Anti-pox viruses antibodies

Abstract

The invention provides monoclonal antibodies which bind and neutralize pox viruses. The invention also provides pharmaceutical compositions comprising the antibodies, and combinations thereof, and their use in methods of prophylaxis, treatment, or amelioration of infections caused by pox viruses, and for reducing side effects of vaccinia-based vaccines.

Description

[0001] Anti-Pox Viruses antibodies

[0002] TECHNOLOGICAL FIELD

[0003] The present invention generally relates to antibodies directed against pox viruses and to methods and uses thereof for treating infections caused by pox viruses, and for reducing side effects of vaccinia-based vaccines.

[0004] BACKGROUND ART

[0005] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0006] 1 Earl, P.L., J.L. Americo, and B. Moss, Lethal monkeypox virus infection of CAST / EiJ mice is associated with a deficient gamma interferon response. J Virol, 2012. 86(17): p. 9105-12.

[0007] 2 Erez N, et al. Diagnosis of imported monkeypox, Israel, 2018. Emerg Infect Dis 25:980-983 (2019).

[0008] 3 Eyal, O. et al. Development of a tissue-culture-based enzyme-immunoassay method for the quantitation of anti -vaccinia-neutralizing antibodies in human sera. J Virol Methods 130, 15-21 (2005).

[0009] 4 Gerichter CH. Preparation of vaccine against smallpox in chorioalantois membranes of chicken embryos. Public Health (Israeli Ministry of Health, Jerusalem) 1966. 9:36-41.

[0010] 5 Gilchuk I, Gilchuk P, Sapparapu G, Lampley R, Singh V, Kose N, Blum DL, Hughes LJ, Satheshkumar PS, Townsend MB, Kondas AV, Reed Z, Weiner Z, Olson VA, Hammarlund E, Raue HP, Sli fka MK, Slaughter JC, Graham BS, Edwards KM, Eisenberg RJ, Cohen GH, Joyce S, Crowe JE Jr. Cross-Neutralizing and Protective Human Antibody Specificities to Poxvirus Infections. Cell. 2016 Oct 20;167(3):684-694.e9. doi: 10.1016 / j .cell .2016.09.049. PMID: 27768891 ; PMCID: PMC5093772. Gu, X. et al. Protective Human Anti-Poxvirus Monoclonal Antibodies Are Generated from Rare Memory B Cells Isolated by Multicolor Antigen Tetramers. Vaccines (Basel) 10, doi: 10.3390 / vaccinesl0071084 (2022). Lantto, J. et al. Capturing the natural diversity of the human antibody response against vaccinia virus. J Virol 85, 1820-1833, doi: 10.1128 / JVI.02127-10 (2011). Lustig, S., Fogg, C., Whitbeck, J. C. & Moss, B. Synergistic neutralizing activities of antibodies to outer membrane proteins of the two infectious forms of vaccinia virus in the presence of complement. Virology 328, 30-35, doi:10.1016 / j.virol.2004.07.024 (2004). Matho, M. H. et al. Structural and biochemical characterization of the vaccinia virus envelope protein D8 and its recognition by the antibody LA5. J Virol 86, 8050-8058 (2012). McCausland, M. M. et al. Combination therapy of vaccinia virus infection with human anti-H3 and anti-B5 monoclonal antibodies in a small animal model. Antivir Ther 15, 661-675, doi : 10.3851 / IMP 1573 (2010). Meng, X. et al. Generation and characterization of a large panel of murine monoclonal antibodies against vaccinia virus. Virology 409, 271-279, doi: 10.1016 / j.virol.2010.10.019 (2011). Noy-Porat, T. et al. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates. Toxins 8, Doi: 10.3390 / toxin s8030064 (2016) . Noy-Porat, T. et al. A panel of human neutralizing mAbs targeting SARS-CoV-2 spike at multiple epitopes. Nat. Commun. 11, 4303. Doi: 10.1038 / s41467-020- 18159-4 (2020). Paran N, et al. Monkeypox DNA levels correlate with virus infectivity in clinical samples, Israel, 2022. Euro Surveill 27:2200636 (2022). Rosenfeld, R. et al. Isolation and chimerization of a highly neutralizing antibody conferring passive protection against lethal Bacillus anthracis infection. PLoS One 4, e6351. Doi: 10.1371, 'journal. pone.0006351 (2009). 16 Townsley AC, et al., Vaccinia virus entry into cells via a low-pH-dependent endosomal pathway. J Virol 80:8899-8908 (2006).

