CHIMERIC PLANT BASED VIRUS-LIKE-PARTICLES, AND USES THEREOF

Title:

CHIMERIC PLANT BASED VIRUS-LIKE-PARTICLES, AND USES THEREOF

Document Type and Number:

WIPO Patent Application WO/2017/051434

Kind Code:

Abstract:

The present disclosure provides chimeric virus-like-particles (VLPs) based on non- human pathogenic plant viruses, which can be used for virus mediated delivery of cargo molecules, such as antibodies. In particular, the instant disclosure provides various chimeric VLPs based on the Sesbania mosaic virus or the Pepper vein banding 10 virus fused with domains from the SpA protein from Staphylocccus.

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Inventors:

SAVITHRI HANDANAHAL SUBBARAO (IN)
NATRAJ USHA (IN)
ABRAHAM AMBILY (IN)

Application Number:

PCT/IN2016/050316

Publication Date:

March 30, 2017

Filing Date:

September 20, 2016

Export Citation:

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Assignee:

INDIAN INST SCIENT (IN)

International Classes:

A61K39/395; A61K39/44; C07K14/005; C07K14/31; C07K16/00; C12N7/00

Foreign References:

EP1764414A1

2007-03-21

Other References:

PALUCHA ANDRZEJ ET AL: "Virus-like particles: Models for assembly studies and foreign epitope carriers", PROGRESS IN NUCLEIC ACID RESEARCH AND MOLECULAR BIOLOGY, ACADEMIC PRESS, US, vol. 80, 1 January 2005 (2005-01-01), pages 135 - 168, XP008111583, ISSN: 0079-6603
GULATI ASHUTOSH ET AL: "Structural studies on chimeric Sesbania mosaic virus coat protein: Revisiting SeMV assembly", VIROLOGY, ELSEVIER, AMSTERDAM, NL, vol. 489, 17 December 2015 (2015-12-17), pages 34 - 43, XP029420184, ISSN: 0042-6822, DOI: 10.1016/J.VIROL.2015.11.029
AMBILY ABRAHAM ET AL: "Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles", SCIENTIFIC REPORTS, vol. 6, 24 February 2016 (2016-02-24), pages 21803, XP055350252, DOI: 10.1038/srep21803
LOKESH ET AL., ARCHIVES OF VIROLOGY, vol. 146, pages 209 - 223

Attorney, Agent or Firm:

PHILLIPS, Prashant et al. (IN)

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Claims:

I/We claim:

1. A virus-like -particle (VLP) comprising:

a. a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119 and,

b. at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9,

wherein said first viral CP is fused to said at least one SpA domain.

2. The VLP as claimed in claim 1 , wherein

a. said at least one SpA domain is fused to N-terminal end of said first viral CP; or

b. said at least one SpA domain is fused to C-terminal end of said first viral CP; or

c. said at least one SpA domain is integrated within the said first viral CP; or

d. said at least one SpA domain is integrated at N and C termini of said first viral CP; or

e. said at least one SpA domain is integrated at N-terminal, C-terminal and within said first viral CP; or

f. said at least one SpA domain is integrated at N-terminal and within said first viral CP; or

g. said at least one SpA domain is integrated at C-terminal and within said first viral CP.

3. The VLP as claimed in any of the claims 1 -2, wherein said first viral CP is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 118 and SEQ ID NO: 120 and said at least one SpA domain is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 8, and SEQ ID NO: 10.

4. A virus-like-particle (VLP) having amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, and SEQ ID NO: 159.

5. The VLP as claimed in claim 4, wherein said VLP is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112. SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158 and SEQ ID NO: 160.

6. A DNA construct comprising a polynucleotide fragment operably linked to a promoter, wherein said polynucleotide fragment encodes a virus-like -particle (VLP) comprising:

a. a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID

NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119 and

b. at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9,

wherein said first viral CP is fused to said at least one SpA domain.

7. The DNA construct as claimed in claim 6, wherein said VLP amino acid sequence is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, and SEQ ID NO: 159.

8. The DNA construct as claimed in claim 6, wherein said polynucleotide fragment sequence is selected from the group consisting of SEQ ID NO: 12, SEQ ID

NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, and SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158 and SEQ ID NO: 160.

9. A recombinant DNA vector comprising a DNA construct, said DNA construct as claimed in any of the claims 6-8.

10. A recombinant host cell comprising a DNA construct as claimed in any of the claims 6-8.

11. A recombinant host cell comprising a recombinant DNA vector as claimed in claim 9.

12. The recombinant host cell as claimed in any of the claim 10-11, wherein said recombinant host cell is of bacterial, fungal, plant, insect, or mammalian origin.

13. A process for purifying an immunoglobulin comprising the steps: a. obtaining a virus-like -particle (VLP) as claimed in any of the claims 1 - 5;

b. contacting said VLP with an immunoglobulin under conditions which allow binding of said VLP with said immunoglobulin; and c. separating said immunoglobulin from said VLP.

14. A process for delivery of antibodies to the surface, inside or both to surface and inside of cells, said process comprising the steps:

a. obtaining a virus-like-particle (VLP) as claimed in any of the claims 1- 5;

b. obtaining at least one antibody;

c. incubating said VLP with said at least one antibody to obtain VLP- antibody complexes; and

d. contacting said VLP-antibody complexes with said cells.

15. A process for imaging a tissue of interest, said process comprising the steps:

a. obtaining a virus-like-particle (VLP) as claimed in any of the claims 1- 5;

b. obtaining at least one antibody;

c. labelling said VLP and said at least one antibody with a fluorophore, wherein the fluorophore for labelling said VLP is different than the fluorophore for labelling said at least one antibody;

d. Incubating said labelled VLP with said labelled at least one antibody to obtain VLP-antibody complexes;

e. administering said VLP-antibody complex to a subject; and f. visualizing said tissue of interest by any known method.

16. A process of targeting a virus-like -particle (VLP) to a cancer cell, said process comprising the steps:

a. obtaining a VLP as claimed in any of the claims 1-5;

b. obtaining at least one molecule which can ameliorate cancer cell activity; C. obtaining at least one antibody which binds preferentially to at least one cell surface moiety present majorly in said cancer cell;

d. encapsulating at least one molecule with said VLP;

e. contacting said VLP from d) with said at least one antibody; and f. contacting said VLP from e) with a tissue comprising cancer wherein said method targets VLP to a cancer cell.

17. The process as claimed in claim 16, further comprising conjugating said VLP from step a) or d) with at least one cell penetrating peptide.

18. A VLP as claimed in any of the claims 1-5 for use in disease diagnosis, cargo delivery, intracellular imaging, or cell specific targeting.

Description:

CHIMERIC PLANT BASED VIRUS-LIKE-PARTICLES, AND USES

THEREOF

FIELD OF INVENTION

[001] The present disclosure relates to the field of virology. The present disclosure provides chimeric plant based virus-like-particles, which can be used for delivery of cargo to cells, and enhanced purification of immunoglobulins.

BACKGROUND OF THE INVENTION

[002] Plant virus based nanoparticles(PVNs) have gained importance as they are part of the food chain (consumed by humans when infected plant material is ingested) and remain non-pathogenic in human host. PVNs can be manufactured by molecular farming and hazards of handling animal viruses can be circumvented.

[003] Sesbania mosaic virus (SeMV) is a non-enveloped, positive sense, single stranded RNA plant virus belonging to genus Sobemovirus. Its4.1kb genome contains40RFsandthe coat protein (CP) is encoded by the sub-genomic RNA encompassing ORF 3. Like many other viruses, SeMV native icosahedral capsids consisting of 180 identical CP subunits are highly stable, and have a high melting temperature of -90 °C.

[004] Pepper vein banding virus(PVBV) is also a non-enveloped positive sense RNA plant virus belonging to the Poty virus genus. It is a flexuous rod shaped virus of length 900 nm, lOnm width with -2000 CP subunits encapsulating the lOkb genome. The N and C terminal domains of the CP are surface exposed.

SUMMARY OF THE INVENTION

[005] In an aspect of the present disclosure, there is provided a virus -like-particle (VLP) comprising: (a) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119 and (b) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain.

[006] In an aspect of the present disclosure, there is provided a virus -like-particle (VLP) having amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, and SEQ ID NO: 159.

[007] In an aspect of the present disclosure, there is provided a DNA construct comprising a polynucleotide fragment operably linked to a promoter, wherein said polynucleotide fragment encodes a virus-like -particle (VLP) comprising: (a) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119 and (b) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain.

[008] In an aspect of the present disclosure, there is provided a recombinant DNA vector comprising a DNA construct, said DNA construct comprising: (a) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119 and (b) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain.

[009] In an aspect of the present disclosure, there is provided a recombinant host cell comprising a DNA construct, said DNA construct comprising: (a) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119 and (b) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain.

[0010] In an aspect of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA vector, said recombinant DNA vector comprising a DNA construct comprising: (a) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119 and (b) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain.

[0011] In an aspect of the present disclosure, there is provided a process for delivery of antibodies to the surface, inside or both to surface and inside of cells, said process comprising the steps: (a) obtaining a virus-like-particle (VLP) comprising : (i) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119; and (ii) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain; (b) obtaining at least one antibody; (c) incubating said VLP with said at least one antibody to obtain VLP-antibody complexes; and (d) contacting said VLP-antibody complexes with said cells.

[0012] In an aspect of the present disclosure, there is provided a process of targeting a virus-like -particle (VLP) to a cancer cell, said process comprising the steps: (a) obtaining a virus-like-particle (VLP) comprising : (i) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119; and (ii) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain; (b) obtaining at least one molecule which can ameliorate cancer cell activity; (c) obtaining at least one antibody which binds preferentially to at least one cell surface moiety present majorly in said cancer cell; (d) encapsulating at least one molecule with said VLP; (e) contacting said VLP from d) with said at least one antibody; and (f) contacting said VLP from e) with a tissue comprising cancer cell, wherein said method targets VLP to a cancer cell.

[0013] In an aspect of the present disclosure, there is provided a process for imaging a tissue of interest, said process comprising the steps: (a) obtaining a virus -like-particle (VLP) comprising : (i) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119; and (ii) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain; (b) obtaining at least one antibody; (c) labelling said VLP and said at least one antibody with a fluorophore, wherein the fluorophore for labelling said VLP is different than the fluorophore for labelling said at least one antibody; (d) incubating said labelled VLP with said labelled at least one antibody to obtain VLP-antibody complexes; (e) administering said VLP-antibody complex to a subject; and (f) visualizing said tissue of interest by any known method. [0014] In an aspect of the present disclosure, there is provided a process for purifying an immunoglobulin comprising the steps: (a) obtaining a virus-like -particle (VLP) comprising : (i) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119; and (ii) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain; (b) contacting said VLP with an immunoglobulin under conditions which allow binding of said VLP with said immunoglobulin; and (c) separating said immunoglobulin from said VLP.

[0015] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0016] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0017] Figure 1A-F depicts the cloning of SLB and SLZ33 in pRSETC vector, in accordance with an embodiment of the present disclosure.

[0018] Figure 2A-C depicts data to show that SLB and SLZ33 form VLPs in-vitro, in accordance with an embodiment of the present disclosure.

[0019] Figure 3A-C depicts the functional analysis of B/Z33 domain in chimeric VLPs, in accordance with an embodiment of the present disclosure. [0020] Figure 4A-C depicts the biochemical analysis of Alexa Fluor 488 labelled VLPs, in accordance with an embodiment of the present disclosure.

[0021] Figure 5A-H depicts the confocal microscopy images of time-dependent entry of VLPs into HeLa cells, in accordance with an embodiment of the present disclosure.

[0022] Figure 6A-E depicts the confocal microscopy images of specificity of CP488 entry into HeLa cells, in accordance with an embodiment of the present disclosure.

[0023] Figure 7A-I depicts the confocal microscopy images of entry of SLB 488 into HeLa and other cells, in accordance with an embodiment of the present disclosure.

[0024] Figure 8A-L depicts the confocal microscopy images of entry of D6F10 by abrin, in accordance with an embodiment of the present disclosure.

[0025] Figure 9A-L depicts the confocal microscopy images showing the entry of D6F10 into cells when delivered by SLB and not CP, in accordance with an embodiment of the present disclosure.

[0026] Figure 10A-P depicts the confocal microscopy images of kinetics of D6F10 delivery by SLB into HeLa cells, in accordance with an embodiment of the present disclosure.

[0027] Figure 11A-H depicts the SLB mediated D6F10 delivery into cells in presence of sheep serum, in accordance with an embodiment of the present disclosure.

[0028] Figure 12A-H depicts the confocal microscopy images of SLB mediated D6F10 delivery into KB and B16-10 cells, in accordance with an embodiment of the present disclosure.

[0029] Figure 13A-C depicts the surface binding of CP, SLB, SLB-D6-F10, and CP- D6F10 by FACS, in accordance with an embodiment of the present disclosure.

[0030] Figure 14A-C depicts the enhanced binding of D6F10 upon pre-incubation with SLB, in accordance with an embodiment of the present disclosure.

[0031] Figure 15A-B depicts the immunoblot analysis of entry of D6F10 into HeLa cells upon pre-incubation with SLB, in accordance with an embodiment of the present disclosure. [0032] Figure 16A-B depicts the functional analysis of internalized D6F10, in accordance with an embodiment of the present disclosure.

[0033] Figure 17A-L depicts the confocal microscopy images of SLB mediated delivery of FITC labelled monoclonal anti-a-tubulin antibody into cells, in accordance with an embodiment of the present disclosure.

[0034] Figure 18A-I depicts the confocal microscopy images of kinetic of SLB mediated anti-a-tubulin delivery into cells, in accordance with an embodiment of the present disclosure.

[0035] Figure 19A-D depicts the confocal microscopy images of Herclon binding to HER2 receptor, in accordance with an embodiment of the present disclosure.

[0036] Figure 20A-O depicts the confocal microscopy images of SLB mediated delivery of Herclon into cells, in accordance with an embodiment of the present disclosure.

[0037] Figure 21A-0 depicts the confocal microscopy images of kinetics of SLB mediated delivery of Herclon into BT-474 cells, in accordance with an embodiment of the present disclosure.

[0038] Figure 22A-L depicts the confocal microscopy images of kinetics of Herclon binding to BT-474 cells as control, in accordance with an embodiment of the present disclosure.

[0039] Figure 23A-B depicts the functional analysis of Herclon delivery by SLB into BT-474 cells, in accordance with an embodiment of the present disclosure.

[0040] Figure 24 depicts the PCR amplification of B domain of SpA, in accordance with an embodiment of the present disclosure.

[0041] Figure 25 depicts the PCR amplification of SeMV CP and ΝΔ65 CP, in accordance with an embodiment of the present disclosure.

