Nanobody against begomoviruses

Abstract

A nanobody directed against begomoviruses is capable of selectively binding to ToLCSDV viral particles, TYLCV particles, and/or other begomoviruses. The nanobody includes an amino acid sequence of SEQ ID NO: 2.

Claims

1. A nanobody directed against a begomovirus, comprising the amino acid sequence of SEQ ID NO.: 2.

2. The nanobody according to claim 1, wherein the begomovirus includes at least one of Tomato Leaf Curl Sudan Virus (ToLCSDV) and Tomato Yellow Leaf Curl Virus (TYLCV).

3. A composition comprising at least one polypeptide having the amino acid sequence of SEQ ID NO: 2.

4. The composition according to claim 3, further comprising a detectable marker.

5. The composition according to claim 3, further comprising an agrochemical or pharmaceutically acceptable carrier.

6. The composition according to claim 3, comprising: about 0.0001% to about 50% by weight of the at least one polypeptide; and an agrochemically or pharmaceutically suitable carrier.

7. A method of inhibiting or attenuating a plant virus, the method comprising applying to a plant or to a part of said plant, the composition of claim 3.

8. A method of detecting infection by a plant virus, the method comprising: applying to a plant or to a part of said plant the composition according to claim 3.

9. A method for protecting or treating a plant or plant part from a biological interaction with a plant pest, the method comprising: applying to the plant or plant part, the composition according to claim 3 under conditions effective to protect or treat the plant or plant part against the biological interaction with the plant pathogenic virus.

10. A method for protecting a harvested plant or a harvested plant part from a biological interaction with a plant virus, the method comprising: applying to the harvested plant or harvested plant part, the composition of claim 3, under conditions effective to protect the harvested plant or harvested plant part against the biological interaction with the plant virus.

11. A transgenic plant comprising at least one polynucleotide having the sequence of SEQ ID NO: 1.

12. The transgenic plant according to claim 11, wherein the transgenic plant is a tomato plant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph depicting results of ELISA tests of an exemplary nanobody of the present application against field samples and control to demonstrate specificity of binding.

(2) FIG. 2 is a graph depicting experimental results confirming exemplary nanobody binding to ToLCSDV by solution competition.

(3) FIG. 3 is a graph depicting experimental results confirming exemplary nanobody binding to ToLCSDV by solution competition.

(4) Similar reference characters denote corresponding features consistently throughout the attached drawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(5) A nanobody against begomoviruses (herein, Nb-To2) in an exemplary embodiment of the present subject matter is capable of specifically binding to Tomato Leaf Curl Sudan Virus (herein, ToLCSDV) and Tomato Yellow Leaf Curl Virus (herein, TYLCV). The nanobody can be used to detect a begomovirus in a plant or plant part. The nanobody may be cloned into transgenic plants to provide resistance to ToLCSDV, TYLCV, and other begomoviruses' infections.

(6) As used herein, the terms nanobody, VHH, VHH antibody fragment and single domain antibody are used indifferently and designate the variable domain of the single heavy chain of antibodies of the type of those found in Camelidae, which are naturally without any light chains. The nanobodies may in particular be nanobodies of camels, dromedaries, llamas or alpacas.

(7) The nanobody directed against begomoviruses has a very strong affinity for ToLCSDV, and also binds very strongly to TYLCV as well as other begomoviruses. A nanobody directed against begomoviruses, as used herein, refers to a nanobody capable of selectively binding to ToLCSDV viral particles, TYLCV particles, and/or other begomoviruses.

(8) The nanobody can include the following amino acid sequence

(9) TABLE-US-00001 (SEQIDNO:2) QVQLQESGGGSVQAGGSLRLSCVASGDTLRLCRMGWYRQAPGKGRELVSS MEIDGTTNYADSVKGRFTISQGDNRNTMYLQMNSLKPEDTAMYYCKMEGG YGGNCRLAIYNYWGQGTQVTVSS;
or a functional fragment thereof.

