CARBOHYDRATE BINDING MODULE AND USE THEREOF
The present invention relates to an antibody mimetic of carbohydrate binding module (CBM) which specifically binds to an epitope on HIV glycoprotein. The present invention also relates to a method of detecting HIV glycoprotein.
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The present invention relates to an antibody mimetic of carbohydrate binding module (CBM) which specifically binds to an epitope on HIV glycoprotein. The present invention also relates to a method of detecting HIV glycoprotein.
BACKGROUND OF THE INVENTIONHuman immunodeficiency virus (HIV) is known to cause acquired immune deficiency syndrome (AIDS), and because the HIV exhibits rapid genetic drift, widely divergent strains are emerging. Thus, detection and treatment of variant strains have proven to be challenging and difficult.
The diagnosis of HIV infection is most commonly achieved by detecting antibody against HIV. Methods for laboratory diagnosis of HIV infection have evolved and offered a large number and a variety of effective methods that can prolong and improve the quality of life for HIV infected patients. In the industrialized countries, enzyme immunoassay (EIA) is the most commonly used method. The assay is comprised of an immobilized viral antigen, which may be comprised of viral lysate, retrovirus proteins or natural or synthetic polypeptides, that reacts with blood or serum components suspected of containing HIV antibodies. Although there is a window stage problem (limiting to the anti-HIV antibody generation time) for the EIA detection, it is still the most popular for HIV diagnosis due to excellent sensitivity, good specificity, and relatively lower cost.
However, there are some disadvantages of the use of isolated viral proteins as antigens for EIA method, such as: the need to grow and handle large quantities of live infectious virus; the possibility that the live virus might be incorporated into test materials; the variable nature of the resulting viral lysate; and the substantial number of false positive and false negative results that require additional confirmatory testing. The use of synthetic polypeptides, which can be engineered to immunologically mimic antigenic epitopes of HIV viruses, may avoid some of the above-mentioned disadvantages, but viral antigenic drift could result in the failure to detect HIV infected sera, presumably due to limited presentation of viral epitopes.
Therefore, there remains a need for a reliable, specific and sensitive test for HIV infection that is affordable and practical on a large scale.
There is also an urgent need to develop an effective prophylactic vaccine and other therapeutic strategies to limit HIV transmission as the epidemic continuous unabated. Most successful vaccines consist of either live-attenuated or inactivated viral particles. However, live-attenuation of the HIV-related simian immunodeficiency virus, resulting in protective responses without resulting pathogenicity has not been accomplished, raising safety concerns that make human trial intractable. Also, HIV has many sophisticated mechanisms to evade envelope glycoprotein-directed antibody responses efficiently, including shrouding well-conserved structures by glycan shielding and masking of vulnerable receptor-binding sites by conformational and steric constraints. Therefore, researches turned to envelope glycoprotein-based immunogens as a means of elicitng antibodies, but the use of monomeric gp120 or peptides derived from the immunodominant V3 loop of gp120 can not generate boardly used antibodies but type-specific antibodies.
As a result, the development of an effective detection and prophylactic vaccine against HIV remains an unrealized goal in the effort to contain the current pandemic.
SUMMARY OF THE INVENTIONThe present invention provides an antibody mimetic of CBM family which specifically binds to an epitope on HIV glycoprotein. The present invention further provides a method of detecting HIV glycoprotein comprising: (a) introducing into contact with a solid surface to which is bound a known quantity of CBM capable of binding to HIV glycoprotein; (b) incubating sample in contact with said surface to form CBM-HIV glycoprotein complexes; and (c) incubating the complexes and subjecting sample to a labelled HIV antigen conjugate capable of yielding a quantitatively measurable signal to score sample as positive or negative for HIV-infection.
The present invention provides an antibody mimetic of CBM which specifically binds to an epitope on HIV glycoprotein.
The term “CBM” used herein refers as a contiguous amino acid sequence within a carbohydrate-active enzyme with a discreet fold having carbohydrate-binding activity. In the primary structure classification of glycoside hydrolases, the CBMs are categorized into 53 families, which include several specificities such as cellulose, xylan, chitin, and starch binding.
The term “antibody mimetic” used herein refers to an object having similar function as the antibody in terms of binding against target structure, but its structure is simpler than an antibody. To produce a large amount of antibodies needs the following steps: (a) fusing single antibody-forming cells to tumor cells grown in culture. The resulting cell is called a hybridoma, (b) each hybridoma produces relatively large quantities of identical antibody molecules, and (c) allowing the hybridoma to multiply in culture, it is possible to produce a population of cells, each of which produces identical antibody molecules. It is labor-consuming and cost-consuming to make real antibodies; however, in the present invention, a broad range of host including bacteria, yeast, insect, and mammalian cells can be used to produce the antibody mimetic of CBM without using animals, which is simpler and more economic.