[0042] Figure 26 depicts the SDS PAGE profile of B-NA65CP fractions obtained by sucrose density gradient centrifugation demonstrating the assembly of chimeric B- NA65CP VLPs, in accordance with an embodiment of the present disclosure. [0043] Figure 27 depicts the SDS PAGE profile of B-CP demonstrating the assembly of B-CP, in accordance with an embodiment of the present disclosure.

[0044] Figure 28A, B depicts the transmission electron micrograph of B-NA65CP, and B-CP respectively, in accordance with an embodiment of the present disclosure.

[0045] Figure 29A, B depicts the SDS PAGE and transmission electron micrograph respectively of NA65CP-B VLPs, in accordance with an embodiment of the present disclosure.

[0046] Figure 30 depicts the DAC ELISA showing the ability of B-NA65-CP VLPs to bind to DAPAL antibodies, in accordance with an embodiment of the present disclosure.

[0047] Figure 31A-L depicts the confocal images showing the cellular entry of NA65CPVLPs, BNA65CP dimer, and BNA65CPVLPs and delivery of D6F10 monoclonal antibody only by BNA65CPVLPs in accordance with an embodiment of the present disclosure.

[0048] Figure 32 depicts the standard graph of doxorubicin, in accordance with an embodiment of the present disclosure.

[0049] Figure 33A-G depicts the confocal images of SeMV mediated DAPI entry into

HeLa cells, in accordance with an embodiment of the present disclosure.

[0050] Figure 34 depicts the SeMV mediated Dox toxicity in HeLa cells, in accordance with an embodiment of the present disclosure.

[0051] Figure 35A-E depicts the cloning of PVBVCP and PVBV BCP in pRSETC expression vector, in accordance with an embodiment of the present disclosure.

[0052] Figure 36A-C depicts the expression and purification of PVBVVLPs and chimeric VLPs, in accordance with an embodiment of the present disclosure.

[0053] Figure 37A-B depicts the transmission electron micrograph of PVBVVLPs and chimeric VLPs, in accordance with an embodiment of the present disclosure.

[0054] Figure 38A-C depicts the western blot analysis of PVBVVLPs and chimeric VLPs with functional B domain, in accordance with an embodiment of the present disclosure. [0055] Figure 39A-C depicts the ELISA for functionality of B domain in PVBVVLPs, in accordance with an embodiment of the present disclosure.

[0056] Figure 40 depicts the sandwich ELISA of PVBVVLPs, in accordance with an embodiment of the present disclosure.

[0057] Figure 41A-F depicts the entry of PVBVVLPs at a fixed concentration of 1.73 nM at varying time points into HeLa cells, in accordance with an embodiment of the present disclosure.

[0058] Figure 42A-F depicts the entry of chimeric VLPs at a fixed concentration of 1 nM at varying time point into HeLa cells, in accordance with an embodiment of the present disclosure.

[0059] Figure 43A-F depicts the entry of PVBV VLPs and chimeric VLPs into various cancerous mammalian cells, in accordance with an embodiment of the present disclosure.

[0060] Figure 44A-B depicts the entry of PVBV VLPs and chimeric VLPs into 3T3 cells, in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0061] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

Definitions

[0062] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0063] The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0064] The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only".

[0065] Throughout this specification, unless the context requires otherwise the word

"comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0066] The term "including" is used to mean "including but not limited to".

"Including" and "including but not limited to" are used interchangeably.

[0067] 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0068] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

[0069] In an embodiment of the present disclosure, there is provided a virus-like- particle (VLP) comprising: (a) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119 and (b) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain.

[0070] In an embodiment of the present disclosure, there is provided a VLP as described herein, wherein said first viral CP is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 118 and SEQ ID NO: 120 and said at least one SpA domain is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 8, and SEQ ID NO: 10.

[0071] In an embodiment of the present disclosure, there is provided a VLP as described herein, wherein said at least one SpA domain is fused to N-terminal end of said first viral CP.

[0072] In an embodiment of the present disclosure, there is provided a VLP as described herein, wherein said at least one SpA domain is fused to C-terminal end of said first viral CP.

[0073] In an embodiment of the present disclosure, there is provided a VLP as described herein, wherein said at least one SpA domain is integrated within the said first viral CP.

[0074] In an embodiment of the present disclosure, there is provided a VLP as described herein, wherein said first viral CP having amino acid sequence as set forth in SEQ ID NO: 1 is encoded by SEQ ID NO: 2.

[0075] In an embodiment of the present disclosure, there is provided a VLP as described herein, wherein said first viral CP having amino acid sequence as set forth in SEQ ID NO: 2 is encoded by SEQ ID NO: 4.

[0076] In an embodiment of the present disclosure, there is provided a VLP as described herein, wherein said first viral CP having amino acid sequence as set forth in SEQ ID NO: 3 is encoded by SEQ ID NO: 5.

[0077] In an embodiment of the present disclosure, there is provided a VLP as described herein, wherein said first viral CP having amino acid sequence as set forth in SEQ ID NO: 117 is encoded by SEQ ID NO: 118. [0078] In an embodiment of the present disclosure, there is provided a VLP as described herein, wherein said first viral CP having amino acid sequence as set forth in SEQ ID NO: 119 is encoded by SEQ ID NO: 120.

[0079] In an embodiment of the present disclosure, there is provided a VLP as described herein, wherein said at least one SpA domain having amino acid sequence as set forth in SEQ ID NO: 7 is encoded by SEQ ID NO: 8.

[0080] In an embodiment of the present disclosure, there is provided a VLP as described herein, wherein said at least one SpA domain having amino acid sequence as set forth in SEQ ID NO: 9 is encoded by SEQ ID NO: 10.

[0081] In an embodiment of the present disclosure, there is provided a virus-like- particle (VLP) having amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, and SEQ ID NO: 159.

[0082] In an embodiment of the present disclosure, there is provided a VLP as described herein, wherein said VLP is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112. SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158 and SEQ ID NO: 160.

[0083] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 11 encoded by SEQ ID NO: 12.

[0084] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 13 encoded by SEQ ID NO: 14.

[0085] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 15 encoded by SEQ ID NO: 16.

[0086] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 17 encoded by SEQ ID NO: 18.

[0087] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 19 encoded by SEQ ID NO: 20.

[0088] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 21 encoded by SEQ ID NO: 22. [0089] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 23 encoded by SEQ ID NO: 24.

[0090] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 25 encoded by SEQ ID NO: 26.

[0091] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 27 encoded by SEQ ID NO: 28.

[0092] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 29 encoded by SEQ ID NO: 30.

[0093] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 31 encoded by SEQ ID NO: 32.

[0094] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 33 encoded by SEQ ID NO: 34.

[0095] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 35 encoded by SEQ ID NO: 36.

[0096] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 37 encoded by SEQ ID NO: 38.

[0097] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 39 encoded by SEQ ID NO: 40.

[0098] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 41 encoded by SEQ ID NO: 42.

[0099] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 43 encoded by SEQ ID NO: 44.

[00100] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 45 encoded by SEQ ID NO: 46.

[00101] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 47 encoded by SEQ ID NO: 48.

[00102] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 49 encoded by SEQ ID NO: 50. [00103] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 51 encoded by SEQ ID NO: 52.

[00104] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 53 encoded by SEQ ID NO: 54.

[00105] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 55 encoded by SEQ ID NO: 56.

[00106] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 57 encoded by SEQ ID NO: 58.

[00107] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 59 encoded by SEQ ID NO: 60.

[00108] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 61 encoded by SEQ ID NO: 62.

[00109] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 63 encoded by SEQ ID NO: 64.

[00110] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 65 encoded by SEQ ID NO: 66.

[00111] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 67 encoded by SEQ ID NO: 68.

[00112] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 69 encoded by SEQ ID NO: 70.

[00113] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 71 encoded by SEQ ID NO: 72.

[00114] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 73 encoded by SEQ ID NO: 74.

[00115] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 75 encoded by SEQ ID NO: 76.

[00116] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 77 encoded by SEQ ID NO: 78. [00117] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 79 encoded by SEQ ID NO: 80.

[00118] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 81 encoded by SEQ ID NO: 82.

[00119] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 83 encoded by SEQ ID NO: 84.

[00120] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 85 encoded by SEQ ID NO: 86.

[00121] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 87 encoded by SEQ ID NO: 88.

[00122] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 89 encoded by SEQ ID NO: 90.

[00123] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 91 encoded by SEQ ID NO: 92.

[00124] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 93 encoded by SEQ ID NO: 94.

[00125] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 95 encoded by SEQ ID NO: 96.

[00126] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 97 encoded by SEQ ID NO: 98.

[00127] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 99 encoded by SEQ ID NO: 100.

[00128] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 101 encoded by SEQ ID NO: 102.

[00129] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 103 encoded by SEQ ID NO: 104.

[00130] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 105 encoded by SEQ ID NO: 106. [00131] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 107 encoded by SEQ ID NO: 108.

[00132] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 109 encoded by SEQ ID NO: 110.

[00133] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 111 encoded by SEQ ID NO: 112.

[00134] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 113 encoded by SEQ ID NO: 114.

[00135] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 115 encoded by SEQ ID NO: 116.

[00136] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 121 encoded by SEQ ID NO: 122.

[00137] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 123 encoded by SEQ ID NO: 124.

[00138] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 125 encoded by SEQ ID NO: 126.

[00139] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 127 encoded by SEQ ID NO: 128.

[00140] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 129 encoded by SEQ ID NO: 130.

[00141] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 131 encoded by SEQ ID NO: 132.

[00142] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 133 encoded by SEQ ID NO: 134.

[00143] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 135 encoded by SEQ ID NO: 136.

[00144] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 137 encoded by SEQ ID NO: 138. [00145] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 139 encoded by SEQ ID NO: 140.

[00146] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 141 encoded by SEQ ID NO: 142.

[00147] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 143 encoded by SEQ ID NO: 144.

[00148] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 145 encoded by SEQ ID NO: 146.

[00149] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 147 encoded by SEQ ID NO: 148.

[00150] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 149 encoded by SEQ ID NO: 150.

[00151] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 151 encoded by SEQ ID NO: 152.

[00152] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 153 encoded by SEQ ID NO: 154.

[00153] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 155 encoded by SEQ ID NO: 156.

[00154] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 157 encoded by SEQ ID NO: 158.

[00155] In an embodiment of the present disclosure, there is provided a VLP having amino acid sequence as set forth in SEQ ID NO: 159 encoded by SEQ ID NO: 160.

[00156] In an embodiment of the present disclosure, there is provided a DNA construct comprising a polynucleotide fragment operably linked to a promoter, wherein said polynucleotide fragment encodes a virus-like -particle (VLP) comprising: (a) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119 and (b) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain.

[00157] In an embodiment of the present disclosure, there is provided a DNA construct as described herein, wherein said first viral CP is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 118 and SEQ ID NO: 120 and said at least one SpA domain is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 8, and SEQ ID NO: 10.

[00158] In an embodiment of the present disclosure, there is provided a DNA construct as described herein, wherein said at least one SpA domain is fused to N- terminal end of said first viral CP.

[00159] In an embodiment of the present disclosure, there is provided a DNA construct as described herein, wherein said at least one SpA domain is fused to C- terminal end of said first viral CP.

[00160] In an embodiment of the present disclosure, there is provided a DNA construct as described herein, wherein said at least one SpA domain is integrated within the said first viral CP.

[00161] In an embodiment of the present disclosure, there is provided a DNA construct as described herein, wherein said at least one SpA domain is integrated at N and C termini of said first viral CP.

[00162] In an embodiment of the present disclosure, there is provided a DNA construct as described herein, wherein said at least one SpA domain is integrated at N- terminal, C-terminal and within said first viral CP.

[00163] In an embodiment of the present disclosure, there is provided a DNA construct as described herein, wherein said at least one SpA domain is integrated at N- terminal and within said first viral CP.

[00164] In an embodiment of the present disclosure, there is provided a DNA construct as described herein, wherein said at least one SpA domain is integrated at C- terminal and within said first viral CP. [00165] In an embodiment of the present disclosure, there is provided a DNA construct as described herein, wherein said VLP amino acid sequence is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:SEQ ID NO: 13, SEQ ID NO

15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO

35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO

55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO

65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO

85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, and SEQ ID NO: 159.

[00166] In an embodiment of the present disclosure, there is provided a DNA construct as described herein, wherein said polynucleotide fragment sequence is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO

16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO

36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO

56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO

66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO

86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158 and SEQ ID NO: 160.

[00167] In an embodiment of the present disclosure, there is provided a recombinant DNA vector comprising a DNA construct, said DNA construct comprising a polynucleotide fragment operably linked to a promoter, wherein said polynucleotide fragment encodes a virus-like -particle (VLP) comprising: (a) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119 and (b) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain.

[00168] In an embodiment of the present disclosure, there is provided a recombinant DNA vector as described herein, wherein said first viral CP is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 118 and SEQ ID NO: 120 and said at least one SpA domain is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 8, and SEQ ID NO: 10.

[00169] In an embodiment of the present disclosure, there is provided a recombinant DNA vector as described herein, wherein said at least one SpA domain is fused to N- terminal end of said first viral CP.

[00170] In an embodiment of the present disclosure, there is provided a recombinant DNA vector as described herein, wherein said at least one SpA domain is fused to C- terminal end of said first viral CP. [00171] In an embodiment of the present disclosure, there is provided a recombinant DNA vector as described herein, wherein said at least one SpA domain is integrated within the said first viral CP.

[00172] In an embodiment of the present disclosure, there is provided a recombinant DNA vector as described herein, wherein said at least one SpA domain is integrated at N and C termini of said first viral CP.

[00173] In an embodiment of the present disclosure, there is provided a recombinant DNA vector as described herein, wherein said at least one SpA domain is integrated at N-terminal, C-terminal and within said first viral CP.

[00174] In an embodiment of the present disclosure, there is provided a recombinant DNA vector as described herein, wherein said at least one SpA domain is integrated at N-terminal and within said first viral CP.

[00175] In an embodiment of the present disclosure, there is provided a recombinant DNA vector as described herein, wherein said at least one SpA domain is integrated at C-terminal and within said first viral CP.

[00176] In an embodiment of the present disclosure, there is provided a recombinant DNA vector as described herein, wherein said VLP amino acid sequence is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, and SEQ ID NO: 159.

[00177] In an embodiment of the present disclosure, there is provided a recombinant DNA vector as described herein, wherein said polynucleotide fragment sequence is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158 and SEQ ID NO: 160.

[00178] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a DNA construct, said DNA construct comprising a polynucleotide fragment operably linked to a promoter, wherein said polynucleotide fragment encodes a virus-like -particle (VLP) comprising: (a) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119 and (b) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain.