(10) According to an embodiment, the nanobody is a nanobody comprising or consisting of the amino acid sequence of SEQ ID NO: 2.

(11) The nanobody is encoded by a polynucleotide comprising a sequence of:

(12) TABLE-US-00002 (SEQIDNO.:1) TACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCC GGCCATGGCCCAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGG CTGGAGGGTCTCTGAGACTCTCCTGTGTAGCCTCTGGAGACACCTTGCGT CTATGTCGCATGGGCTGGTACCGCCAGGCTCCAGGGAAGGGGCGYGAGTT GGTCTCAAGTATGGAGATTGATGGTACTACAAACTATGCGGACTCCGTGA AGGGCCGATTCACCATCTCCCAAGGCGACAATAGGAACACGATGTATCTG CAAATGAACAGCCTGAAACCTGAGGACACGGCCATGTATTACTGCAAGAT GGAAGGGGGATACGGTGGTAATTGTCGGCTTGCTATTTATAACTATTGG GGCCAGGGGACCCAGGTCACCGTCTCCTCA;
or a functional fragment thereof.

(13) An embodiment of the present subject matter relates to a composition comprising at least one polypeptide encoding a variable domain of a heavy-chain antibody (VHH) or a functional fragment thereof capable of specifically binding to a begomovirus.

(14) The begomovirus can include at least one of Tomato Leave Curl Sudan Virus (ToLCSDV) and Tomato Yellow Leaf Curl Virus (TYLCV). Specifically, the at least one polypeptide of the composition may include a sequence of:

(15) TABLE-US-00003 (SEQIDNO:2) QVQLQESGGGSVQAGGSLRLSCVASGDTLRLCRMGWYRQAPGKGRELVSS MEIDGTTNYADSVKGRFTISQGDNRNTMYLQMNSLKPEDTAMYYCKMEGG YGGNCRLAIYNYWGQGTQVTVSS.

(16) The composition may include an agrochemically or pharmaceutically suitable carrier. For example, the composition can include about 0.0001% to about 50% by weight of the at least one polypeptide and an agrochemically or pharmaceutically suitable carrier. The composition may include a detectable marker. The composition may be applied to a plant or part to protect or treat the plant or plant part from a biological interaction with the begomovirus. The composition may be administered under conditions effective to protect or treat the plant or plant part against the biological interaction with the begomovirus. The plant can include harvested and unharvested plants. The plant can include a tomato plant.

(17) The at least one polypeptide of the composition may be encoded by a polynucleotide comprising a sequence of:

(18) TABLE-US-00004 (SEQIDNO:1) TACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCC GGCCATGGCCCAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGG CTGGAGGGTCTCTGAGACTCTCCTGTGTAGCCTCTGGAGACACCTTGCGT CTATGTCGCATGGGCTGGTACCGCCAGGCTCCAGGGAAGGGGCGYGAGTT GGTCTCAAGTATGGAGATTGATGGTACTACAAACTATGCGGACTCCGTGA AGGGCCGATTCACCATCTCCCAAGGCGACAATAGGAACACGATGTATCTG CAAATGAACAGCCTGAAACCTGAGGACACGGCCATGTATTACTGCAAGAT GGAAGGGGGATACGGTGGTAATTGTCGGCTTGCTATTTATAACTATTGG GGCCAGGGGACCCAGGTCACCGTCTCCTCA;
or a functional fragment thereof.