[0011] 17 Zaitseva M, Kapnick SM, Meseda CA, Shotwell E, King LR, Manischewitz J, Scott J, Kodihalli S, Merchlinsky M, Nielsen H, Lantto J, Weir JP, Golding H. Passive immunotherapies protect WRvFire and IHD-J-Luc vaccinia virus- infected mice from lethality by reducing viral loads in the upper respirator}- tract and internal organs. J Virol. 201 1 Sep;85(17):9147-58. doi: 10.1128 / JVI.00121- 11. Epub 2011 Jun 29. PMID: 21715493; PMCID: PMC3165812.

[0012] Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

[0013] BACKGROUND

[0014] Members of the Orthopoxvirus (OPV) genus can cause mild to severe infections in humans. Among them: Variola virus (VARV), the causative agent of smallpox, monkeypox virus (MPXV), the causative agent of monkeypox, cowpox virus (CPXV), Vaccinia virus (VACV), and others, which exhibit sporadic occurrences worldwide.

[0015] Tecovirimat (also known as TPOXX) is an FDA approved anti-viral drug for treating human smallpox disease caused by Variola virus. However, its use for other orthopoxvirus infections is not approved by the FDA. Another FDA approved smallpox drug is Tembexa (brincidofovir). Still, the best way to prevent orthopoxvirus diseases is by vaccination using vaccinia virus (VACV). VACV has been used as a live-attenuated vaccine to eradicate smallpox.

[0016] This vaccination, however, has rare but severe adverse reactions, which can be treated today using Vaccinia immune globulins (VIG). VIG is prepared from the plasma of vaccinated human donors but is in short supply due to the decreasing numbers of vaccinated people and suffers from other disadvantages such as batch-to-batch variability, limited potency, and the risk of transmitting other viral agents.

[0017] Lustig et al (2004) describe synergistic neutralizing activities of antibodies to outer membrane proteins of the two infectious forms of vaccinia virus in the presence of complement. McCausland et al (2010) describe a combination therapy of vaccinia virus infection with human anti-H3 (against the mature virion) and anti-B5 (against an extracellular virion) monoclonal antibodies in a small animal model.

[0018] Lantto et al (2010) isolated several VACV-specific antibodies that recognize a variety of different VACV antigens from immunized volunteers and generated a fully human, recombinant analogue to plasma-derived vaccinia immunoglobulin (VIG).

[0019] Meng et al (2011) studied the antibody response to vaccinia virus and generated a panel of murine monoclonal antibodies against the virus.

[0020] Gu et al (2022) describe the generation of human anti-Poxvirus monoclonal antibodies directed against the virus’ extracellular envelope protein A33.