[00179] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a DNA construct as described herein, wherein said first viral CP is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 118 and SEQ ID NO: 120 and said at least one SpA domain is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 8, and SEQ ID NO: 10.

[00180] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a DNA construct as described herein, wherein said at least one SpA domain is fused to N-terminal end of said first viral CP.

[00181] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a DNA construct as described herein, wherein said at least one SpA domain is fused to C-terminal end of said first viral CP.

[00182] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a DNA construct as described herein, wherein said at least one SpA domain is integrated within the said first viral CP.

[00183] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a DNA construct as described herein, wherein said at least one SpA domain is integrated at N and C termini of said first viral CP.

[00184] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a DNA construct as described herein, wherein said at least one SpA domain is integrated at N-terminal, C-terminal and within said first viral CP.

[00185] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a DNA construct as described herein, wherein said at least one SpA domain is integrated at N-terminal and within said first viral CP.

[00186] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a DNA construct as described herein, wherein said at least one SpA domain is integrated at C-terminal and within said first viral CP. [00187] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a DNA construct as described herein, wherein said VLP amino acid sequence is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, and SEQ ID NO: 159.

[00188] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a DNA construct as described herein, wherein said polynucleotide fragment sequence is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 1 10, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158 and SEQ ID NO: 160.

[00189] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA vector, said recombinant DNA vector comprising a DNA construct, said DNA construct comprising a polynucleotide fragment operably linked to a promoter, wherein said polynucleotide fragment encodes a virus-like -particle (VLP) comprising: (a) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119 and (b) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain.

[00190] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA vector as described herein, wherein said first viral CP is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 118 and SEQ ID NO: 120 and said at least one SpA domain is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 8, and SEQ ID NO: 10.

[00191] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA vector as described herein, wherein said at least one SpA domain is fused to N-terminal end of said first viral CP.

[00192] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA vector as described herein, wherein said at least one SpA domain is fused to C-terminal end of said first viral CP. [00193] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA vector as described herein, wherein said at least one SpA domain is integrated within the said first viral CP.

[00194] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA vector as described herein, wherein said at least one SpA domain is integrated at N and C termini of said first viral CP.

[00195] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA vector as described herein, wherein said at least one SpA domain is integrated at N-terminal, C-terminal and within said first viral CP.

[00196] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA vector as described herein, wherein said at least one SpA domain is integrated at N-terminal and within said first viral CP.

[00197] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA vector as described herein, wherein said at least one SpA domain is integrated at C-terminal and within said first viral CP.

[00198] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA vector as described herein, wherein said VLP amino acid sequence is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, and SEQ ID NO: 159.

[00199] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA vector as described herein, wherein said polynucleotide fragment sequence is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158 and SEQ ID NO: 160.

[00200] In an embodiment of the present disclosure, there is provided a recombinant host cell as described herein, wherein said recombinant host cell is of bacterial, fungal, plant, insect, or mammalian origin.

[00201] In an embodiment of the present disclosure, there is provided a recombinant host cell as described herein, wherein said recombinant host cell is bacterial cell. [00202] In an embodiment of the present disclosure, there is provided a recombinant host cell as described herein, wherein said recombinant host cell is E.coli.

[00203] In an embodiment of the present disclosure, there is provided a recombinant host cell as described herein, wherein said recombinant host cell is fungal cell.

[00204] In an embodiment of the present disclosure, there is provided a recombinant host cell as described herein, wherein said recombinant host cell is plant cell.

[00205] In an embodiment of the present disclosure, there is provided a recombinant host cell as described herein, wherein said recombinant host cell is insect cell.

[00206] In an embodiment of the present disclosure, there is provided a recombinant host cell as described herein, wherein said recombinant host cell is mammalian cell.

[00207] In an embodiment of the present disclosure, there is provided a process of for purifying an immunoglobulin comprising the steps: (a) obtaining a virus-like-particle (VLP) comprising : (i) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119; and (ii) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain; (b) contacting said VLP with an immunoglobulin under conditions which allow binding of said VLP with said immunoglobulin; and (c) separating said immunoglobulin from said VLP.

[00208] In an embodiment of the present disclosure, there is provided a process of for purifying an immunoglobulin as described herein, wherein said first viral CP is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 118 and SEQ ID NO: 120 and said at least one SpA domain is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 8, and SEQ ID NO: 10.

[00209] In an embodiment of the present disclosure, there is provided a process of for purifying an immunoglobulin as described herein, wherein said at least one SpA domain is fused to N-terminal end of said first viral CP. [00210] In an embodiment of the present disclosure, there is provided a process of for purifying an immunoglobulin as described herein, wherein said at least one SpA domain is fused to C-terminal end of said first viral CP.

[00211] In an embodiment of the present disclosure, there is provided a process of for purifying an immunoglobulin as described herein, wherein said at least one SpA domain is integrated within the said first viral CP.

[00212] In an embodiment of the present disclosure, there is provided a process of for purifying an immunoglobulin as described herein, wherein said at least one SpA domain is integrated at N and C termini of said first viral CP.

[00213] In an embodiment of the present disclosure, there is provided a process of for purifying an immunoglobulin as described herein, wherein said at least one SpA domain is integrated at N-terminal, C-terminal and within said first viral CP.

[00214] In an embodiment of the present disclosure, there is provided a process of for purifying an immunoglobulin as described herein, wherein said at least one SpA domain is integrated at N-terminal and within said first viral CP.

[00215] In an embodiment of the present disclosure, there is provided a process of for purifying an immunoglobulin as described herein, wherein said at least one SpA domain is integrated at C-terminal and within said first viral CP.

[00216] In an embodiment of the present disclosure, there is provided a process of for purifying an immunoglobulin as described herein, wherein said VLP amino acid sequence is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, and SEQ ID NO: 159.

[00217] In an embodiment of the present disclosure, there is provided a process of for purifying an immunoglobulin as described herein, wherein said polynucleotide fragment sequence is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158 and SEQ ID NO: 160.

[00218] In an embodiment of the present disclosure, there is provided a process of for delivery of antibodies to the surface, inside or both to surface and inside of cells, said process comprising the steps: (a) obtaining a virus-like-particle (VLP) comprising : (i) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119; and (ii) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain; (b) obtaining at least one antibody; (c) incubating said VLP with said at least one antibody to obtain VLP-antibody complexes; and (d) contacting said VLP-antibody complexes with said cells.

[00219] In an embodiment of the present disclosure, there is provided a process of for delivery of antibodies to the surface, inside or both to surface and inside of cells as described herein, wherein said first viral CP is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 118 and SEQ ID NO: 120 and said at least one SpA domain is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 8, and SEQ ID NO: 10.

[00220] In an embodiment of the present disclosure, there is provided a process of for delivery of antibodies to the surface, inside or both to surface and inside of cells as described herein, wherein said at least one SpA domain is fused to N-terminal end of said first viral CP.

[00221] In an embodiment of the present disclosure, there is provided a process of for delivery of antibodies to the surface, inside or both to surface and inside of cells as described herein, wherein said at least one SpA domain is fused to C-terminal end of said first viral CP.

[00222] In an embodiment of the present disclosure, there is provided a process of for delivery of antibodies to the surface, inside or both to surface and inside of cells as described herein, wherein said at least one SpA domain is integrated within the said first viral CP.

[00223] In an embodiment of the present disclosure, there is provided a process of for delivery of antibodies to the surface, inside or both to surface and inside of cells as described herein, wherein said at least one SpA domain is integrated at N and C termini of said first viral CP.

[00224] In an embodiment of the present disclosure, there is provided a process of for delivery of antibodies to the surface, inside or both to surface and inside of cells as described herein, wherein said at least one SpA domain is integrated at N-terminal, C- terminal and within said first viral CP.

[00225] In an embodiment of the present disclosure, there is provided a process of for delivery of antibodies to the surface, inside or both to surface and inside of cells as described herein, wherein said at least one SpA domain is integrated at N-terminal and within said first viral CP.

[00226] In an embodiment of the present disclosure, there is provided a process of for delivery of antibodies to the surface, inside or both to surface and inside of cells as described herein, wherein said at least one SpA domain is integrated at C-terminal and within said first viral CP.

[00227] In an embodiment of the present disclosure, there is provided a process of for delivery of antibodies to the surface, inside or both to surface and inside of cells as described herein, wherein said VLP amino acid sequence is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, and SEQ ID NO: 159.

[00228] In an embodiment of the present disclosure, there is provided a process of for delivery of antibodies to the surface, inside or both to surface and inside of cells as described herein, wherein said polynucleotide fragment sequence is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158 and SEQ ID NO: 160.

[00229] In an embodiment of the present disclosure, there is provided a process for imaging a tissue of interest, said process comprising the steps: (a) obtaining a viruslike-particle (VLP) comprising : (i) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119; and (ii) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain; (b) obtaining at least one antibody; (c) labelling said VLP and said at least one antibody with a fluorophore, wherein the fluorophore for labelling said VLP is different than the fluorophore for labelling said at least one antibody; (d) incubating said labelled VLP with said labelled at least one antibody to obtain VLP-antibody complexes; (e) administering said VLP-antibody complex to a subject; and (f) visualizing said tissue of interest by any known method.

[00230] In an embodiment of the present disclosure, there is provided a process for imaging a tissue of interest as described herein, wherein said first viral CP is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 118 and SEQ ID NO: 120 and said at least one SpA domain is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 8, and SEQ ID NO: 10.

[00231] In an embodiment of the present disclosure, there is provided a process for imaging a tissue of interest as described herein, wherein said at least one SpA domain is fused to N-terminal end of said first viral CP.

[00232] In an embodiment of the present disclosure, there is provided a process for imaging a tissue of interest as described herein, wherein said at least one SpA domain is fused to C-terminal end of said first viral CP.

[00233] In an embodiment of the present disclosure, there is provided a process for imaging a tissue of interest as described herein, wherein said at least one SpA domain is integrated within the said first viral CP.

[00234] In an embodiment of the present disclosure, there is provided a process for imaging a tissue of interest as described herein, wherein said at least one SpA domain is integrated at N and C termini of said first viral CP.

[00235] In an embodiment of the present disclosure, there is provided a process for imaging a tissue of interest as described herein, wherein said at least one SpA domain is integrated at N-terminal, C-terminal and within said first viral CP. [00236] In an embodiment of the present disclosure, there is provided a process for imaging a tissue of interest as described herein, wherein said at least one SpA domain is integrated at N-terminal and within said first viral CP.

[00237] In an embodiment of the present disclosure, there is provided a process for imaging a tissue of interest as described herein, wherein said at least one SpA domain is integrated at C-terminal and within said first viral CP.

[00238] In an embodiment of the present disclosure, there is provided a process for imaging a tissue of interest as described herein, wherein said VLP amino acid sequence is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, and SEQ ID NO: 159.

[00239] In an embodiment of the present disclosure, there is provided a process for imaging a tissue of interest as described herein, wherein said polynucleotide fragment sequence is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158 and SEQ ID NO: 160.

[00240] In an embodiment of the present disclosure, there is provided a process for imaging a tissue of interest as described herein, wherein said tissue is a tissue.

[00241] In an embodiment of the present disclosure, there is provided a process for imaging a tissue of interest as described herein, wherein said tissue is embryonic tissue.

[00242] In an embodiment of the present disclosure, there is provided a process for imaging a tissue of interest as described herein, wherein said tissue is a tumour.

[00243] In an embodiment of the present disclosure, there is provided a process of targeting a virus-like -particle (VLP) to a cancer cell, said process comprising the steps: (a) obtaining a virus-like -particle (VLP) comprising : (i) a first viral coat protein (CP) having amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 117 and SEQ ID NO: 119; and (ii) at least one Staphylococcus protein A (SpA) domain having amino acid sequence selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 9, wherein said first viral CP is fused to said at least one SpA domain; (b) obtaining at least one molecule which can ameliorate cancer cell activity; (c) obtaining at least one antibody which binds preferentially to at least one cell surface moiety present majorly in said cancer cell; (d) encapsulating at least one molecule with said VLP; (e) contacting said VLP from d) with said at least one antibody; and (f) contacting said VLP from e) with a tissue comprising cancer cell, wherein said method targets VLP to a cancer cell.

[00244] In an embodiment of the present disclosure, there is provided a process of targeting a virus-like-particle (VLP) to a cancer cell as described herein, wherein said first viral CP is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 118 and SEQ ID NO: 120 and said at least one SpA domain is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 8, and SEQ ID NO: 10.

[00245] In an embodiment of the present disclosure, there is provided a process of targeting a virus-like-particle (VLP) to a cancer cell as described herein, wherein said at least one SpA domain is fused to N-terminal end of said first viral CP.

[00246] In an embodiment of the present disclosure, there is provided a process of targeting a virus-like-particle (VLP) to a cancer cell as described herein, wherein said at least one SpA domain is fused to C-terminal end of said first viral CP.

[00247] In an embodiment of the present disclosure, there is provided a process of targeting a virus-like-particle (VLP) to a cancer cell as described herein, wherein said at least one SpA domain is integrated within the said first viral CP.

[00248] In an embodiment of the present disclosure, there is provided a process of targeting a virus-like-particle (VLP) to a cancer cell as described herein, wherein said at least one SpA domain is integrated at N and C termini of said first viral CP.

[00249] In an embodiment of the present disclosure, there is provided a process of targeting a virus-like-particle (VLP) to a cancer cell as described herein, wherein said at least one SpA domain is integrated at N-terminal, C-terminal and within said first viral CP.

[00250] In an embodiment of the present disclosure, there is provided a process of targeting a virus-like-particle (VLP) to a cancer cell as described herein, wherein said at least one SpA domain is integrated at N-terminal and within said first viral CP. [00251] In an embodiment of the present disclosure, there is provided a process of targeting a virus-like-particle (VLP) to a cancer cell as described herein, wherein said at least one SpA domain is integrated at C-terminal and within said first viral CP.

[00252] In an embodiment of the present disclosure, there is provided a process of targeting a virus-like-particle (VLP) to a cancer cell as described herein, wherein said VLP amino acid sequence is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, and SEQ ID NO: 159.

[00253] In an embodiment of the present disclosure, there is provided a process of targeting a virus-like-particle (VLP) to a cancer cell as described herein, wherein said polynucleotide fragment sequence is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158 and SEQ ID NO: 160.

[00254] In an embodiment of the present disclosure, there is provided a process of targeting a virus-like-particle (VLP) to a cancer cell as described herein, wherein said process further comprises the step of conjugating said VLP from step a) or d) with at least one cell penetrating peptide.

[00255] In an embodiment of the present disclosure, there is provided a process of targeting a virus-like-particle (VLP) to a cancer cell as described herein, wherein said at least one molecule is an anti-cancer drug.