(19) In another embodiment of the present subject matter, a transgenic plant, plant tissue or plant cell comprises at least one polynucleotide a sequence of:

(20) TABLE-US-00005 (SEQIDNO:1) TACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCC GGCCATGGCCCAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGG CTGGAGGGTCTCTGAGACTCTCCTGTGTAGCCTCTGGAGACACCTTGCGT CTATGTCGCATGGGCTGGTACCGCCAGGCTCCAGGGAAGGGGCGYGAGTT GGTCTCAAGTATGGAGATTGATGGTACTACAAACTATGCGGACTCCGTGA AGGGCCGATTCACCATCTCCCAAGGCGACAATAGGAACACGATGTATCTG CAAATGAACAGCCTGAAACCTGAGGACACGGCCATGTATTACTGCAAGAT GGAAGGGGGATACGGTGGTAATTGTCGGCTTGCTATTTATAACTATTGG GGCCAGGGGACCCAGGTCACCGTCTCCTCA;
or a functional fragment thereof.

(21) The transgenic plant may be a tomato plant. The transgenic plant may be a harvested or unharvested plant. The plant tissue or plant cell may be that of a tomato plant or other plant.

(22) Another embodiment of the present subject matter includes a method of inhibiting or attenuating the begomovirus in a plant. The plant may be a harvested or unharvested plant. The plant may be a tomato plant. The method includes at least applying to a plant or to a plant part a composition including the amino acid of SEQ ID NO.:2 in an amount sufficient to attenuate an infection of the begomovirus in the plant or plant part.

(23) Another embodiment of the present subject matter includes a method of detecting a plant virus in a plant. The plant may be a harvested or unharvested plant. The plant may be a tomato plant. The method includes applying to a plant or plant part a composition including the amino acid of SEQ ID NO.:2 and a detectable marker.

(24) The following examples illustrate the present teachings.

Example 1

Exemplary Nanobody Isolation and Evaluation

(25) Tomato plants exhibiting yellowing, leaf curl and stunting from Usfan, Saudi Arabia were collected and characterized using biological, serological and molecular methods. Agro-infectious clone of ToLCSDV was cloned from the collected plants. Following the inoculation of tobacco plants, the ToLCSDV viral particles were purified from symptomatic plants using sucrose density gradient. The purified antigen (ToLCSDV particles) was injected into Arabian camel (Camelus dromedaries) to generate immune response. The blood lymphocytes of the immunized Arabian camel were used to build VHH phage display library from the heavy chain antibodies (HCAbs). A library of around 210.sup.7 independent transformants was obtained. The purified viral particles were used to isolate specific nanobodies from the VHH library using phage display technology. Here, we summarize the results of the isolated Nb-To2.

(26) The DNA sequence of the isolated Nb-To2 gene is as follows:

(27) TABLE-US-00006 (SEQIDNO:1) TACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCC GGCCATGGCCCAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGG CTGGAGGGTCTCTGAGACTCTCCTGTGTAGCCTCTGGAGACACCTTGCGT CTATGTCGCATGGGCTGGTACCGCCAGGCTCCAGGGAAGGGGCGYGAGTT GGTCTCAAGTATGGAGATTGATGGTACTACAAACTATGCGGACTCCGTGA AGGGCCGATTCACCATCTCCCAAGGCGACAATAGGAACACGATGTATCTG CAAATGAACAGCCTGAAACCTGAGGACACGGCCATGTATTACTGCAAGAT GGAAGGGGGATACGGTGGTAATTGTCGGCTTGCTATTTATAACTATTGG GGCCAGGGGACCCAGGTCACCGTCTCCTCA.

(28) The resulting amino acid sequence encoded by the above Nb-To2 gene is:

(29) TABLE-US-00007 (SEQIDNO:2) QVQLQESGGGSVQAGGSLRLSCVASGDTLRLCRMGWYRQAPGKGRELVSS MEIDGTTNYADSVKGRFTISQGDNRNTMYLQMNSLKPEDTAMYYCKMEGG YGGNCRLAIYNYWGQGTQVTVSS.

(30) Nb-To2 was purified by IMAC affinity using the 6His tag. Successful purification was confirmed by SDS-PAGE analyses with commasie staining and western blotting using alkaline phosphatase conjugated monoclonal anti-polyHistidine antibody (Sigma) for detection.