[0021] GENERAL DESCRIPTION

[0022] In a first of its aspects, the present invention provides a pharmaceutical composition comprising as an active ingredient at least two isolated monoclonal antibodies or antigen-binding fragments thereof which bind to VACV, and a pharmaceutically acceptable carrier, excipient, or diluent, wherein one of said at least two isolated monoclonal antibodies comprises complementarity determining regions selected from a group consisting of: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 89, CDRH2 denoted by SEQ ID NO. 90, CDRH3 denoted by SEQ ID NO. 91, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 92, a CDRL2 denoted by SEQ ID NO. 93, and a CDRL3 denoted by SEQ ID NO. 94 (designated MV33); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 59, CDRH2 denoted by SEQ ID NO. 60, CDRH3 denoted by SEQ ID NO. 61, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 62, a CDRL2 denoted by SEQ ID NO. 63, and a CDRL3 denoted by SEQ ID NO. 64 (designated MV7); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 71, CDRH2 denoted by SEQ ID NO. 72, CDRH3 denoted by SEQ ID NO. 73, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 74, a CDRL2 denoted by SEQ ID NO. 75, and a CDRL3 denoted by SEQ ID NO. 76 (designated MV26); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 77, CDRH2 denoted by SEQ ID NO. 78, CDRH3 denoted by SEQ ID NO. 79, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 80, a CDRL2 denoted by SEQ ID NO. 81, and a CDRL3 denoted by SEQ ID NO. 82 (designated MV31); e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 83, CDRH2 denoted by SEQ ID NO. 84, CDRH3 denoted by SEQ ID NO. 85, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 86, a CDRL2 denoted by SEQ ID NO. 87, and a CDRL3 denoted by SEQ ID NO. 88 (designated MV32); f. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 101, CDRH2 denoted by SEQ ID NO. 102, CDRH3 denoted by SEQ ID NO. 103, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 104, a CDRL2 denoted by SEQ ID NO. 105, and a CDRL3 denoted by SEQ ID NO. 106 (designated MV49); g. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 119, CDRH2 denoted by SEQ ID NO. 120, CDRH3 denoted by SEQ ID NO. 121, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 122, a CDRL2 denoted by SEQ ID NO. 123, and a CDRL3 denoted by SEQ ID NO. 124 (designated MV57); h. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 125, CDRH2 denoted by SEQ ID NO. 126, CDRH3 denoted by SEQ ID NO. 127, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 128, a CDRL2 denoted by SEQ ID NO. 129, and a CDRL3 denoted by SEQ ID NO. 130 (designated MV60); and a second of said at least two isolated monoclonal antibodies comprises complementarity determining regions selected from a group consisting of: i. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 95, CDRH2 denoted by SEQ ID NO. 96, CDRH3 denoted by SEQ ID NO. 97, and a light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 98, a CDRL2 denoted by SEQ ID NO. 99, and a CDRL3 denoted by SEQ ID NO. 100 (designated EV42); j . a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 53, CDRH2 denoted by SEQ ID NO. 54, CDRH3 denoted by SEQ ID NO. 55, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 56, a CDRL2 denoted by SEQ ID NO. 57, and a CDRL3 denoted by SEQ ID NO. 58 (designated EV4); k. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 65, CDRH2 denoted by SEQ ID NO. 66, CDRH3 denoted by SEQ ID NO. 67, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 68, a CDRL2 denoted by SEQ ID NO. 69, and a CDRL3 denoted by SEQ ID NO. 70 (designated EVI 5); l. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 107, CDRH2 denoted by SEQ ID NO. 108, CDRH3 denoted by SEQ ID NO. 109, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 110, a CDRL2 denoted by SEQ ID NO. 11, and a CDRL3 denoted by SEQ ID NO. 112 (designated EV55); and m. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 113, CDRH2 denoted by SEQ ID NO. 114, CDRH3 denoted by SEQ ID NO. 115, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 116, a CDRL2 denoted by SEQ ID NO. 117, and a CDRL3 denoted by SEQ ID NO. 118 (designated EV56).

[0023] In one embodiment, one of said at least two isolated monoclonal antibodies comprises: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 89, CDRH2 denoted by SEQ ID NO. 90, CDRH3 denoted by SEQ ID NO. 91, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 92, a CDRL2 denoted by SEQ ID NO. 93, and a CDRL3 denoted by SEQ ID NO. 94; and a second of said at least two isolated monoclonal antibodies comprises: b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 95, CDRH2 denoted by SEQ ID NO. 96, CDRH3 denoted by SEQ ID NO. 97, and a light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 98, a CDRL2 denoted by SEQ ID NO. 99, and a CDRL3 denoted by SEQ ID NO. 100.

[0024] In one embodiment, one of said at least two isolated monoclonal antibodies comprises a heavy chain variable region and a light chain variable region, wherein: a. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 13 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 14; or b. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 3 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 4; or c. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 7 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 8; or d. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 9 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 10; or e. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 11 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 12; or f. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 17 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 18; or g. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 23 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 24; or h. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 25 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 26; and a second of said at least two isolated monoclonal antibodies comprises a heavy chain variable region and a light chain variable region, wherein: i. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 15 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 16; or j. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 1 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 2; or k. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 5 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 6; or l. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 19 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 20; or m. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 21 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 22.

[0025] In one embodiment, one of said at least two isolated monoclonal antibodies comprises: a. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 39 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 40, or a variant thereof; or b. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 29 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 30, or a variant thereof; or c. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 33 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 34, or a variant thereof; or d. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 35 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 36, or a variant thereof; or e. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 37 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 38, or a variant thereof; or f. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 43 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 44, or a variant thereof; or g. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 49 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 50, or a variant thereof; or h. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 51 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 52, or a variant thereof and a second of said at least two isolated monoclonal antibodies comprises: i. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 41 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 42, or a variant thereof; or j . a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 27 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 28, or a variant thereof; or k. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 31 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 32, or a variant thereof; or l. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 45 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 46, or a variant thereof; or m. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 47 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 48, or a variant thereof.