[00256] In an embodiment of the present disclosure, there is provided a process of targeting a virus-like-particle (VLP) to a cancer cell as described herein, wherein said at least one anti-cancer drug is selected from the group (but not limited to) consisting of doxorubicin, methotrexate, vincristine, cytarabine, daunamycin, cyclophosphamide, and combinations thereof.

[00257] In an embodiment of the present disclosure, there is provided a process of targeting a virus-like-particle (VLP) to a cancer cell as described herein, wherein said at least one molecule is siRNA.

[00258] In an embodiment of the present disclosure, there is provided VLPs or chimeric VLPs as described herein, for use in tissue imaging.

[00259] In an embodiment of the present disclosure, there is provided VLPs or chimeric VLPs as described herein, for use in cell imaging. [00260] In an embodiment of the present disclosure, there is provided VLPs or chimeric VLPs as described herein, for use in targeted intracellular drug delivery.

[00261] In an embodiment of the present disclosure, there is provided VLPs or chimeric VLPs as described herein, which are capable of intracellular disassembly.

[00262] Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible.

EXAMPLES

[00263] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. Example 1

Materials and methods

[00264] Chemicals: General chemicals, antibiotics and gel elution kits used in the study were obtained from Sigma-Aldrich USA; Merck, Germany; Calbiochem, USA; Novagen, Germany and Spectrochem Mumbai, India. Media (for E. coli) were procured from Hi-Media, Mumbai, India. Restriction enzymes, modifying enzymes and molecular markers were obtained from MBI Fermentas Inc., USA; New England Biolabs Inc., USA and Finnzymes, USA. Formvar coated copper grids (Code: 3440C- MB) were procured from SPI Supplies, USA. TMB and secondary antibodies were obtained from Bangalore Genei, India. FITC labeled monoclonal anti-a-tubulin antibody (F2168), sucrose (Cat no: 15925) and nuncimmiunomodules were purchased from Thermo Fischer Scientific, USA. Herclon was obtained from Roche Product (India) Pvt Ltd. Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute (RPMI) media, formaldehyde, Fluorosheild (F6182), DAPI (D9542), Propidium iodide (P4170), protease inhibitor cocktail (P8340) were obtained from Sigma, USA. Fetal bovine serum (FBS) was purchased from PAN biotech, Germany. Alexa Fluor 488, Alexa Fluor 633, almar blue, amino acid mixture, penicillin, streptomycin and Glutamax were procured from Invitrogen Corporation, USA. Radiolabeled tritiated leucine was obtained from BRIT, India.

[00265] Cloning, expression and purification of chimeric VLPs with B and Z33 domain: SeMV CP was cloned at the Xhol and Bglll sites of pRSETC vector (data not provided). B domain of SpA was cloned at the Nhel and BamHI site of pRSETC NA65CP to create pRSETC ΝΔ65Β CP. For cloning the B domain in HI loop region of SeMV CP to generate SeMV loop B (SLB), first the nucleotides corresponding to Ser 242 present in the middle of HI loop, were mutated to alanine using CP sdmAfel sense and antisense primers (Table 1) by site directed mutagenesis with pRSETC CP as template, such that a new restriction site Afel was generated at the midpoint of the HI loop. The sequence TTCC in pRSETC CP was changed to CGCT in recombinant clone pRSETC CP sdm. This plasmid was digested with Afel enzyme and the linear plasmid was used for cloning the PCR product of B domain gene segment obtained by amplification using pRSETC ΝΔ65Β CP as template and B domain specific primers as mentioned in Table 1. The PCR product was li gated to Afel digested pRSETC CP sdm using T4 DNA ligase. DH5a cells were transformed with the resultant plasmid (pRSETC SLB His) and clones were screened for insertion of B domain gene segment by PCR using CP mid sense and B antisense primers (expected size of the PCR product 324 bp). For removal of the N terminal Histidine tag, the entire construct was PCR amplified using CP specific primers (Table 1) digested with EcoRI and inserted in Ndel end filled and EcoRI cut pRSETC vector. The clones (pRSETC SLB) were confirmed by PCR amplification using CP specific primers. Z33 PCR product was obtained by using Z33 dsDNA (annealed Z33 full length sense and antisense oligos) as the template and Z33 sense and antisense primers (Table 1). A strategy similar to that used for cloning of pRSETC SLB was used in cloning pRSETC SLZ33.

[00266] Table 1

Z33 anti 169 22 of Z33 domain to be cloned at the HI loop

CP sen 170 27 PCR amplification of SLB from

CP anti 171 30

pRSETC SLB His for creating pRSETC SLB

[00267] All the clones were expressed in E. coli Rosetta cells. VLPs were purified by sucrose density gradient ultracentrifugation as described elsewhere. Briefly, Rosetta cells transformed with pRSETC CP, pRSETC SLB and pRSETC SLZ33 were grown in 500 ml LB broth at 37 °C till O.D.600 reached 0.4 and induced overnight at 16 °C with 0.3 mM IPTG. Cells were harvested and resuspended in ice cold 50 mMTris pH 7.5 containing 0.1 % Triton X-100. After sonication, the insoluble pellet was discarded after centrifugation of the suspension at 13,000 rpm, 30 min, 4 °C. The supernatant was ultrapelleted at 26,000 rpm, 3 hr, 4 °C using SW32 rotor (Beckman Coulter). The ultrapellet was resuspended in 2 ml of 50 mMTrisHCl pH 7.5 by keeping on end-to- end rotor, overnight at 4 °C. The supernatant obtained after low speed centrifugation was layered on to 10-40% sucrose density gradient and centrifuged at 26,000 rpm for 3 h at 4 °C. 1.5 ml fractions were collected from the bottom of the tube and 20 μΐ of each fraction was subjected to SDS PAGE analysis. The peak fractions were collected and once again centrifuged at 26000 rpm for 3 hr at 4 °C. The ultrapellet was dissolved in 50 mM TrisHCl pH 7.5, 5 % glycerol. Western blot analysis was done using CP specific and DAPAL non-specific antibodies (polyclonal antibodies raised against Diaminopropionate ammonia lyase (DAPAL)). While anti-CP antibodies can bind to all the chimera and SeMV CP VLPs, DAPAL antibodies can bind only to those with a functional B/Z33 domain. For ease of representation SeMV CP VLPs and SLB VLPs will be referred to as CP and SLB respectively.

[00268] Transmission Electron Microscopy: 0.2 mg/ml protein was adsorbed on formvar coated copper grids (SPI Supplies, USA, Code:3440C-MB) for 2 min followed by wash in filter sterile 50 mM Tris-HCl buffer pH 7.5 for 30 sec. The grids were finally stained with 1 % uranyl acetate for 1 min followed by buffer wash and then air dried for 4 hr. The grids were viewed in Tecnai G2 Spirit (Biotwin FEI, USA) at 120 kx. The diameters of 50 particles were measured and analyzed using ImageJ software.

[00269] Enzyme Linked Immunosorbent Assay: In order to analyze if the chimeric VLPs have a functional B/Z33 domain, direct antigen coating (DAC) ELISA was performed using VLPs as antigen and non-specific DAPAL antibody sera as primary antibody. Briefly, varying concentration of VLPs (1-10000 nM (assuming 180 subunits)) were coated on nunc immune modules and incubated at 4 °C overnight. 10 mM phosphate buffered saline pH 7.5 (1 X PBS -137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂HP0 ₄, 2 mM KH ₂PO ₄) alone was used as negative control. Following three times wash with 1 X PBS, all wells were blocked with 5 % skimmed milk in 1 X PBS buffer for 2 hr at 37 °C. Henceforth, three times wash with 1 X PBST (1 X PBS containing 0.05 % Tween-20) and 1 X PBS was followed after each step. DAPAL antibody (1:3000) was incubated for 1 hr. For detection, 1:7500 dilution of goat anti rabbit IgG HRP conjugate was used. The A450 was monitored and the reaction was stopped after significant colour development using 100 μΐ 2 N H2S04. All readings were taken in microplate reader (Tecan infinite M200 pro) at 450 nm. The data obtained were plotted and analyzed using GraphPad Prism software. In order to analyze IgG binding efficiency, similar DAC ELISA was performed using SpA (Protein A) as positive control. [00270] Mammalian cells and their maintenance: The different kinds of mammalian cells used in this study include Human colon cancer cells (HeLa), Keratinized HeLa (KB) (kind gift from Prof. Anjali Karande, IISc, India), Mus musculus melanoma (B16-F10) (kind gift from Prof. P. N. Rangarajan, IISc, India) and human breast carcinoma-derived cells (BT-474, ATCC No.HTB-20). Cancerous breast epithelial cells (CB) 704 and primary cultures of human mammary epithelial cells 704 (HMECs) were obtained from Prof Annapoorni Rangarajan, IISc, India. All cells were maintained in DMEM media, supplemented with 10 % FBS, 100 I.U./ml penicillin, 100 I.U./ml streptomycin and 2 mM Glutamax at 37 °C in a humidified 5 % C02 incubator. For BT-474 cells, 1.2 g/L of sodium bicarbonate and 1 mM sodium pyruvate were supplemented to the medium. Cells were passaged every 3-4 days.

[00271] Alexa labeling of proteins: Labeling of proteins was carried out as per manufacturers' protocol [122]. Before labeling, all proteins were dialyzed against IX PBS buffer pH 7.5. The pH of 500 μΐ 2 mg/ml protein was adjusted to 8.5 using bicarbonate buffer pH 9.0. Each protein was incubated with 10 μΐ of 1 mg/ml Alexa 488 or Alexa 633 for 2 hr at room temperature and purified from free dye via gravity based gel permeation chromatography using PD 10 columns packed with Sephadex G25 column (Ge Healthcare) equilibrated with 1 X PBS pH 7.5. Twenty five 500 μΐ fractions were collected and the A494 or A633 were quantified using Nanodrop 2000 (Thermo Fischer, USA). A494 was used because Alexa Flour 488 dye shows maximum fluorescence when excited at 494 nm. Hereafter, labeled proteins will be represented with their name suffixed with the conjugated dye. The protein concentration was estimated by Bradford assay and the dye absorbance was calculated using Alexa Fluor 488 labeling kit (Invitrogen). Moles dye per mole protein = (A494)/(71,000 x protein concentration (M)), where 71,000 cm-1 M-l is the molar extinction coefficient of Alexa Flour 488.

[00272] Confocal microscopy: Briefly, 20,000 HeLa cells were adhered overnight on sterile coverslips and incubated with 0.79-7.9 nM CP 488 for 2 hr in fresh DMEM media containing 10 % FBS. Time course analysis was also done by incubating 1.58 nM CP 488 for 2, 4, 8 and 10 hr. The cells were washed twice with IX PBS pH 7.5, followed by fixation using 4 % paraformaldehyde for 10 min. The cells were thoroughly washed and permeabilized with 1 X PBS buffer containing 0.01 % saponin for 15 min and stained with 2.5 μg/μl DAPI (binds to AT region of DNA and emits blue fluorescence) for 10 min. The cells were finally washed and mounted on coverslides in presence of Fluorosheild. Images were acquired using Plan-Neofluar 100 x/1.3 oil objective of Zeiss 510 Meta microscope and analyzed using Zeiss LSM image browser version 4.2.0.121. In order to check the specificity of VLP entry, 10 nM of unlabeled CP was incubated along with 1.58 nM CP 488 for 2 hr. Also to check VLP entry in presence of non-specific proteins or serum, 1.58 nM CP 488 were added on HeLa cells that were pre -blocked with 10 μg BSA or 1 :50 dilution of sheep serum/rabbit serum for 1 hr. CP 488 entry at 4 °C was monitored by incubation of adhered HeLa cells with 1.58 nM CP 488 at 4 °C for 2 hr. Chimeric SLB 488 were also incubated in HeLa cells in a similar manner. SLB 488 entry was also checked with KB, B16-F10, BT-474, CB 704 and HMECs 704 using the same protocol. The concentration of VLP and antibodies were fixed based on preliminary concentration variation experiments.

[00273] Antibody internalization was monitored with pre -incubation of 1.58 nM SLB 488 with 6.6 nM D6F10 633 for 1 hr followed by addition onto HeLa cells adhered on sterile coverslips. The colocalization efficiency was calculated using Zeiss LSM image browser software. As positive control, abrin 488 internalized cells were fixed, permeabilized, blocked with 50 mM PBS pH 7.5 buffer containing 3 % BSA and probed with 6.6 nM D6F10 633 in 50 mM PBS pH 7.5 containing 0.1 % BSA. Antibodies alone were used as negative control. Cells were washed, fixed and stained with DAPI as mentioned earlier. For checking abrin and abrin-D6F10 complex entry, 1.5 nM abrin 488 alone and complexed with 6.6 nM D6F10 633 was used. Delivery of D6F10 633 by SLB 488 was also checked in KB and B16-F10 cells in a similar manner. [00274] Similar protocol was followed for examining FITC labeled monoclonal anti- a-tubulin antibody delivery using SLB in HeLa cells. Fixed cells were permeabilized and probed with the antibody to demonstrate the specificity of the an ti -tubulin antibody. Antibody alone was used as negative control. The antibodies were diluted from the stock (1.5 mg/ml) and the concentration of each antibody used is represented as final dilution added on to cells. 1.58 nM SLB mediated tubulin antibody (1:200) delivery was also monitored at varying time points (2, 4, 6 hr) and varying concentration (1:200, 1: 100, 1 :50).

[00275] Herclon internalization was checked in a similar manner on adhered BT-474 cells usingl.58 nM SLB 488 preincubated with 46 nM Herclon 633 for varying time points (1-3 hr) in presence of assay media (DMEM/F12 with 2 mM glutamine, 1.2 g/L of sodium bicarbonate, 3.15 g/L of glucose, 15 mM HEPES, 1 mM sodium pyruvate, and 2% FBS). CP-Herclon (1.58 nM, 46 nM) and Herclon alone (46 nM) were used as controls. The expression of HER2 receptor was checked by immunostaining of adhered and fixed BT-474 cells with 46 nM Herclon 633 for 1 hr. BT-474 cells after each treatment were processed in a similar manner as mentioned above.

[00276] Fluorescence-activated cell sorting (FACS) analysis: For checking of VLP binding to HeLa cells, varying concentration 0.18-3 nM CP 488 and SLB 488 were incubated separately for 1 hr with 0.1 million HeLa cells at 4 °C, followed by centrifugation at 300xg, 4 °C and washed twice with ice cold 1 X PBS buffer pH 7.5. Ten μg unlabeled CP and SLB were used to compete out labeled CP and SLB respectively. Buffer treated cells were taken as control for all experiments. To examine antibody binding, varying dilutions of CP488/SLB 488:D6F10 633 (1 : 1, 1:5, 1: 10) were pre-incubated in ice for 1 hr, followed by incubation with 0.1 million HeLa cells for 1 hr at 4 °C. All data were acquired by BD FACS Verse (Alexa 488: Excitation - 488 nm, Emission - 527±16 nm; Alexa 633: Excitation - 649 nm, Emission - 660±5 nm) and analyzed using BD FACSuite software.

