present invention now allows the preparation of monospecific polyclonal and monoclonal antisera or antibodies by standard techniques well known to one of ordinary skill in the art. As shown by the results presented herein supra, the antibodies are useful in detecting the presence of retroviruses which have substantial degree of homology to the SIVMno. It is pointed out that HIV-2 has substantially greater homology with SIVs than HIV- 1.
The availability of the isolated antigens of the present invention also allows the detection of anti-SIV antibodies in a biological sample. This is accomplished by simply reacting a serum with the antigen (SIV gp120 or gp32). The formation of an antigen-antibody complex would be indicative of the presence of anti-SIV antibodies in the test sample. The antigens and the antibodies can, of course, also be employed for in situ immuno-histological tests for detecting the presence of SIVs following standard methodologies for such tests.
A kit in accordance with the present invention comprises containers separately containing the substantially pure antigens and antibodies of the present invention and instructional material for performing routine immunological tests. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
N -Terminal amino acid sequence of SIVmne gp120 and gp32
gp120env . 5 10 15
Ile-Gln-Tyr-Val-Thr-Val-Phe-Tyr-Gly-Val-Pro-Ala-Trp-Arg-XXX-Ala- - -> - -> - -> - -> - -> - -> - -> - -> - -> - -> - -> - ->(- ->) - -> - ->
Thr-Ile-Pro- - -> - -> - -> gp32env 5 10 15
- -> - -> - -> - -> - -> - -> - -> - -> - -> - -> - -> - -> - -> - -> - -> - ->
- -> - -> - -> - -> - ->
H-terminal amino add sequences of gp120env and gp32env were
determined by automated Edman degradation of protein purified
directly from virlons. Arrow heads to the right [ - ->] Indicate residues positively identified; arrow heads 1n brackets [( - ->)] indicate residues tentatively identified. Steps where residues were not Identified are indicated by XXX. Edman steps 1 and 17 for gp32 gave two residues per step as Indicated by residues in parentheses ( ).
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
|Patente citada||Fecha de presentación||Fecha de publicación||Solicitante||Título|
|US4839288 *||3 Mar 1986||13 Jun 1989||Institut Pasteur||Retrovirus capable of causing AIDS, antigens obtained from this retrovirus and corresponding antibodies and their application for diagnostic purposes|
|1||*||JOURNAL OF VIROLOGY, Volume 60, No. 2, issued November 1986, R.E. BENVENISTE et al., "Isolation of a Lentivirus from a Macoque with Lymphoma Comparison with HTLV-III/LAV and other Lentiviruses", pages 483-490.|
|2||*||NATURE, Volume 321, issued 22 May 1986, M. MURPHY-CORB et al., "Isolation of an HTLV-III-related retrovirus from macaques with simian AIDS and its possible origin in asympotomatic mangabeys", pages 435-437.|
|3||*||NATURE, Volume 335, issued 15 September 1988, X.F. YU et al., "A naturally immunogenic Virion-associated protein specific for HIV-2 and SIV", pages 262-265.|
|4||*||SCIENCE, Volume 232, issued 11 April 1986, P.J. KANKI et al., "New Human T-Lymphotopic Retrovirus Related to Simian T-Lymphotropic Virus Type III (STLV-IIIAGM)", pages 238-243.|
|Patente citante||Fecha de presentación||Fecha de publicación||Solicitante||Título|
|EP0452319A4 *||12 Jun 1989||2 Sep 1991||Us Secretary United States Dep||Novel protein and coding sequence for detection and differentiation of siv and hiv-2 group of viruses.|
|Clasificación internacional||C07K16/10, C07K14/16|
|Clasificación cooperativa||C12N2740/16222, C07K14/005, C07K16/1063|
|Clasificación europea||C07K14/005, C07K16/10K1D|
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