In the present invention, the preferable CBM is starch binding domain (SBD). The term “SBD” used herein refers to a functional domain that can bind granular or soluble starch, increasing the local concentration of substrate at the active site of the enzyme, and that may also disrupt the structure of the starch surface, thereby enhancing the amylolytic rate. At present, there are nine starch-binding CBM families: CBM20, CBM21, CBM25, CBM26, CBM34, CBM41, CBM45, CBM48 and CBM53. In a preferred embodiment of the invention, the SBD is the member of the CBM families 20 and 21, which is derived from Aspergillus niger glucoamylase (AnSBD) and Rhizopus oyzae glucoamylase (RoSBD), respectively. Even though CBM20 and CBM21 share fairly low identity in their amino acid sequences (approximately 13.5%), they have similar secondary and tertiary structures as well as the role in enhancing enzymatic activity in terms of hydrolyzing granular starch (Tung J Y et al., Biochem. (2008) 416: 27-36).
The antibody mimetic of the present invention, wherein the RoSBD analyses by Glycan microarray screening shows that it has the ability to bind a glycan, Manα(1,2)Manα(1,2)Manα(1,2)Manα(1,3), which is a special glycan present in the epitope of the heavily glycosylated glycoprotein gp120 of HIV.
Human monoclonal antibody 2G12 was first isolated and characterized at 1996, and proved to neutralize the clade A and B strains of HIV1 (Trkola A, et al., J. Virol. (1996) 70: 1100-1108). Site-directed alanine scanning mutagenesis has shown that the 2G12 epitope covers mainly on the high-mannose or hybrid glycans of residue N295, N332, N339, N386, N392, and N448 on gp120 (Sanders R W, et al., J. Virol. (2002) 76: 7293-7305 and Scanlan C N, et al., J. Virol. (2002) 76: 7306-7321), unlike most antibodies which recognize protein backbones of viral components. The crystal structure of 2G12 (PDB ID: 1ZLS) and its complexes with the oligosaccharide MangGlcNAc2 (PDB ID: IOP5) reveal that two Fabs assemble into a VH domain-swapped dimer (Chalarese D A et al., Science (2003) 300: 2065-2071). In the present invention, the RoSBD and AnSBD also recognize glycan moieties of HIV As
The present invention also provides a method of detecting HIV glycoprotein comprising: (a) introducing into contact with a solid surface to which is bound a known quantity of CBM capable of binding to HIV glycoprotein; (b) incubating sample in contact with said surface to form CBM-HIV glycoprotein complexes; and (c) incubating the complexes and subjecting same to a labelled HIV antigen conjugate which is capable of yielding a quantitatively measurable signal to score sample as positive or negative for HIV-infection. In a preferable embodiment, the CBM includes but is not limited to SBD. The method of the present invention, wherein said antigen of step (c) is labelled with an enzyme which is capable of producing the said signal when contact with an enzyme substrate. The enzyme comprises but not limited to horse radish peroxidase (HRP), alkali phosphatases, peroxidases, β-galactosidase, glucoamylase, urease and chloramphenicol acetyltransferase. In the preferable embodiment, the emzyme is HRP. Appropriate examples and the use of necessary substrates for the detection by means of enzymatic reactions are known to the person skilled in the art, and in the preferable embodiment, the substrate includes but is not limited to 3,3′,5,5′-tetramethylbenzidibe (TMB).
Those skilled in the art will recognize the foregoing outline as a description of an ELISA procedure. They will recognize also that the generalized outline omits certain of the specific steps such as serial dilution and washing with appropriate buffers which are standard in the ELISA procedure. Although buffers and other reagents will be described hereinafter, and specific dilutions will be employed to llustrate the invention, the skilled artisan will recognize that these are illustrative only and that many equivalents are possible.
In the present invention, the major targets of the antibody mimetic are the exterior envelope glycoprotein of HIV, gp120 and the transmembrane glycoprotein, gp140. These proteins are generated by cleavage of a heavily glycosylated precursor protein, gp160, by furin-like enzymes during transport through the Golgi apparatus. Each new infectious cycle is initiated when the external envelope glycoprotein gp120 binds the primary recetor, CD4, which is embedded in the plasma membrane on the surface of potential target cells. In this invention, the antibody mimetic of CBM, which is not only can bind to the glycan structure of the envelope glycoprotein of HIV to serve as a potential therapeutic agent to block the interaction between HIV and target cells, but also can be a prophylactic vaccine against conserved but exposed epitopes on the HIV envelope glycoprotein to limit newly acquired infections.
As described above, the CBM has the ability to use as an antibody mimetic which can be appled to HIV detection and may further be applied as a prevention and treatment agent for HIV infection.