[00277] Immunoblot analysis of HeLa lysate: Since D6F10 was purified in large amount, immunoblot analysis of HeLa cells incubated with SLB-D6F10 complex was carried out. Briefly, 3 million HeLa cells were incubated with 250 μg CP and SLB individually or pre-incubated (1 hr) with 140 μg D6F10 for 5 hr at 37 °C, followed by three time wash with ice cold PBS buffer pH 7.5. The spent media was also collected for analysis. The cells were trypsinized, pelleted, washed and finally dissolved in 1 X PBS buffer, 5 % glycerol, 0.1 % Triton X-100, 1 mM PMSF and 10 μΐ of protease inhibitor cocktail. The cells were lysed by suspension using a syringe three times followed by incubation at 4°C for 5 min. The supernatant collected after centrifugation at 1000 x g for 5 min of the lysate was used for detection of nanocarrier and/or antibody. To detect CP and SLB, CP polyclonal antibody followed by goat anti rabbit HRP conjugate were used, while goat anti mouse IgG HRP conjugate was used to identify D6F10. β actin (detected using monoclonal anti β actin IgG HRP conjugate) was used as internal control. 250 μg SLB preincubated with 140 μg of D6F10 was also incubated on cells for 2, 4, 8 and 10 hr and processed in a similar manner as above.

[00278] Protein synthesis assay using tritiated Leucine: As mentioned in S. Bagaria et. al. 2013, 0.2 million HeLa cells were plated on a 24 well plate (Nunc cell culture plates) and allowed to adhere for overnight at 37 °C, 5 % C02. 10 ng/ml abrin and abrin pre-incubated with varying concentration of D6F10 (1:25, 1 :50) were used as positive controls. 1 μg SLB was pre incubated (1 hr) with varying concentration of D6F10 and internalized into HeLa cells for 2 hr. After washing with PBS, abrin treatment was done for 7 hr in all assays. The DMEM media was replaced with RPMI Leucine free media for 2 hr and pulse chased with 0.4 mCi of [3H] leucine for 2 hr. Overnight precipitation was carried in presence of 5 % TCA at 4 °C. The precipitate was washed once with 200 μΐ of 20 % ethanol, dried and dissolved in 1% SDS, 0.1 N NaOH and incubated with 4 ml scintillation liquid (Cocktail T, Spectrobiochem) for 5 hr. Radioactivity was measured using Beckman scintillation counter. To ensure that internalization of SLB or CP didn't affect protein synthesis, the respective proteins were used as control. Buffer treated cells are referred to as untreated sample. To ensure D6F10 entry via SLB alone, CP pre-incubated (1 hr) with D6F10 was also used as control. [00279] Cell cycle progression analysis: For cell progression, briefly, 60,000 cells were incubated with 10 nM of CP and SLB for 36 hr. In order to check whether SLB- D6F10 can rescue the effect of abrin induced apoptosis, HeLa cells were treated with 10 ng of abrin and abrin pre- incubated with D6F10 (Molar ratio=l: 10) for 36 hr. SLB (0.1 nM)-D6F10 (1.5 nM) (pre-incubated for 1 hr) (Molar ratio=l: 10) were incubated for 4 hr, washed with PBS twice, followed by abrin treatment for 36 hr. All cells were trypsinized, washed and then fixed using 1 ml 70% ethanol in -20 °C overnight. The cells were pelleted, washed thrice with ice cold PBS and treated with 10 μg/ml RNase (Sigma) for 30 min followed by 15 min incubation with 0.3 μg/μl Propidium Iodide (Sigma). For each reaction, 10,000 events for single cells were recorded in triplicates. All data were acquired by BD FACS Verse (Propidium iodide: Excitation -488 nm, Emission-586±21 nm) and analyzed by BD FACS Diva software.

[00280] Anti-proliferation/Cell viability assay: The biological activity of Herclon was tested using anti-proliferation assay by resazurin sodium staining method. Briefly, 0.1 million BT-474 cells/ml were adhered onto 96 well plates (Nunc immuno modules) in assay media (DMEM/F12 with 2 mM glutamine, 1.2 g/L of sodium bicarbonate, 3.15 g/L of glucose, 15 mM HEPES, 1 mM sodium pyruvate, and 2% FBS). BT-474 cells were treated with varying concentration of 100 μΐ Herclon (2.5 - 0.004 μg/ml) (diluted in assay medium) alone or preincubated with 0.166 nM SLB for 48 hr in a humidified incubator with 5% C02, followed by addition of 30 μΕ of pre-warmed resazurin sodium to each well. After 7 hr, the plate is cooled to room temperature and fluorescence was measured at an excitation wavelength of 530 nm and emission wavelength of 590 nm. The percentage of cell viability was calculated as the fraction of fluorescence (sample)/ fluorescence (untreated cells), multiplied by 100. Similar assay were performed with 0.166 nM CP, 0.166 nM SLB, Herclon (0.664 nM), SLB- Herclon (Molar ratio=l:4) and CP-Herclon (Molar ratio=:4) at 48 & 90 hr.

[00281] Construction of PVBV CP and chimeric PVBV BCP: For the construction of pRCP, PVBV CPgene was amplified from pUC CP (CP cloned in pUC 19 vector earlier) using forward primer containing Nhel site (SEQ ID NO: 181)and reverse primer containing BamHI site (SEQ ID NO: 182) primers.

[00282] Purified PCR product (Figure 35A) and pRSETC vector were double digested with Nhel and BamHI restriction enzymes. The digested PCR product was ligated into double digested pRSETC vector. The clones confirmed by restriction digestion (Figure 35B) and later by PCR using CP specific forward and reverse primer (Figure 35C) followed by DNA sequencing. The cloning of CP gene at Nhel and BamHI sites adds 36 extra nucleotides from the vector. Similarly, pRBCP was also constructed. B domain of SpA was cloned earlier at the Nhel and BamHI site of pRSETC (pRB). The amplified CP gene product obtained using Xhol forward and Kpnl reverse CP specific primers.

[00283] was first digested with Kpnl and Xhol and ligated with the double digested (with Xhol and Kpnl) pRB vector (Figure 35D). The positive clone was named as pRB CP having B domain at the N terminus of PVBV CP which was confirmed by PCR (Figure 35D) and restriction digestion (Figure 35E) followed by DNA sequencing. B- domain has 174 nucleotides and along with it, 36 nucleotides extra comes from the pRSETC vector. In figure 35 A, lanes 1-3 depict the amplified CP gene. In figure 35B, lanes 1, 2, 5, 8, and 9 are false positive clones, while lanes 3, 4, 6, and 7 demonstrate release of 1 kb CP gene, hence positive clones. In figure 35C, the various lanes (1-6) depict confirmation of the positive clones by PCR amplification (1 kbp) with gene specific primers. In figure 35D, lanes 1-3 are undigested plasmids, and lanes 4-7 are positive clones showing amplified PCR product corresponding to 1 kbp. Figure 35E depicts confirmation of clones by restriction digestion. Lanes 1-4 are false positive clones, while lane 5 is a positive BCP clone showing release of the CP gene.

[00284] Assembly of PVBV CP and chimeric BCP to form VLPs: Competent BL21 (DE3) pLysS cells were separately transformed with pRSETC CP and pRSETC BCP by heat shock method and protein expression was checked by SDS PAGE analysis of respective induced cultures. CP(32kDa) and BCP(42kDa) proteins were clearly observed in induced cultures as seen in figure 36A and B respectively. Virus like particles (VLPs) were purified by 10-40% sucrose density gradient ultracentrifugation as mentioned previously. The gradient profile indicates that recombinant CP and BCP are able to form higher oligomers as seen in figure 36C. CP and BCP were spread through out the gradient indicating heterogeneity in the higher oligomers.. The major peak of BCP was found in fractions 6-12 indicating that BCP indeed forms VLPs whereas CP was present predominantly in fraction 2-9 indicating that these VLPs might have a higher s value. In figure 36A (BL21 (DE3) pLysS cells harboring pRSETC CP), lanes 1, 3, and 5 are uninduced samples, while lanes 2, 4, and 6 are induced samples. In figure 36B, lanes 1, 3 are induced samples of pLysS cells harboring pRSETC BCP, while lanes 2, 4 are uninduced samples of pLysS cells. In figure 36C the top panel shows sucrose density purification profile of CP, while the bottom panel shows sucrose density purification profile of BCP on 12% SDS PAGE. Lane 1 is 40% sucrose, while lane 17 is 10% sucrose.

[00285] In order to confirm the VLP formation of these higher oligomers, purified proteins were analyzed by transmission electron microscopy. EM analysis of CP and BCP showed that they assembled into flexuous rods with an average length of 130nm and 180nm respectively as show in in figure 37A, and B respectively.

Example 2

Results

[00286] Cloning of CP with B and Z33 domain: For the construction of pRSETC SLB and pRSETC SLZ33 clones, site directed mutagenesis on pRSETC CP was carried out using CP sdm sense and antisense primers (Table 1). The mutant was confirmed by Afel restriction digestion (Fig 1A). Purified B PCR product (-174 bp) (Fig IB) and Z33 PCR product (-99 bp) (Fig 1C) were ligated to Afel digested pRSETC CP sdm separately to obtain pRSETC SLB His (SLB expressed along with hexa-histidine tag at the N-terminus) and pRSETC SLZ33 His (SLZ33 expressed along with hexa histidine tag at the N-terminus). The clones were confirmed by PCR using CP mid sense and B/Z33 antisense primers (Table 1) (Fig ID). Using these confirmed clones as template, the SLB/SLZ33 chimeric CP genes were PCR amplified using CP specific primers (Table 1) (-981 bp and -906 bp respectively) (Fig IE), digested with EcoRI and ligated onto Ndel end filled and EcoRI digested pRSETC vector to create pRSETC SLB and pRSETC SLZ33 lacking nucleotides coding for hexa-Histidine at their 5' end. All clones were confirmed by PCR amplification using CP sense and B/Z33 antisense primers (Table 1) (Fig IF) and also by DNA sequencing.

[00287] In fig 1A, Lane 1 -pRSETC CP sdm, Lane 2- pRSETC CP sdm after Afel digestion. In fig IB, PCR amplification of B domain gene segment {- 174 bp} (Lane 1, 2) and CP (~ 809 bp) (lane 3). In fig 1C, PCR amplification of Z33 DNA segment (lane 1) {- 99 bp}. In fig ID, Screening for pRSETC SLB His and pRSETC SLZ33 His clones by PCR using CP specific and B/Z33 antisense primers: pRSETC SLB his (lanes 1-7) (Lane 1 positive clone) and pRSETC SLZ33 his (lanes 8-13) (lanes 9, 10, 13 positive clones). Positive clones for pRSETC SLB his will yield -324 bp PCR product while that of pRSETC SLZ33 his will yield -250 bp PCR product. In fig IE, PCR amplification of CP using pRSETC SLB His as template and CP specific primers (lane 1 - 981 bp) and pRSETC SLZ33 His (lanes 4, 5; -906 bp) as template.in fig IF, Clone confirmation of pRSETC SLB and pRSETC SLZ33 using CP specific primers after ligation of PCR products shown in E) to pRSETCNdel end filled and EcoRI digested plasmid. Positive pRSETC SLB and pRSETC SLZ33 clones yield 981 bp (lane 1) and 906 bp product (lane 2, 3) respectively. Lane M-Gene 1 kb marker.

[00288] SLB and SLZ33 can self-assemble to form VLPs: For protein expression analysis, competent Rosetta cells were separately transformed with 20 ng pRSETC CP, pRSETC SLB and pRSETC SLZ33 by heat shock method and protein expression was checked by SDS PAGE analysis of respective induced cultures. CP (-30 kDa), SLB (-36 kDa) and SLZ33 (-33 kDa) were clearly observed in the induced lanes (Fig 2A). Virus like particles were purified by 10-40 % sucrose density gradient ultracentrifugation as mentioned in the methods section. The gradient profile indicated that like CP, SLB and SLZ33 were able to form higher oligomers. The major peak of SLB was found in fractions 7-10 (Fig 2.B middle row) and that of SLZ33 was found from 8th fraction onwards (Fig 2B last row). CP was present majorly in fractions 4-7 (Fig 2B first row). This indicates that SLB forms oligomers which were slightly larger than CP. In contrast, SLZ33 spread throughout the gradient from fraction 8, implying a heterogeneous population. For final pelleting of SLZ33, only 8-11 fractions were chosen. Converse to our expectation, covalent fusion of a smaller domain (33 aa) resulted in more heterogeneous population as compared to a 58 aa insertion. In order to check whether the higher oligomers were indeed VLPs, we analyzed purified proteins by Electron microscopy (EM). EM analysis of CP, SLB and SLZ33 (Fig 2C) showed that they assembled into VLPs with an average diameter of 37 nm for SLB VLPs and 30 nm for SLZ33 VLPs. CP VLPs showed an average diameter of 30 nm as reported earlier. As expected, the heterogeneity of SLZ33 VLPs was higher than that of SLB VLPs (Fig 2C). However, the SLB VLPs formed slightly heterogeneous VLPs unlike that reported in chimeric HBV containing B domain. Mutation of Serine 242 to Alanine did not affect the VLP formation by CP (data not shown). For ease of representation, CP VLPs and SLB VLPs will be henceforth represented as CP and SLB.

[00289] In fig 2A, Expression analysis of CP, SLB and SLZ33. Ul-uninduced, I- induced, M-unstained protein marker. Arrows indicate the position of induced protein. In fig 2B, Sucrose density gradient profile of CP (top), SLB (middle) and SLZ33 (bottom) from top (lane 1, 10 % sucrose) to bottom (lane 18, 40 % sucrose). In fig 2C, Transmission electron micrographs of CP, SLB and SLZ33. The table represents the average diameter of 50 capsids and its standard deviation (SD) analyzed using ImageJ software.