[0026] In another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragments thereof which binds to the MV form of VACV, wherein said isolated monoclonal antibody comprises complementarity determining regions selected from a group consisting of: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 89, CDRH2 denoted by SEQ ID NO. 90, CDRH3 denoted by SEQ ID NO. 91, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 92, a CDRL2 denoted by SEQ ID NO. 93, and a CDRL3 denoted by SEQ ID NO. 94 (designated MV33); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 59, CDRH2 denoted by SEQ ID NO. 60, CDRH3 denoted by SEQ ID NO. 61, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 62, a CDRL2 denoted by SEQ ID NO. 63, and a CDRL3 denoted by SEQ ID NO. 64 (designated MV7); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 71, CDRH2 denoted by SEQ ID NO. 72, CDRH3 denoted by SEQ ID NO. 73, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 74, a CDRL2 denoted by SEQ ID NO. 75, and a CDRL3 denoted by SEQ ID NO. 76 (designated MV26); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 77, CDRH2 denoted by SEQ ID NO. 78, CDRH3 denoted by SEQ ID NO. 79, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 80, a CDRL2 denoted by SEQ ID NO. 81, and a CDRL3 denoted by SEQ ID NO. 82 (designated MV31); e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 83, CDRH2 denoted by SEQ ID NO. 84, CDRH3 denoted by SEQ ID NO. 85, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 86, a CDRL2 denoted by SEQ ID NO. 87, and a CDRL3 denoted by SEQ ID NO. 88 (designated MV32); f. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 101, CDRH2 denoted by SEQ ID NO. 102, CDRH3 denoted by SEQ ID NO. 103, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 104, a CDRL2 denoted by SEQ ID NO. 105, and a CDRL3 denoted by SEQ ID NO. 106 (designated MV49); g. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 119, CDRH2 denoted by SEQ ID NO. 120, CDRH3 denoted by SEQ ID NO. 121, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 122, a CDRL2 denoted by SEQ ID NO. 123, and a CDRL3 denoted by SEQ ID NO. 124 (designated MV57); and h. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 125, CDRH2 denoted by SEQ ID NO. 126, CDRH3 denoted by SEQ ID NO. 127, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 128, a CDRL2 denoted by SEQ ID NO. 129, and a CDRL3 denoted by SEQ ID NO. 130 (designated MV60).

[0027] In one embodiment, said isolated monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein: a. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 13 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 14; or b. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 3 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 4; or c. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 7 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 8; or d. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 9 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 10; or e. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 11 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 12; or f. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 17 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 18; or g. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 23 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 24; or h. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 25 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 26.

[0028] In one embodiment, said isolated monoclonal antibody comprises: a. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 39 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 40, or a variant thereof; or b. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 29 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 30, or a variant thereof; or c. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 33 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 34, or a variant thereof; or d. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 35 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 36, or a variant thereof; or e. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 37 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 38, or a variant thereof; or f. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 43 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 44, or a variant thereof; or g. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 49 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 50, or a variant thereof; or h. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 51 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 52, or a variant thereof.

[0029] In one embodiment, said antibody or antigen-binding fragment thereof binds to at least one surface protein of the MV form of VAC V. In one embodiment, said at least one surface protein of the MV form of VAC V is selected from a group consisting of H3, D8, A27, and LI.

[0030] In another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragments thereof which binds to the EV form of VAC V, wherein said isolated monoclonal antibody comprises complementarity determining regions selected from a group consisting of a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 95, CDRH2 denoted by SEQ ID NO. 96, CDRH3 denoted by SEQ ID NO. 97, and a light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 98, a CDRL2 denoted by SEQ ID NO. 99, and a CDRL3 denoted by SEQ ID NO. 100 (designated EV42); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 53, CDRH2 denoted by SEQ ID NO. 54, CDRH3 denoted by SEQ ID NO. 55, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 56, a CDRL2 denoted by SEQ ID NO. 57, and a CDRL3 denoted by SEQ ID NO. 58 (designated EV4); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 65, CDRH2 denoted by SEQ ID NO. 66, CDRH3 denoted by SEQ ID NO. 67, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 68, a CDRL2 denoted by SEQ ID NO. 69, and a CDRL3 denoted by SEQ ID NO. 70 (designated EVI 5); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 107, CDRH2 denoted by SEQ ID NO. 108, CDRH3 denoted by SEQ ID NO. 109, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 110, a CDRL2 denoted by SEQ ID NO. 11, and a CDRL3 denoted by SEQ ID NO. 112 (designated EV55); and e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 113, CDRH2 denoted by SEQ ID NO. 114, CDRH3 denoted by SEQ ID NO. 115, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 116, a CDRL2 denoted by SEQ ID NO. 117, and a CDRL3 denoted by SEQ ID NO. 118 (designated EV56). In one embodiment, said isolated monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein: a. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 15 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 16; or b. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 1 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 2; or c. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 5 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 6; or d. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 19 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 20; or e. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 21 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 22.