The examples below are non-limiting and are merely representative of various aspects and features of the present invention.
EXAMPLE Example 1Purification of RoSBD and AnSBD by Amylose Affinity Chromatography
The RoSBD and AnSBD were produced by E. coli system using pET23a and pET15Bb expression vector, respectively. The cell pellets of E. coli containing the recombinant protein was resuspended in binding buffer (50 mM sodium acetate, pH 5.5), and then homogenized (EmulsiFlex-C5 homogenizer). The cell debris was removed by centrifugation at 16,000×g for 20 min at 4° C., and the supernatant was subjected to purification by affinity chromatography equipped with amylose resin (New England Biolabs, Ipswich, Mass.). After the resin was washed with 5 column volumes of binding buffer, the supernatant was loaded to the column at a flow rate of 1 mL/min. The recombinant protein was then eluted by elution buffer (10 mM glycine/NaOH, pH 11.0) after the column was washed with another 5 column volumes of the binding buffer. The purified RoSBD/AnSBD was dialyzed against sodium acetate buffer (50 mM, pH 5.5) using an Amicon® Ultra-15 centrifugal filter devices (Millipore) PL-10 (10 kDa cutoff) after 30 kDa cutoff (Lin S C, et al., BMC Biochem. (2007) 8: 9-21 and Liu W T, et al., Biochem. Biophys. Res. Commun. (2008) 377: 966-970).
Example 2Glycan Array Screening of ROSBD
RoSBD was produced in E. coli system using pET23a expression vector and purified using amylose resin as previously described. Glycan microarray analyses were conducted by the Consortium for Functional Glycomics, Core H facility. The array contained a total of 377 different natural and synthetic glycans and version 3.1 (http//:www.functionalglycomics.org/static/consortium/resources/resourcecoreh11.shtml) was used for the analyses reported here. Briefly, RoSBD was diluted to 200 mg/mL in sodium acetate binding buffer (50 mM sodium acetate pH 5.5, 1% BSA and 0.05% Tween-20). Seventy microliters was applied to the printed surface of the array, coverslipped, and incubated at room temperature in a humidified chamber away from light for 1 hr. After the incubation, the coverslip was removed and rinsed four times in TSM buffer [50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 0.5 M sucrose] and four times in TSM buffer. Seventy microliters of Anti-RoSBD monoclonal antibody, diluted to 1:200 in PBS binding buffer, was applied to the printed surface of the microarray and incubated in a humidified chamber for 1 hr. Washes were performed as above. To detect binding, the secondary antibody incubation was performed with Alexa488-labeled goat anti-mouse IgG at 5 mg/mL in PBS buffer for one hour in a humidified chamber, followed by wash steps. The binding image was read in a Perkin-Elmer Microarray XL4000 scanner and analyzed using Imagene (V6) image analysis software.
Structure Modeling and Sequence Alignment between VH Domain of 2G12 and RoSBD
HIV-RoSBD Binding Analysis
The ELISA procedurefor preparation of AnSBD/RoSBD coated plates At first 100 μL of 100 nM AnSBD/RoSBD were coated on individual well of a 96-well plate (Greiner-Bio One GmbH, Frickenhausen, Germany) in sodium bicarbonate buffer (pH 9.5)/Tris-HCl buffer (pH 8) via 16-hr incubation at 4° C. The plate was washed with PBST [10 mM Phosphate Buffered Saline (pH 7.0) plus 0.05% Tween-20], then 200 μL blocking buffer [0.01 M Phosphate Buffered Saline (pH 7.0) plus 5% BSA] was added to each well and incubated at 37° C. for 2 hr. The blocking buffer was removed and the plate was dried at 25° C. for 1 hr. One hundred microliter per well of HIV Ag & Ab positive panel (ID#9144532, SeraCare Life Sciences, Milford, Mass.) and Anti-HCV mixed titer performance panel (ID# PHV205-24, SeraCare Life Sciences, Milford, Mass.) was separately added and reacted at 37° C. for 1 hr. This HIV Ag & Ab positive panel was identified as HIV antigen positive by Perkin Elmer EIA and anti-HIV antibody positive by Abbott EIA. Secondly, HRP coupled E. coli HIV1 recombinant gp120 antigen conjugate (0.05 μg/mL) was added and reacted at 37° C. for 30 min. This conjugate could bind with the human anti-HIV1 antibody in HIV Ag & Ab positive panel directly. To test the specificity of the SBD binding to HIV, HCV test sample and HCV recombinant antigen (Core+NS3+NS5)-HRP conjugate (0.33 μg/mL) were analyzed following the same protocol in parallel. Finally, the plates were developed by adding 100 μL 3,3′,5,5′-tetramethylbenzidibe (TMB) at 37° C. for 30 min. The absorbance was measured at 450 nm in ELISA reader after the reaction was stopped by addition of 100 μL 2 N H2SO4 in each well. These absorbance values were compiled as a statistical value (Cut off value; COV) and described in terms of cut off index (COI) value. The COV was calculated as the OD value of negative control (Normal Human Serum) plus 0.1 (COV=NC+0.1). The COI value was calculated as the OD value of test sample divided by COV. If a sample has an absorbance higher than the COV, i.e. COI value is greater than 1, it is considered to be positive in the assay.