[00290] B/Z33 domain is functional in chimeric VLPs: Upon western analysis of purified VLPs using polyclonal antibodies to CP, it was observed that all three proteins bound to CP specific antibodies and gave a positive band (Fig 3A left blot). On the other hand, when non-specific DAPAL antibody was used, only SLB and SLZ33 showed positive signal (Fig 3A right blot) while CP did not show any corresponding band. These results demonstrate that B/Z33 domain inserted in HI loop of CP can recognize IgG molecules and are therefore functional. In order to examine if these domains were exposed in the assembled VLPs in solution, DAC ELISA was performed using DAPAL antibodies. For this purpose, the VLPs (CP, SLB and SLZ33) were coated at different concentrations (1-10000 nM) and tested for their ability to bind to IgG. As can be seen in Fig 3B, both the chimeric VLPs bound to nonspecific DAPAL antibody while CP showed no binding, confirming that both B and Z33 domains inserted in CP retain antibody binding ability after assembly into VLPs. Interestingly, SLB showed better binding as compared to SLZ33 indicating that the all the Z33 domains inserted in the HI loop of CP may not have the same conformation as the Z33 domain alone. Since SLB exhibited higher antibody affinity, it was used for all further studies. In comparison with SpA (Fig 3C), SLB (~Kd = 10 nM) bound to antibody -45 times better than SpA (~Kd = 457 nM) while CP showed no binding, clearly indicating the presence of multiple functional B domains in SLB in contrast with two IgG binding domains of SpA dimer. The low Kd of SpA (standard being 5-10 nM) could be due to use of polyclonal sera for ELISA. Further work using surface plasmon resonance with purified IgGs is in progress. Such high antibody affinity elevates the potential for use of these chimeric VLPs as an alternative for SpA or B/Z33 fused proteins.

[00291] In fig 3A, Western blot analysis of CP, SLZ33, SLB using CP polyclonal antibody (left blot) and DAPAL polyclonal antibody (right blot). In fig 3B, DAC ELISA using CP, SLZ33 and SLB as antigen and DAPAL as primary antibody. In fig 3C, DAC ELISA using CP, SLB and SpA as antigen and DAPAL as primary antibody.

[00292] Fluorescent labeling does not alter structural integrity of VLPs: CP and SLB were labeled with Alexa Fluor 488 as per manufacturer's protocol and the absorbance A494 of the fractions collected after sephadex G-25 column chromatography is shown in Fig 4A. Maximum labeling of CP 488 (0.33 mg/ml) showed fluorescence equivalent to 30.5 μΜ Alexa 488 and SLB 488 (0.98 mg/ml) showed fluorescence equivalent to 72.3 μΜ Alexa 488. Such high labeling efficiency (on an average ~> 85 %) is an indication of efficient labeling of exposed lysines and imply that the labeled VLPs could serve as good imaging agents. EM and western analysis confirmed that the structural integrity and antibody binding ability of the SLB are not altered by addition of Alexa Fluor 488 (Fig 4B & C). Similarly, the antibodies (D6F10 and Herclon) were labeled with Alexa Fluor 633 (data not shown).

[00293] In fig 4A, Elution profile of CP 488 and SLB 488 after labeling with Alexa Fluor 488. Absorbance at 280 and 494 of the respective proteins are represented. In fig4B, Electron micrographs of CP 488 and SLB 488. The table represents the average diameter of 50 capsids and its standard deviation analyzed using ImageJ software. In fig 4C, Western blot analysis of CP, CP 488, SLB and SLB 488 using CP polyclonal antibody (left blot) and DAPAL polyclonal antibody (right blot).

[00294] Demonstration of entry of VLPs into mammalian cells: VLP entry into mammalian cells was examined by incubating Alexa Fluor 488 labeled VLPs with HeLa cells at 37 °C, followed by confocal microscopy. The intensity of alexa 488 fluorescence in HeLa cells directly co-related with increase in concentration and time (Fig 5A & B). No VLP entry was detected at a 30 min time point (data not shown). CP is found in the cytoplasm of HeLa cells and does not enter nucleus, like other known plant virus nanoparticles. It can be noted that at higher concentrations, an aggregation pattern is observed (Fig 5A, last panel), probably due to CP self-interaction. After 8 hr, the fluorescence of the particles decreased indicating possible degradation of VLPs.

[00295] In fig 5A, the images of HeLa cells incubated with varying concentrations of CP 488 (0.79-7.9 nM) for 2 hr at 37 °C followed by fixing, permeabilization and DAPI staining. The images were taken as described in the methods section. In fig 5B, The images of HeLa cells treated with 1.58 nM CP 488 for 2, 4, 8, 10 hr at 37 °C followed by processing of cells as mentioned in A). Green=CP 488, Blue=DAPI stained nucleus.

[00296] When 10 nM unlabeled CP is incubated along with 1.58 nM CP 488 before internalization, there was a decrease in green fluorescence (Fig 6B) as compared to the fluorescence observed with 1.58 nM CP 488 (Fig 6A). This indicates the specificity of the internalization of CP, as the unlabeled CP competed out CP 488. VLP entry was also observed when cells were pre-incubated with non-specific protein BSA (Fig 6C) and sheep serum (Fig 6D), indicating entry of these VLPs even in presence of other non-specific proteins or serum. Similar results were obtained when HeLa cells were incubated with CP in presence of rabbit serum (data not shown). Cytoplasmic distribution observed at 37 °C (Fig 6A) was reduced to a large extent to surface binding at 4 °C (Fig 6E). Thus for the first time, it is shown that SeMV CP and its chimeric SLB can enter HeLa cells.

[00297] In Fig 6A and B, confocal images of HeLa cells treated with 1.58 nM CP 488 for 2 hr at 37 °C in the absence (A) and in the presence of 10 nM CP (unlabeled) (B). fig 6C and D represent confocal images of cells incubated with 1.58 nM CP 488 in presence of 0.1 mg/ml BSA (C) and sheep serum (1:50) (D) respectively. E) Images of cells incubated with 1.58 nM CP 488 at 4 °C for 2 hr. Green=CP 488, Blue=DAPI stained nucleus.

[00298] Interestingly, SLB 488 (Fig 7A) also showed an internalization profile similar to that observed with CP 488 at varying time points. Though 7 nm wider than CP (Fig 7B & C), SLB can also enter HeLa cells. Furthermore, SLB entry was also observed in KB, B16-F10, BT-474, CB 704 and most importantly, HMECs 704 (primary breast cancer cells), demonstrating lack of cell target specificity (Fig 7B) with respect to entry of the VLPs into cells. CP also showed as similar profile of cellular entry in the above mentioned cells like SLB (data not shown).The versatility of cellular entry is a striking feature of CP and SLB.

[00299] In fig 7A, confocal images of HeLa cells treated with 1.58 nM SLB 488 for 2, 4, 8, 10 hr followed by fixing, permeabilization and DAPI staining. In fig 7B, Confocal images showing the entry of SLB 488 in KB, B16-F10, BT-474, CB 704 and HMECs 704 cells.

[00300] In a similar fashion, entry of PVBV VLPs, and chimeric VLPs was also checked in different cancerous cell lines such as Hela, Hep G2, MDA-MB 231, and BT-474 cells. Entry was also checked in normal cells. Figure 41A-F kinetics of entry of PVB VCPVLPs at a concentration of 1.73 nM into Hela cells at various time points (panel A-30 minutes; panel B-l hour; panel C-2hours; panel D-3 hours; panel D-4 hours; and panel E-6hours). Figure 42A-F shows the kinetics of entry of PVBV BCPVLPs at a concentration of 1 nM into Hela cells at various time points (panel A- 30 minutes; panel B-l hour; panel C-2hours; panel D-3 hours; panel D-4 hours; and panel E-6hours). Figure 43 panel A depicts the entry of PVBV VLPs into HepG2 cells at 1 hour. Panel B shows entry of PVBVBCP VLPs into HepG2 cells at 1 hour. Panel C shows entry of PVBV VLPs into MDA MB -231 cells at 1 hour. Panel D shows entry of PVBVBCP VLPs into MDA-MB-231 cells at 1 hour. Panel E shows entry of PVBV VLPs into BT474 cells at 1 hour, while panel F shows entry of PVBVBCP VLPs into BT474 cells at 1 hour. In Figure 43, and 44, the concentrations of VLPs is as per Figure 42. Figure 44 depicts the specificity of internalization of VLP and chimeric VLP into normal mammalian cells. Panel A shows the entry of PVBV VLPs into 3T3 cells, while panel B shows the entry of PVBVBCP VLPs into 3T3 cells. In each of the figures above, green signal is Alexa 488 conjugated goat anti-rabbit secondary antibody, while blue is DAPI stained nucleus.

[00301] Antibody delivery by SLB in mammalian cells: Three different monoclonal antibodies - D6F10 (anti-abrin), anti-a-tubulin and Herclon (humanized antibodies against HER2 receptor) were used as cargo for delivery using SLB.

[00302] D6F10 delivery by SLB onfocal microscopy of SLB mediated delivery of D6F10:The ability of D6F10 to bind to abrin was tested by incubating HeLa cells with 1.5 nMabrin 488 internalized for 2 hr, followed by fixing, permeabilizing, blocking with 1 X PBS 7.5 containing 3 % BSA and probing with 6.6 nM D6F10 633 in 50 mM PBS pH 7.5 buffer containing 0.1 % BSA. It is clear from the yellow color seen upon merging the abrin 488 (green fluorescence) with D6F10 633 (red fluorescence) that D6F10 633 binds to intracellular abrin 488 (Fig 8A) confirming specificity of D6F10 binding to abrin epitope. To check whether D6F10 can internalize by itself or via preincubation with abrin, HeLa cells were treated with 6.6 nM D6F10 633 alone or pre- incubated with 1.5 nMabrin and the images were taken after fixing, permeabilizing and staining with DAPI. It was seen that D6F10 cannot enter cells by itself (Fig 8B) but can enter via its binding to abrin with 85 % co-localization seen as yellow color in the merged image (Fig 8C).

[00303] Fig 8A, The ability of D6F10 to bind to abrin was tested by incubating HeLa cells with 1.5 nM abrin 488 internalized for 2 hr, followed by fixing, permeabilizing, blocking with 1 X PBS 7.5, 3 % BSA and probing with 6.6 nM D6F10 633 in 50 mM PBS pH 7.5, 0.1 % BSA buffer. To check whether D6F10 can internalize by itself or via pre-incubation with abrin, HeLa cells were treated with 6.6 nM D6F10 633 alone B) or pre-incubated with 1.5 nM abrin 488 C) and the images were taken after fixing, permeabilizing and staining with DAPI. Green =Abrin 488 (first column), red=D6F10 633 (second column), blue=DAPI stained nuclei (third column) and merge of first three columns is shown in the last column.

[00304] In order to test if SLB, which can enter HeLa cells, also can deliver D6F10 like abrin, similar assay was done by pre-incubating D6F10 with SLB. D6F10 cannot enter cells by itself as no red fluorescence was observed in Fig 9A. Nanocarrier- antibody complex was made by 1 hr pre-incubation of 1.58 nM SLB 488 with 6.6 nM of D6F10 633 and allowed to enter HeLa cells (Fig 9B). The images obtained indicate that D6F10 was successfully internalized by SLB as compared to the D6F10 alone (Fig 9B & A). Similar incubation with CP failed to do so as shown by the absence of yellow color due to lack of entry of D6F10 633 (Fig 9C), confirming the ability of SLB to deliver the antibodies and not CP.

[00305] Kinetics of D6F10 entry when bound with SLB showed maximum internalization of D6F10 between 4-8 hr, after which SLB started to degrade (Fig 10). SLB was completely degraded by 12 hr as the green fluorescence due to SLB 488 could not be observed at this time point (Fig 10 bottom row). Further, the intensity of red fluorescence was also slightly reduced, also indicating degradation of D6F10.

[00306] In Fig 10, SLB 488 pre-incubated with D6F10 633 was incubated with adhered HeLa cells for 2, 4, 8 and 12 hr and processed for confocal microscopy as mentioned in methods section. Green = SLB 488 (first column), red = D6F10 633 (second column), blue = DAPI stained nuclei (third column) and merge of first three columns is shown in the last column.

[00307] Once bound, D6F10 was not displaced when incubated with sheep serum for 3 hr as confirmed by the red fluorescence in Fig 11 A & B. Similar results were obtained when the same experiment was performed in presence of rabbit polyclonal serum (data not shown). This implies that the nanocarrier antibody complex is highly stable and the antibodies were not displaced in presence of other IgGs in the serum. The ability of SLB to deliver antibodies into cells was also examined by using other cell lines such as KB and B16-F10 (Fig 12A & B). In both these cases, the antibodies were indeed delivered, although not as efficiently as in HeLa cells, in the case of B16-F10 cells. However, the SLB entry is also slightly reduced as compared to KB or HeLa cells.

[00308] In Fig 11, A) SLB 488 pre-incubated with D6F10 633 was incubated on to HeLa cells in the absence A) and in the presence B) of sheep serum (1:50). Green = SLB 488 (first column), red = D6F10 633 (second column), blue = DAPI stained nuclei (third column) and merge of first three columns is shown in the last column.

[00309] In Fig 12, A) Images of KB cells treated with SLB 488 pre-incubated with D6F10 633 and processed as described earlier. B) Images of B16-F10 cells treated with SLB-D6F10 for delivery of the antibody into the cells. Green = SLB 488 (first column), red = D6F10 633 (second column), blue = DAPI stained nuclei (third column) and merge of first three columns is shown in the last column.

[00310] FACS analysis: The proportion of VLP and antibody bound to HeLa cells were quantified by FACS analysis. Firstly, varying concentration of CP 488 and SLB 488 binding to HeLa cells were quantified (Fig 13A, B). Black profile represents untreated cells and different colored profile represents the varying concentrations used. A right shift indicates increase in the fluorescence of the respective samples. CP 488 and SLB 488 bound equally well to -85 % of the HeLa cells. ΙΟηΜ unlabeled VLPs were used as competitor to respective labeled VLPs. A leftward shift (dark green profile in Fig 13A and light green profile in Fig 13 B) indicates lesser binding of CP 488 or SLB 488 as compared to respective controls (navy blue profile in Fig 13A &B). The results summarized in the bar diagram (Fig 13C), shows a dose dependent VLP binding to HeLa cell surface, which was reduced in presence of unlabeled VLPs (last bar), indicating specific binding of CP or SLB.

[00311] In Fig 13, Varying molar concentration of CP 488 A) and SLB 488 B) were incubated with 0.1 million HeLa cells for 1 hr at 4 °C. To check the specificity 3nM CP 488 / SLB 488 were incubated with 10 nM unlabeled CP/SLB. Untreated cells are represented in black profile and the different coloured profile indicates different concentration of CP 488 or SLB 488 as shown in the inset. The graph C) represents the mean fluorescence of cells in three independent experiments.