[0031] In one embodiment, said isolated monoclonal antibody comprises: a. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 41 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 42, or a variant thereof; or b. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 27 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 28, or a variant thereof; or c. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 31 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 32, or a variant thereof; or d. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 45 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 46, or a variant thereof; or e. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 47 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 48, or a variant thereof.

[0032] In one embodiment, said antibody or antigen-binding fragment thereof binds to at least one surface protein of the EV form of VAC V.

[0033] In one embodiment the at least one surface protein of the EV form of VACV is A33 or B5.

[0034] In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof of the invention that binds at least one surface protein of the EV form of VACV, is for use in combination with at least one isolated monoclonal antibody or antigen-binding fragment thereof of the invention that binds at least one surface protein of the MV form of VACV.

[0035] In one embodiment, said antibody or antigen-binding fragment thereof binds VACV with a KD of about 1 pM to about 10 nM.

[0036] In one embodiment, said antibody or antigen-binding fragment thereof inhibits the ability of a pox virus to infect mammalian cells.

[0037] In one embodiment, said antibody or antigen-binding fragment thereof is a singlechain Fv-Fc (scFv-Fc) molecule, single-chain Fv (scFv), Fv, heavy chain variable region, light chain variable region, Fab, F(ab)2(or any combination thereof. In one embodiment, said antibody or antigen-binding fragment thereof is a scFv- Fc molecule.

[0038] In one embodiment, said antibody is a neutralizing antibody.

[0039] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or any antigen-binding fragment thereof according to the invention.

[0040] In another aspect, the present invention provides an expression vector comprising the isolated nucleic acid molecule of the invention.

[0041] In another aspect, the present invention provides a host cell transfected with the expression vector of the invention.

[0042] In one embodiment, said pharmaceutical composition further comprises an additional therapeutic agent.

[0043] In another aspect, the present invention provides a method of prophylaxis, treatment, or amelioration of infections caused by pox viruses, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition, at least one, or at least two of the isolated monoclonal antibodies or antigenbinding fragments thereof of the invention.

[0044] In another aspect, the present invention provides a method of reducing side effects of vaccinia-based vaccines, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition, at least one, or at least two of the isolated monoclonal antibodies or antigen-binding fragments thereof of the invention.

[0045] In one embodiment, said method further comprises administering to a subject in need thereof an additional therapeutic agent.

[0046] In another aspect, the present invention provides a method of inhibiting the ability of VAC V to infect mammalian cells in a subject comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition, at least one, or at least two of the isolated monoclonal antibodies or antigen-binding fragments thereof of the invention.

[0047] In one embodiment, said pharmaceutical composition, said at least one, or at least two isolated monoclonal antibodies or antigen-binding fragment thereof, are administered to said subject before or after exposure to a pox virus. In another aspect, the present invention provides a method of detecting VACV in a sample, said method comprising: a. contacting said sample with at least one isolated monoclonal antibody or antigen-binding fragment thereof according to the invention; and b. detecting said at least one isolated monoclonal antibody or any antigenbinding fragment thereof; wherein the presence of said isolated monoclonal antibody or any antigenbinding fragment thereof indicates the presence of VACV in said biological sample.

[0048] In another aspect, the present invention provides a method of detecting VACV in a sample, said method comprising: a. immobilizing at least one isolated monoclonal antibody or antigen-binding fragment thereof according to the invention (the "capture antibody") onto a substrate, wherein said substrate is placed in a vessel; b. contacting said capture antibody with said sample; and c. adding to said vessel at least one isolated monoclonal antibody or antigenbinding fragment thereof of the invention (the "reporter antibody"), wherein said at least one reporter antibody is different from said at least one capture antibody, and wherein said reporter antibody is optionally detectably labelled; wherein detecting the presence of said at least one reporter antibody indicates the presence of VACV in said sample.