Example 2

Nanobody Reactivity

(31) Purified Nb-To2 was able to recognize not only purified Tomato Leave Curl Sudan Virus (ToLCSDV) but also purified Tomato Yellow Leave Curl Virus (TYLCV) begomovirus in both ELISA analysis (FIG. 1) and dot blot analysis (not shown). Additionally, Nb-To2 showed reactivity with extracts from tomato leaves showing the typical symptoms of begomovirus-infected plants collected from different locations in Riyadh, Saudi Arabia.

(32) FIG. 1 depicts results of ELISA tests of Nb-To2 against field samples. FS1-6 are separate samples taken from tomato leaves showing the typical symptoms of Geminiviruses from different locations. FS is a control sample of healthy tomato leave. Purified particles of ToLCSDV and TYLCV were also tested as controls.

(33) Using Surface Plasmon Resonance (SPR) Analysis, the affinity and kinetic rate constants of Nb-To2 were determined (Table 1). The results of the single-cycle-kinetics of Nb-To2 to captured ToLCSDV shows high affinity of K.sub.D=290 pM with fast association rate of k.sub.ass=4.7 10.sup.6.Math.M.sup.1s.sup.1 and medium dissociation rate of k.sub.diss=1.4 10.sup.3.Math.s.sup.1.

(34) TABLE-US-00008 TABLE 1 Affinity and kinetic rate constants of Nb-To2 binding to captured ToLCSDV. R.sub.Capture k.sub.ass k.sub.diss K.sub.D R.sub.max C hi.sup.2 185 RU 4.65 10.sup.6 .Math. 1.35 292 pM 6.82 RU 0.022 M.sup. 1.sub.S 1 10.sup. 3.sub..Math.S 1 310 RU 4.05 10.sup.6 .Math. 1.51 374 pM 11.8 4 RU 0.053 M.sup. 1.sub.S 1 10.sup. 3.sub..Math.S 1 405 RU 3.72 10.sup.6 .Math. 1.61 432 pM 17.1 2 RU 0.118 M.sup. 1.sub.S 1 10.sup. 3.sub..Math.S 1

(35) FIGS. 2-3 depict results from a competition experiment performed using soluble Nb-To2 and soluble Nb-To5 (another ToLCSDV-specific nanobody) to inhibit the binding of ToLCSDV to immobilized Nb-To5. Nb-To2 and Nb-To5 showed similar IC.sub.50, or to be more precise ID.sub.50 of 3.775 and 3.811 and p values of 1.377 and 1.423 respectively, meaning that 50% inhibition was observed using a 1:5956 and 1:6471 dilutions of the nanobody sample. Assuming a concentration of the nanobodies of 0.2 mg/mL this translate to an IC.sub.50 concentration value of 30-35 ng/mL. These results further confirm binding of the Nb-To2 to ToLCSDV.

(36) Specifically, FIGS. 2-3 show results of a 1:25 dilution of ToLCSDV incubated with a two-fold serial dilution of the nanobodies and the subsequent inhibition of binding to immobilized Nb-To5. The binding data was evaluated using a dose-response model with the equation Y=Al+(A2A1)/(1+10^((log x.sub.0x).sup.p)), where p represents a measure for positive or negative cooperativity and log x.sub.0 denotes the concentration/dilution at which 50% of the binding signal is inhibited.

(37) The results demonstrate that the two nanobodies, Nb-To2 and Nb-To5, specifically bind to ToLCSDV with high affinity. Note that one can reasonably expect that nanobodies accumulate to 10 g/mL or higher when expressed in the secretory pathway in plants, which, given the high affinity of the nanobody, would be high enough to efficiently neutralize ToLCSDV.

(38) It is to be understood that the nanobody against begomoviruses is not limited to the specific embodiments described above, but encompasses any and all embodiments within the scope of the generic language of the following claims enabled by the embodiments described herein, or otherwise shown in the drawings or described above in terms sufficient to enable one of ordinary skill in the art to make and use the claimed subject matter.