[00312] Similar binding assay with pre-incubated 1.58nM CP 488 or SLB 488 with varying concentration of D6F10 633 (Fig 14A & B) was performed and the Alexa 633 fluorescence was quantified. Fig 14A clearly shows a dose dependent increase in the D6F10 binding when pre-incubated with SLB. However, when incubated with CP 488 (Fig 2.22 B), no rightward shift was observed indicating lack of D6F10 binding to HeLa cells when pre-incubated with CP. D6F10 633 alone (dark green, light green and orange profile in Fig 14A & B) showed similar profile as untreated cells (black profile), confirming that D6F10 cannot bind to cells by itself. All the results are summarized in bar diagram (Fig 14C). It can be clearly seen that the when HeLa cells were incubated with CP 488 and SLB 488 pre-incubated with varying concentration of D6F10, there was enhanced 488 fluorescence, indicating VLP binding to cells but 633 fluorescence was observed only when D6F10 was incubated with SLB. Thus, in confirmation with the confocal experiments, D6F10 was able to bind to HeLa cells using SLB as a nanocarrier.

[00313] In Fig 14, A) FACS profile of D6F10 633 (DF) when preincubated with varying concentration of SLB 488 (SLB). B) FACS profile of D6F10 633 (DF) when preincubated with varying concentration of CP 488 (CP). C) Bar diagram of two independent experiments is represented with Mean 488 and 633 fluorescence of CP- DF (D6F10 633 preincubated with CP 488) and SLB-DF (D6F10 633 preincubated with SLB 488) analyzed by FACS. [00314] Immunoblot analysis of HeLa cell lysates: VLP and antibody internalization was also confirmed by immunoblot analysis of HeLa cell lysates. CP (250 μg), CP- D6F10 (Molar ratio=l:20), SLB (250 μg), SLB-D6F10 (1 :20) and D6F10 (140 μg) were incubated on 3 million HeLa cells for 5 hr at 37 °C. The whole cell lysate was prepared as mentioned in the methods section and analyzed by 12 % SDS-PAGE followed by western analysis with CP polyclonal antibody (Fig 15 row I), anti mouse IgG HRP conjugate (Fig 15 row II) and anti β actin IgG HRP conjugate (Fig 15 row III) (loading control). CP polyclonal antibodies can detect CP and SLB, goat anti- mouse IgG for detection of D6F10 antibody and anti β actin IgG HRP conjugate for detecting β actin. Recombinant CP (Fig 15 A. lane 1), SLB (Fig 15 A. lane 2) and D6F10 (Fig 15A, lane 3) were used as positive controls. Interestingly, CP (Fig 15A lanes 4, 5) and SLB were degraded once inside the cells (Fig 15 A. row I lanes 6, 7) while D6F10 remained intact (Fig 15 row II, lane 7). D6F10 cannot by itself enter cells (Fig 15 A, row II lane 8) nor when preincubated with CP (Fig 15A row II, lane 5). β actin was taken as internal control (Fig 15 A, row III). Varying time points also confirmed VLP entry and enhanced degradation (Fig 15B row I) at later time points, while D6F10 remained intact till 10 hr (Fig. 15B row II).

[00315] Functional analysis of antibody delivery: Function of the internalized antibodies was tested based on the effect each antibody has within cells. D6F10 is known to neutralize abrin mediated protein synthesis inhibition as well as apoptosis. To check the effect of the delivered antibody on the inhibition of protein synthesis caused by abrin, tritiated leucine based protein synthesis assay was performed as described previously in the presence of CP, SLB, abrin and abrin-D6F10, SLB- D6F10 and CP-D6F10 (Fig 16A). Untreated cells were taken as control (Fig 16A bar 1). Total cpm indicated that entry of CP and SLB alone have no effect on protein synthesis (Fig 16A, bars 2, 3) while 0.16 nMabrin (Fig 16A, bar 4) resulted in 75 % protein synthesis inhibition. Abrin pre incubated with varying concentration of D6F10 (Fig 16A, bars 5, 6) lead to 2 fold rescue, which was elevated to 3.5 fold when D6F10 was delivered by SLB nanocarriers (Fig 16A, bar 7, 8). As expected, no rescue of protein synthesis inhibition was observed when cells were treated with D6F10 pre- incubated with CP (Fig 16A, bar 9), confirming that the enhanced protein synthesis is due to the antibody delivered by chimeric SLB and not CP. The increase in the rescue of protein synthesis inhibition caused by abrin when the antibody was delivered by SLB shows that nanocarrier mediated delivery is more effective as compared to antibody pre bound with toxin. Apoptosis of cells treated in a similar manner was scored using PI staining method. Dead cells will be stained less by propidium iodide while maximum staining will be of cells in the G2/M phase (cell division phase). Untreated cells were taken as control. The percentage of dead population (Y axis) is plotted with respect to the different treatment (X axis) as shown in bar diagram (Fig 16B). 10 nM CP and SLB (Fig 16B, bar 1-3) incubation with the HeLa cells for 36 hr did not result in any apparent change in the dead population as compared to untreated cells, indicating that CP and SLB do not cause any adverse cytotoxicity. Abrin caused ~ 54 % apoptosis (Fig 16B, bar 4) when treated for 36 hr, while abrin-D6F10 (Molar ratio=l: 10) caused 18 % apoptosis (Fig 16B, bar 5). When D6F10 was delivered by SLB for 4 hr, prior to abrin treatment for 36 hr, the percentage of dead cells dropped to 10 % (Fig 16B, bar 6), indicating that D6F10 delivered by SLB was functional and holds better potential in preventing abrin mediated cytotoxicity than abrin pre- incubated with D6F10. Thus SLB mediated delivery holds promise for using such a nanocarrier mediated neutralizing antibody delivery prior to toxin exposure in general.

[00316] Anti-tubulin antibody delivery by SLB: Fixed HeLa cells when immune- probed with FITC labeled anti a-tubulin antibody showed a tubular network (Fig 17A), indicating that antibody is functional. Antibody alone is unable to cross membrane barrier (Fig 17B). To test the ability of SLB to deliver FITC labeled tubulin antibodies, unlabeled SLB (1.58 nM) preincubated with FITC labeled tubulin antibodies for 1 hr, were then incubated with HeLa cells for 2 hr and the confocal images were captured. As evident from Fig 17C, a cytoplasmic appearance of tubulin antibodies were observed instead of a network arrangement (Fig 17A), confirming the entry of the antibodies and disruption of the network by their interaction with tubulin. At 1:50 dilution of antibody, aggregates similar to that reported previously elsewhere were observed (Fig 17D).

[00317] Time course of SLB mediated anti-tubulin antibody delivery in HeLa cells (Fig 18E) also showed an aggregation pattern after 4 hr (Fig 2.26 middle row). Large spherical aggregates were observed at 6 hr time point (Fig 181ast row), indicating extensive tubulin depolymerization. Similar results were observed for varying concentration of anti-tubulin antibody delivery via fixed concentration of SLB. These results confirm that not only were the antibodies delivered within the cells but also were able to interact with intracellular tubulin monomers and sequestering them, thereby preventing further polymerization. The aggregation pattern observed also implies that although antibodies are complexed with SLB before entry, they are still able to interact with their specific antigen via the Fab portion of IgGs and co-localize with the antigen.

[00318] Herclon delivery by SLB: In order to explore the possibility of using SLB for delivery of therapeutic antibodies, the delivery of Herclon, a humanized anti HER2 (a tyrosine kinase receptor over- expressed in breast cancer cells) monoclonal antibody used for effective treatment for certain breast cancer patients, was used. Herclon was conjugated with Alexa Fluor 633 (Herclon 633) as described in materials and methods section and antibody delivery into BT-474 cells was examined by confocal microscopy. To detect total HER2 expression, fixed cells were permeabilized and later probed with Herclon 633 (Fig 19). Herclon bound to surface expressed HER2 receptor as well as intracellular HER2 protein. Red= Herclon 633, blue= DAPI stained nuclei, merge=combination of both the fluorophores and enlarged= enlarged version of merged image. The white arrow indicates enhanced surface binding of Herclon.

[00319] When Herclon 633 alone was incubated with BT-474 cells for 1 hr, red fluorescence was majorly observed on the cell surface, indicating binding to cell surface expressed HER2 receptor (Fig 20A). Preincubation of Herclon 633 (46 nM) with 1.58 nM SLB 488 for 1 hr followed by incubation on BT-474 for 1 hr, showed that Herclon 633 was internalized along with SLB 488 (Fig 20B) with 39 % co-localization efficiency. This localization was minimal when CP 488 and Herclon 633 were preincubated and added onto BT-474 cells (Fig 20C) and the co-localization efficiency reduced to 12 %. This again confirms that only SLB (because of the presence of functional B domain) is able to internalize Herclon in BT-474 cells while CP failed to do so.

[00320] In Fig 20, A) Confocal images showing surface binding to Herclon 633 (46 nM) when incubated with adhered BT-474 cells for 1 hr. Confocal images of B) SLB 488-Herclon 633 and C) CP 488-Herclon 633 preincubated for 1 hr followed by addition onto adhered BT-474 cells for 1 hr. The cells were processed as mentioned in the methods section. White arrow represents enhanced cytoplasmic localization of SLB 488 and Herclon 633 in B) and cytoplasmic localization of CP 488 and membrane localization of Herclon 633 in C). Green=SLB 488/CP 488, red= Herclon 633, blue= DAPI stained nuclei, merge=combination of all three fluorophores and enlarged= enlarged version of merged image.

[00321] When Herclon 633 alone was incubated with BT-474 cells for 1 hr, red fluorescence was majorly observed on the cell surface, indicating binding to cell surface expressed HER2 receptor (Fig 20A). Preincubation of Herclon 633 (46 nM) with 1.58 nM SLB 488 for 1 hr followed by incubation on BT-474 for 1 hr, showed that Herclon 633 was internalized along with SLB 488 (Fig 20B) with 39 % co-localization efficiency. This localization was minimal when CP 488 and Herclon633 were preincubated and added onto BT-474 cells (Fig 20C) and the co- localization efficiency reduced to 12 %. This again confirms that only SLB (because of the presence of functional B domain) is able to internalize Herclon in BT-474 cells while CP failed to do so.

[00322] Kinetics of Herclon internalization in the presence (Fig 21) and absence of SLB (Fig 22) shows time dependent increase in Herclon within BT-474 cells when delivered by SLB. Both SLB 488 and Herclon 633 (green and red fluorescence respectively) was enhanced in the cytoplasm of BT-474 cells (Fig 21) with increase in time of incubation, although Herclon 633 alone did not show much internalization (Fig 22). At a 3 hr time point, Herclon by itself showed slight internalization (Fig 22 bottom row), however, the effective concentration was low as compared to SLB mediated delivery (Fig 21 bottom row). This is in conformity with earlier report of Herclon binding profiles when complexed with gold nanoparticles. Thus SLB can effectively deliver Herclon within BT-474 cells. It is also clear that both the nanocarrier and the antibody do not enter the nucleus, perhaps due to the size of the nuclear pores.

[00323] In Fig 21, Confocal microscopic images of time course of 46 nM Herclon 633 entry when preincubated with 1.58 nM SLB for 1 hr (top row), 2 hr (middle row) and 3 hr (last row) as mentioned in the methods section. The different columns represent the fluorescence label used (mentioned at the top of the column). The colours and white arrows represented are similar to Fig 20.

[00324] In Fig 22, confocal images of time course of 46 nM Herclon binding to BT- 474 cells for 1 hr (top row), 2 hr (middle row) and 3 hr (bottom row). The cells were processed as mentioned in the methods section and observed under Plan-Neofluar 100 x/1.3 oil objective of Zeiss 510 Meta microscope. The colours are similar to those shown in Fig 20.

Cell viability assay: It was hypothesized that Herclon delivered by SLB can heighten Herclon cytotoxicity. BT-474 cells were treated with varying concentration of Herclon alone or pre-incubated with 0.166 nM SLB for 48 hr. As expected, decreasing concentration of Herclon showed decrease in cell toxicity (Fig 23 A, bar 1). The maximum cytotoxicity (25 %) was observed at highest concentration of antibody (2.5 ng/ μΐ, 1: 100). Interestingly, SLB mediated delivery of the same concentration of Herclon, showed enhanced cytotoxicity till Herclon concentration was 0.02 ng/μΐ (1:0.8) (Fig 23 A, bar 2). Herclon cytotoxicity was enhanced to 83 % at highest antibody concentration when delivered by SLB and was also effective even at lower concentrations where there was minimal effect by Herclon alone. CP and SLB by themselves had minimum effect on cell viability (Fig 23B, bars 1, 2, 6, 7) at 48 and 90 hr. Antibody (0.1 ng/ μΐ) pre-incubated with CP (Fig 23B, bars 5, 10) showed similar cell viability as antibody alone control at both time points (Fig 23B, bars 3, 8). Interestingly, at 90 hr time point, the cytotoxic effect of Herclon delivered via SLB (Fig 23B, bar 9) was decreased (20 %) as compared to antibody alone control (88 %) (Fig 23B, bar 8). This would be of prime importance in an in vivo scenario where Herclon will be cleared via body fluid clearance and the amount of Herclon that can effectively act on cells becomes crucial. SLB can effectively improve the cytotoxic effects of Herclon at a lower concentration as well as lower time point, highlighting its potential for delivering therapeutic antibodies.

[00325] In Fig 23, A) Cell viability assay of BT-474 cells incubated with Herclon or SLB-Herclon for 48 hr. B) Cell viability assay of BT-474 cells incubated with 0.166 nM CP (bars 1, 6), 0.166 nM SLB (bars 2, 7), Herclon (0.1 ng/ml) (bars 3, 8), SLB- Herclon (0.166 nM-0.664 nM,l :4) (bars 4, 9)and CP-Herclon (1 :4) (bars 5, 10) for 48 and 90 hr.

Example 3

Cloning and expression of B-domain fused at the N and C terminus of NA65CP

[00326] B-domain of protein A, (a helical polypeptide of 58 aa) was fused covalently to the N and C termini of ΝΔ65 CP (SeMV CP in which N-terminal 65 amino acid) using recombinant DNA technology. Briefly, B-domain was amplified from the Staphylococcus aureus COL strain genomic DNA using the Nhe I restriction enzyme contained in the sense primer (SEQ ID NO: 172), and BamH I site contained in the anti-sense primer (SEQ ID NO: 173). The amplified product was double digested with Nhe I and BamH I and subsequently ligated in the double digested (with Nhe I and BamH I) linear pRSETA vector. This clone was named pRB. This strategy adds 36 nucleotides from the vector and 174 nucleotides corresponding to the B domain. As seen in Figure 24, lane 1 is the DNA ladder, lane 2 is the template control, while lane 3 shows the expected 174 bp PCR amplicon.