[0049] In one embodiment, said at least one reporter antibody is a biotinylated antibody or is being associated with a detectable molecule (e.g., a fluorophore or an enzyme such as horse radish peroxidase or alkaline phosphatase).

[0050] In one embodiment, said sample is a biological sample obtained from a subject.

[0051] In another aspect, the pharmaceutical composition, or the isolated monoclonal antibody or antigen-binding fragment thereof of the invention are for use in a method of prophylaxis, treatment, or amelioration of infections caused by pox viruses, or for use in reducing side effects of vaccinia-based vaccines, said methods comprising administering to a subject in need thereof a therapeutically effective amount of said pharmaceutical composition, at least one, or at least two of said isolated monoclonal antibodies or antigen-binding fragments thereof. In one embodiment, said methods further comprise administering to a subject in need thereof an additional therapeutic agent.

[0052] In another aspect, the pharmaceutical composition, or the isolated monoclonal antibody or antigen-binding fragment thereof of the invention are for use in a method of inhibiting the ability of VACV to infect cells in a subject, said method comprising administering to a subject in need thereof a therapeutically effective amount of said pharmaceutical composition, at least one or at least two of said isolated monoclonal antibodies or antigen-binding fragments thereof.

[0053] In one embodiment, said pharmaceutical composition, said at least one, or at least two isolated monoclonal antibodies or antigen-binding fragment thereof, are administered to said subject before or after exposure to a pox virus.

[0054] In another aspect, the present invention provides a kit for detecting VACV infection comprising:

[0055] (a) at least one isolated monoclonal antibody or antigen-binding fragment thereof of the invention;

[0056] (b) means for detection of said isolated monoclonal antibody; and optionally

[0057] (c) instructions for use of said kit.

[0058] BRIEF DESCRIPTION OF THE DRAWINGS

[0059] To better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0060] Figure l is a bar graph showing results of an ELISA binding assay (expressed in O.D. (405nM) of the antibodies EV4, MV7, EV15, MV26, MV31, MV32, MV33, EV42, MV49, EV55, MV56 and VIG to vaccinia and to its major surface proteins H3, D8, A27, LI, B5, and A33.

[0061] Figures 2A-2G are pictures showing results of an EV-comet inhibition assay. This in vitro assay measures neutralization of BSC-1 cells’ infection with vaccinia virus by monoclonal antibodies. Each antibody: EV4 (Fig. 2A), EV42 (Fig. 2B), EV55 (Fig. 2C), EV15 (Fig. 2D), and EV56 (Fig. 2F) was tested at four concentrations (2.5-20 pg / ml), and the two lowest concentrations exhibiting effective comet-inhibition, are presented. A VIG sample (Fig. 2E) was used as a reference in the test, and a virus-only sample (No Ab) was used as a negative control (Fig. 2G). The assay was repeated twice for each mAh.

[0062] Figures 3A-3B are graphs showing Mpox in vitro neutralization. Representative mAbs, MV32 (marked by circles) and MV33 (marked by triangles), were tested for their ability to neutralize Mpox strains 2018 (A) and 2022 (B) in an in vitro MV neutralization assay. VIG samples (marked by squares) were used as control. Data represent mean ± SEM of duplicates.

[0063] Figures 4A-4F are graphs showing the percentage of animal survival over time following infection with ectromelia virus. The animals were treated on day 1, 2, 3, 4, or 5 post infection. Fig. 4A: treatment at 1 day post infection with the antibodies MV33, EV42, MV49, VIG, or left untreated; Fig. 4B: treatment at 1 day post infection with a combination of MV33+EV42 (0.2pg + 0.2pg of each, or with 0.1 pg + 0.1 pg of each), or left untreated; Figs. 4C-4F: treatment at days 2-5 post infection, respectively, with the antibodies MV33, EV42, a combination of MV33+EV42, VIG, or BLN12 (a non-relevant antibody). Mortality was monitored for 21 days post infection.

[0064] Figure 5 is a graph showing the percentage of animal survival over time following infection with ectromelia virus. The animals were treated on 1 day post infection with the antibodies EV55, EV56, MV57 and MV60 or left untreated. Naive (non-infected) mice showed a 100% survival.

[0065] Figure 6 is a graph showing bioluminescent signal / noise intensity of total photon flux (photon / second / cm2 / steradian) calculated by region of interest (ROI) analysis on the chest and abdomen cavity. The same ROI was used for all mice examined. The signal / noise intensity is shown as a function of time (days post infection). Treatment commenced at 3 dpi. Each of EV42 and MV33+EV42 showed a significant difference vs. BLN12 - p<0.05 by One-way ANOVA. Measurement data are expressed as mean ± standard error (SE).