Glycan Effects on RoSBD/AnSBD Binding to HIV
Ten millimolar maltoheptaose (G7 glycan) and β-cyclodextrin (βCD) (Sigma-Aldrich, St. Louis, Mo.) were dissolved in PBS plus 5% BSA and mixed with the same volume of HIV Ag & Ab positive panel at 37° C. for 1 hr. After adding the mixture to RoSBD coated plate (100 nM in 0.05 M Tris-Hcl, pH 8) or AnSBD coated plate (100 nM in 0.05 M sodium bicarbonate buffer, pH 9.5) at 37° C. for 1 hr, 100 μL HRP coupled HIV1 recombinant gp120 antigen conjugate (0.05 μg/mL) was used for detection.
Similarly, the competition occurred when 10 mM maltoheptaose or β-cyclodextrin (βCD) was added to AnSBD (
Competition Effects of 2G12 and gp140 to HIV-RoSBD and HIV-AnSBD Binding
HIV Ag & Ab positive panel was separately mixed with the same volume of 500 nM human IgG1 secondary antibody, 2G12 monoclonal antibody, and 250 nM HIV1 gp140 antigen at 37° C. for 1 hr. After adding the mixture to (a) RoSBD coated (100 nM in 0.05 M Tris-Hcl, pH 8) and (b) AnSBD coated plate (100 nM in 0.05 M sodium bicarbonate buffer, pH 9.5) individually at 37° C. for 1 hr, 100 pL HRP coupled HIV1 recombinant gp120 antigen conjugate (0.05 μg/mL) was used for detection.
Amino Acid Sequence Alignment of 2G12 Epitope Region in HIV1 Isolates
Claims
1. An antibody mimetic of carbohydrate binding module (CBM) which specifically binds to an epitope on HIV glycoprotein.
2. The antibody mimetic of claim 1, wherein the CBM specifically binds to a glycan structure present in the epitope of the HIV glycoprotein.
3. The antibody mimetic of claim 1, wherein the CBM processes Immunoglobulin-like (Ig-like) domain similar to classical antibodies.
4. The antibody mimetic of claim 1, wherein the CBM is starch binding domain (SBD).
5. The antibody mimetic of claim 1, wherein the SBD is a member selected from the group consisting of CBM families 20, 21, 25, 26, 34, 41, 45, 48 and 53.
6. The antibody mimetic of claim 5, wherein the SBD is CBM family 20.
7. The antibody mimetic of claim 5, wherein the SBD is CBM family 21.
8. The antibody mimetic of claim 5, wherein the SBD is derived from Rhizopus oryzae glucoamylase (RoSBD).
9. The antibody mimetic of claim 5, wherein the SBD is derived from Aspergillus niger glucoamylase (AnSBD).
10. A method of detecting HIV glycoprotein comprising:
- (a) introducing into contact with a solid surface to which is bound a known quantity of carbohydrate binding module (CBM) capable of binding to HIV glycoprotein;
- (b) incubating sample in contact with said surface to form CBM-HIV glycoprotein complexes; and
- (c) incubating the complexes and subjecting same to a labelled HIV antigen conjugate which is capable of yielding a quantitatively measurable signal yielding a quantitatively measurable signal to score sample as positive or negative for HIV-infection.
11. The method of claim 10, wherein said antibody of step (c) is labelled with an enzyme which is capable of producing the said signal when contacted with an enzyme substrate.
12. The method of claim 10, wherein the CBM is starch binding domain (SBD).
13. The method of claim 10, wherein the SBD is derived from Rhizopus oyzae glucoamylase (RoSBD).
14. The method of claim 10, wherein the SBD is derived from Aspergillus niger glucoamylase (AnSBD).
Type: Application
Filed: May 12, 2009
Publication Date: Nov 18, 2010
Applicant: NATIONAL TSING HUA UNIVERSITY (Hsinchu)
Inventors: Margaret Dah-Tsyr Chang (Hsinchu), Yuan-Chuan Lee (Hsinchu), Rong-Yuan Huang (Hsinchu), Shu-Chuan Lin (Hsinchu), Wei-I Chou (Hsinchu), Shi-Hwei Liu (Hsinchu)
Application Number: 12/464,788
International Classification: C12Q 1/70 (20060101); C07K 16/08 (20060101);