[00327] Simultaneously, Sesbania Mosaic Virus Coat protein (CP) and ΝΔ65 CP DNA was amplified using Xhol containing sense primer (SEQ ID NO: 174) and Bglll containing antisense primer (SEQ ID NO: 1745. SeMV CP and ΝΔ65 CP PCR products were digested with Xhol and cloned into pRB vector double digested with Xhol (SEQ ID NO: 180) and PvuII (SEQ ID NO: 175). As seen in Figure 25, lane 1 is DNA ladder, lane 2 is CP amplicon (800 bp), and lane 3 is NA65CP amplicon (605 bp).

[00328] The clones were transformed in BL-21 - Rossetta cells and proteins were purified by differential ultracentrifugation. For purification of VLPs, cells were harvested in buffer containing 50 mM of sodium acetate pH 5.5 and 0.02% sodium thioglycolate (SAT buffer) and sonicated. VLPs were pelleted from the clear lysate by spinning the supernatant at 26,000 rpm for 3 h using a SW28 rotor in a Beckman coulter optima L-90 ultracentrifuge. The pellet obtained was resuspended in a minimum volume of SAT buffer and layered over 10^-0% sucrose gradient and spun again at 26,000 rpm for 3 h. Fractions of 1.5 ml were collected after the run and the presence of protein was examined using SDS-PAGE. Fractions containing the protein of interest were pooled and sucrose was diluted by adding SAT buffer. VLPs were finally pelleted using ultra-centrifugation (26,000 rpm, 3 h, SW28 rotor) and resuspended in a suitable volume of SAT buffer. The proteins could enter the sucrose gradient, which suggested that they could assemble into VLPs. As seen in Figure 26, the SDS PAGE profile of sucrose density gradient fractions of B-NA65CP shows the assembly of chimeric B-NA65CP VLPs. Fractions 6-11 were pooled, ultrapelleted, dissolved in a buffer containing 5% glycerol and used for further studies.

[00329] Figure 27 shows the SDS PAGE profile of sucrose density gradient fractions of SeMVB-CP, demonstrating the assembly of SeMV BCP VLPs. As seen in Figure 27, the yield and purity of the VLPs was poor, and hence not used for further studies.

[00330] A fraction of the purified protein was also loaded on the EM-grids and stained with 1% uranyl acetate. Examination under electron microscope clearly showed that the proteins had assembled into VLPs, as shown in Figure 28 A, and B. Figure 28A shows the TEM of B-NA65CP SeMVVLPs, while Figure 28B shows the TEM of B-CP SeMV VLPs. Example 4

Cloning, expression of B domain fused at the C-terminus of NA65CP

[00331] For B domain fused at the C-terminus (NA65CP-B), NA65CP gene was re- amplified with Nhel containing sense and BamHI containing antisense primers (SEQ ID NO: 176, and 177 respectively). The PCR product and the pRSETC vector were double digested with Nhel and BamHI and ligated. The B domain was then PCR amplified with BamHI containing sense (SEQ ID NO: 178) and Xhol containing antisense (SEQ ID NO: 179) primers and the NA65CP-pRSETC were double digested with BamHI and Xhol and ligated to obtain NA65CP-B construct. The recombinant protein was expressed in E.coli and purified using ultra-centrifugation. The protein could enter the sucrose gradient and TEM analysis also suggested that it had assembled to form VLPs (Figure 29A, B). Figure 29A shows the SDS PAGE profile of NA65CP-B, demonstrating the assembly of NA65CP-B VLPs. Figure 29B shows the TEM of NA65CP-B VLPs (Fractions 5-10 were pooled and used for TEM).

Example 5

Antibody binding to VLPs

[00332] Direct antigen coating (DAC) ELISA of NA65-CP/SpA/ Β-ΝΔ65 CP- VLPs/ B- ΝΔ65- CP dimers performed as described in the methods section using non-specific DAPAL antibody. Semi-log plot of antigen concentration versus optical density at 450nm is shown. Β-ΝΔ65- CP particles showed significantly greater (~ 50 times) binding to the antibodies in comparison to SpA and B- NA65-CP dimers. However, interestingly, NA65CP-B VLPs did not react with DAPAL antibodies suggesting that the B-domain is not accessible for IGg binding in this construct.

Example 6

HeLa cell entry by B-NA65CP- VLPs

The ability of B- NA65-CP-VLPs to enter Hela cells and deliver antibodies was tested as described before. The alexafluor 488 labeledBNA65CP dimer, NA65CP VLP and BNA65CP VLP were incubated with alexa flor 633 labelled D6F10 antibody for 2 hours and was checked for its internalization in HeLa cells. The confocal images were taken as described earlier.

[00333] As seen in Figure 31, top panel 488 alexa flor labeled B- NA65-CP dimer could not enter the cells as no green fluorescence was visible and the 633 labelled D6F10 antibody also did not enter the cells .On the other hand, B- NA65-CP-VLPs could enter the cells (middle panel) but could not deliver the 633 labelled D6F10 antibody. Interestingly, only 488 alexa flor labelled B- NA65-CP-VLPs could enter and deliver the 633 alexa flor labelled antibodies as seen in the bottom panel. These results demonstrate that only VLPs have the ability to enter the cells.

Example 7

Use of SeMV in imaging and drug delivery

[00334] SeMV, a plant Sobemovirus, that infects Sesbania grandiflora ,is a single stranded RNA virus made up of 180 copies of a 29 kDa coat protein (CP) and forms a 30 nm-diameter icosahedral particle.

[00335] The use of SeMV as a carrier for drug delivery and imaging molecules was further tested using chemotherapeutic drug doxorubicin and imaging molecule DAPI.

[00336] SeMV propagation and purification: SeMV was purified from SeMV infected Sesbania leaves harvested 20-30 days post inoculation by following established protocols. (Lokesh, et al., Archives of Virology, 146, 209-223).

[00337] The purity of SeMV sample was determined by size exclusion FPLC using a Sepharose 6 column (AKTA Purifier, GE Healthcare). Virus concentration was measured with UV spectrophotometer (NanoVue plus, GE Healthcare).

[00338] Cargo loading via infusion: SeMV VLPs (at 1 mg/mL) were incubated with 1000 fold molar excess of DAPI, or 1000, and 2000 fold molar excess of doxorubicin(Dox) in the presence of 0.1 M phosphate buffer pH 7.2 for two hours at room temperature. Excess of the DAPI and doxorubicin was removed from the infused particles by dialyzing the mixture against 0.1 M phosphate buffer pH 7.2 for 24 hours with two buffer exchanges. The dialyzed sample was concentrated by multiple rounds of centrifugation using molecular weight cut off centrifuge filters and the concentrated sample was used for further experiments.

[00339] Labeling of cargo-infused SeMV: The surface exposed lysines of cargo infused SeMV was labeled with Alexafluor 555 NHS-succinimidyl ester carboxylic acid. The labeled particles were passed through PD-10 column and the fractions were analyzed for the labeled protein.

[00340] UV/Vis spectroscopy: Absorbance scanning was performed for the Dox infused SeMV (SeMV- Dox) between 400-500 nm. Dox has a maximum absorbance at 480 nm. A standard graph was plotted with varying concentrations of Dox and recording the absorbance at 480 nm (Figure 32). Absorbance for SeMV-Dox samples were recorded at 480 nm and the concentration of infused Dox was calculated and the data is as shown in the table below.

[00341] SeMV-DAPI for imaging: 10,000 HeLa cells were seeded on a cover slip and incubated for overnight at 37°C, 5% C0 ₂. The cells were washed with PBS thrice with 5 minutes incubation period. SeMV-DAPI was added at 1 , and 5 μg and incubated for 1, and 3 hours respectively. 1 μg of A555 labeled SeMV-DAPI was added to the cells and incubated for 1 hour. Unlabelled SeMV CP-DAPI VLPs which encapsidate E.coli ribosomal RNA was also tested. After incubation the cells were washed thrice with PBS and fixed with 4% formaldehyde. Cells stained with DAPI alone was kept as a positive control. Later the coverslips were mounted on to clean glass slides by adding a drop of antifade and then sealed. The slides were further processed for confocal fluorescent microscopy. As seen in Figure 33, panel A shows control cells stained with DAPI. Panel B shows unlabelled SeMV VLPs loaded with DAPI at beginning of incubation showing entry of DAPI. Panel C shows the results of incubation of 1 μg Alexa flor 555 labelled SeMV-DAPI with HeLa cells after 1 hour. It can be see seen that the nulcei in the cells have been stained with DAPI and the SeMV has remained in the cytosol. Similar nuclei staining can also be seen in panels D unlabelled SeMV DAPI Ιμ ΐητ, Panel E (SeMV_DAPI ^g 3hours), F (SeMV-DAPI 5 μg 1 hour), and G (SeMV-DAPI 5 μg 3 hours). It can be appreciated from the various panels in Figure 33 that SeMV is able to enter cells and release the load (DAPI).

[00342] SeMV-Dox MTT assay: 10,000 HeLa cells were seeded per well in a tissue culture plate and incubated for overnight. Dox alone or SeMV-Dox was added to the cells in varying concentrations and incubated for 24 hours (Figure 34). MTT was added to the cells and incubated at 37°C for 4 hours. The plates were spun for a short time, supernatant was discarded and DMSO was added to dissolve the formazan crystals. Absorbance was recorded at 590 nm and a histogram was plotted to compare free dox and SeMV-Dox. As seen in Figure 34, the cytotoxicity conferred by SEMV- Dox is about 2 fold more than free Dox, suggesting that SeMV-Dox is more effective in entering the cells than free Dox.

Example 8

B domain functionality in PVBV chimeric VLPs

[00343] To demonstrate that the B domain inserted into PVBV CP VLPs retain their function, their ability to bind to IgG was tested by western blot analysis. After the purified PVBV CP and BCP were run on 12% SDS PAGE, western blot analysis was performed using both PVBV CP specific as well as nonspecific antibodies (DAPAL). It was observed that both PVBV CP and BCP were detected by PVBV CP antibody (Figure 38A and B respectively), however with DAPAL antibody, the positive signal was detected only with PVBV BCP (Figure 38C). This demonstrates that the B domain at the N- terminus of PVBV CP can recognize any IgG and is thus functional.

[00344] Further, to check whether this B domain was exposed in the assembled VLPs in solution, DAC ELISA was performed using DAPAL antibodies. For this purpose varying concentrations of PVBV BCP (1 to 10000 nM) and PVBV CP ( 1 to 10000 nM as a negative control) were coated on to the polystyrene plates and tested for their ability to bind IgG. As can be seen from Figure 39A, CP is not detected with a non- specific antibody (DAPAL Antibody) while BCP is detected by both the specific (PVBV CP Antibody) as well as the non-specific antibody (Figure 39B). Therefore, the chimeric PVBV BCP VLPs retain their antibody binding ability in solution too and the B domain is exposed in the assembled VLP to interact with the Fc region of IgG.

[00345] For comparing the IgG binding affinity of PVBV BCP VLP and SpA (Staphylococcus Protein A), DAC ELISA was carried out using DAPAL antibody. It was observed that PVBV BCP VLP (~ K _d= 40nM) has 10 times better binding ability in comparison with SpA (~ K _d= 400nM) (Figure 39C), while PVBV CP VLP showed no binding, clearly indicating the presence of multiple B domains in PVBV BCP VLP in contrast to the two IgG binding domains of SpA dimer.

Example 9

Use of chimeric VLPs in disease diagnosis

[00346] One of the most common method of diagnosis of any disease is detection of specific antigen that is known to reflect the disease condition, and estimation of the same using Sandwich ELISA. In this method, two different antibodies that recognize two different epitopes are used. In this method, initially, polyclonal antibodies raised in rabbit are coated onto ELISA plates. This is followed by addition of antigen. The bound antigen is detected by using a monoclonal antibody as primary antibody directed at a specific epitope and goat anti-mouse IgG HRP conjugate as the secondary or detecting antibody. If the plates are initially coated with chimeric VLPs expressing B domain, one may expect a more efficient binding of the polyclonal antibodies via this B-domain and improved orientation of the antibodies thereby result increased sensitivity of the method. In this study the poly and monoclonal antibodies to ground nut bud necrosis virus nucleo capsid protein (NP) was used to detect the purified recombinant NP to demonstrate the proof of this concept.

[00347] Sandwich ELISA was performed as follows: Polyclonal antibodies of GBNV NP (1 :5000 dilution, 100 μΐ.) was added to the microtiter wells and incubated overnight at 4 °C. Unbound antibodies were removed and the plate was blocked by adding 300 ul of 5% skimmed milk solution in PBS and incubated for 2 hr at 37 °C. After the removal of blocking agent the plate was washed thrice with PBST and PBS. Various concentrations of GBNV NP (1 ng to 1000 ng) were added to the wells and incubated for 2 hrs at 37°C. Then the sample was removed and the plate was washed as before. This was followed by addition of monoclonal antibody of GBNV NP (A10D10, 1:20 dilution) and incubated for 1 hr at 37°C. Once again the sample was removed and the plate was washed as before. 1: 1000 dilution of secondary antibody, goat antimouse IgG HRP conjugate was added and incubated for 1 hr at 37°C. Then it was washed with PBST and PBS. After that substrate TMB was added and the reaction was stopped by the addition of HC1 and absorbance was measured at 450 nm. In parallel, 500 ng of BCP and CP were coated onto microtiter plates and incubated overnight at 4°C. Then sandwich ELISA was performed as explained above by using 1:5000 dilution of polyclonal antiserum and 1:20 dilution of monoclonal antiserum and 1: 1000 dilution of secondary antibody (goat anti-mouse IgG HRP conjugated) and absorbance was measured at 450 nm. After fixing the concentration of NP and the dilutions for the polyclonal and monoclonal antibodies, sandwich ELISA was performed. As seen in Figure 40, comparison of the antigen dilution curves with and without the initial binding of PVBV BCP VLPs shows that at low concentrations there is a two-fold increase in the sensitivity of detection. The antibodies to NP did not bind to wells precoated with PVBV CPVLPs as it lacks the B-domain and therefore no absorbance at 450 nm was observed. [00348] Overall, the present disclosure provides chimeric VLPs based on the Sesbania mosaic virus, or the Pepper vein banding virus, which can be fused in various combinations with B/Z33 domain of Staphylococcus protein A (SpA) (which can bind Fc fragment of various IgGs). These VLPs disassemble inside cells, which can be exploitedfor intracellular delivery of of various cargo molecules such as antibodies/therapeutics. The VLPs can also be used for imaging applications. The ease of VLP disassembly and reassembly allows for exploitation of the system to load various cargo molecules, and the subsequent intracellular disassembly of the loaded VLPs intracellularly allows for a potent and efficient delivery mechanism of cargo molecules of interest, in particular antibodies for antibody mediated treatment regimes for various diseases.

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