[0066] Figure 7 is a graph showing bioluminescent signal / noise intensity of total photon flux (photon / second / cm2 / steradian) calculated by region of interest (ROI) analysis on the chest and abdomen cavity. The same ROI was used for all mice examined. The signal / noise intensity is shown as a function of time (days post infection).

[0067] Figures 8A-F are graphs showing viral load analysis following ECTV challenge and mAbs treatment. Viral titers were determined in the spleen (Figs 8A and 8B), liver (Figs 8C and 8D), and lung (Figs 8E and 8F). Viral loads are presented as PFU / gr for mice treated at 3 dpi. (Figs. 8A, 8C and 8E) and at 5 dpi. (Figs 8B, 8D and 8F). Horizontal lines represent the mean of each group (the value is indicated above each line). Dashed line represents limit of detection (LOD). Samples below the LOD were assigned a value of half of the LOD. An asterisk denotes a significant reduction in viral load compared to the BLN12 (isotype control treated group) using Mann-Whitney non-parametric unpaired T-test (n=4, *p=0.03). The error bars represent the SEs.

[0068] Figures 9A-B are graphs showing body weight changes (9 A) and mortality (9B) following MPXV challenge and treatment. CastZEiJ mice were challenged with MPXV. At 2 dpi, mice were i.p. treated with lOO+lOOpg of MV33+EV42 or left untreated (Infected untreated). Fig. 9A shows body weight changes presented as % of initial weight over time (Days post infection). Fig. 9B shows mortality presented as % survival over time (Days post infection); n=4 mice per group. For A, statistical analysis was performed using one unpaired t-test per row, with correction for multiple comparisons using the Holm-Sidak method vs. infected untreated (*p < 0.003). Measurement data are expressed as mean ± standard error ( SE). For B, Log-rank (Mantel-Cox) Test vs. infected untreated (*p<0.05.) was used.

[0069] Figures 9C-D are graphs showing body weight changes (9C) and mortality (9D) following MPXV challenge and treatment. CastZEiJ mice were challenged with MPXV. At 3, 4 or 5 dpi, mice were i.p. treated with lOOpg +100pg of MV33+EV42 or left untreated (Infected untreated). Fig. 9C shows body weight changes presented as % of initial weight over time (Days post infection). Fig. 9D shows mortality presented as % survival over time (Days post infection); n=6 mice per group. For C, statistical analysis was performed using one unpaired Ltest per row, with correction for multiple comparisons using the Holm-Sidak method vs. infected untreated, * p < 0.003 of day 3 treatment vs. the infected untreated group. Measurement data are expressed as mean ± standard error (SE).

[0070] DETAILED DESCRIPTION OF EMBODIMENTS

[0071] The present invention concerns the production of antibodies directed to pox viruses. Specific and high-affinity antibodies were prepared from B cells of Macaque monkeys immunized with VACV, by efficient screening methods using phage-display libraries. Based on the construction of a combinatorial single-chain (scFv) library, and selection based on binding to VACV, 13 high-affinity antibodies were identified, and constructed to full length antibodies, termed herein EV4, MV7, EV15, MV26, MV31, MV32, MV33, EV42, MV49, EV55, EV56, MV57, and MV60. The anti-pox antibodies described herein were capable of binding to VACV and neutralizing it, as demonstrated by an in vitro neutralization assay presented below. The antibodies were also capable of neutralizing Mpox virus in an in vitro neutralization assay. Furthermore, the anti-pox antibodies described herein were shown to protect mice from infection with the pox virus Ectromelia, to increase animal survival rate, to reduce virus dissemination in the animals’ body, to reduce viral load in target organs, and to protect against MPXV challenge in mice.

[0072] In the host, the OPV exists in two distinct forms, the intracellular mature virion (MV) and the extracellular enveloped virion (EV). The outer membrane of each form displays a distinct set of proteins, hence, an efficient antibody-based treatment against poxviruses should contain antibodies against both forms.

[0073] Some of the antibodies of the invention were found to bind purified VACV protein components, namely H3 and D8, proteins which are major surface proteins of the MV form of the virus, and others were found to bind the A33 protein which is known as one of the major surface proteins of the EV form of the virus. The antibodies were therefore divided into two groups, one, including MV7, MV26, MV31, MV32, MV33, MV49, MV57 and MV60, is directed to the MV form of the virus and one, including EV4, EVI 5, EV42, EV55 and EV56, is directed to the EV form of the virus. The invention thus provides a unique combination of at least two antibodies, at least one antibody specific to the MV form of the virus and at least one additional antibody specific to the EV form of the virus.

[0074] A combination of the antibodies MV33 and EV42 was found to be highly efficacious in in vivo protection experiments.

[0075] The antibodies of the present invention can be used inter alia as a therapy for pox infections and for the treatment of side effects and / or undesirable symptoms of VACV- based vaccination.

[0076] Therefore, in a first of its aspects, the present invention provides a pharmaceutical composition comprising as an active ingredient at least two isolated monoclonal antibodies or antigen-binding fragments thereof which bind to vaccinia virus (VACV) and a pharmaceutically acceptable carrier, excipient, or diluent, wherein at least one of said monoclonal antibodies recognized the MV form of VACV and at least one of said monoclonal antibodies recognized the EV form of VACV and wherein said at least two isolated monoclonal antibodies are selected from a group consisting of the antibodies denoted EV4, MV7, EV15, MV26, MV31, MV32, MV33, EV42, MV49, EV55, EV56, MV57, and MV60.

[0077] The term “vaccinia virus (VACV)” refers to a virus of the genus Orthopoxvirus (OPV). The terms “Orthopoxvirus (OPV)” and “pox virus” are used interchangeably herein and refer to a genus of viruses comprising Vaccinia virus (VACV), Ectromelia virus (ECTV), Variola virus (VARV), Monkeypox virus (MPXV), and Cowpox virus (CPXV). These viruses are the causative agents of various epidemic diseases, e.g., smallpox and monkeypox. Live attenuated vaccinia virus was used as a vaccine to eradicate smallpox which is caused by VARV, thus antibodies directed to vaccinia virus are effective against other members of the OPV genus. Therefore, anti VACV antibodies can be used as anti-pox antibodies to fight pox infections prophylactically or therapeutically.

[0078] As used herein the term OPV or pox virus also encompasses OPV variants.

[0079] The term "OPV variants" refers to OPV that have undergone a change (e.g., by a spontaneous mutation or by deliberate engineering aimed for example at bioterrorism).

[0080] The outer membrane of each form of OPV (MV or EV) displays a distinct set of proteins.

[0081] A33 protein (GenPept P68617.1) is a vaccinia virus extracellular envelope protein. A33 is required for efficient cell binding by EV particles, serves as a chaperon for other EV proteins, and is involved in the production of actin tails, essential for effective cell-to-cell spread within the host.

[0082] D8 protein (GenPept AAA48233.1) is a vaccinia virus mature virion protein, involved in the infection of cells through its binding to Chondroitin sulfate (CS-E) receptor.

[0083] H3 protein (GenPept P07240.1) is a vaccinia virus mature virion protein. It is involved in binding and fusion to host cells through binding to heparan sulfate, and also involved in MV morphogenesis.

[0084] Specifically, the present invention provides a pharmaceutical composition comprising as an active ingredient at least two isolated monoclonal antibodies or antigen- binding fragments thereof which bind to VACV and a pharmaceutically acceptable carrier, excipient, or diluent, wherein one of said at least two isolated monoclonal antibodies comprises complementarity determining regions selected from a group consisting of: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 89, CDRH2 denoted by SEQ ID NO. 90, CDRH3 denoted by SEQ ID NO. 91, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 92, a CDRL2 denoted by SEQ ID NO. 93, and a CDRL3 denoted by SEQ ID NO. 94 (designated MV33); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 59, CDRH2 denoted by SEQ ID NO. 60, CDRH3 denoted by SEQ ID NO. 61, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 62, a CDRL2 denoted by SEQ ID NO. 63, and a CDRL3 denoted by SEQ ID NO. 64 (designated MV7); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 71, CDRH2 denoted by SEQ ID NO. 72, CDRH3 denoted by SEQ ID NO. 73, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 74, a CDRL2 denoted by SEQ ID NO. 75, and a CDRL3 denoted by SEQ ID NO. 76 (designated MV26); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 77, CDRH2 denoted by SEQ ID NO. 78, CDRH3 denoted by SEQ ID NO. 79, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 80, a CDRL2 denoted by SEQ ID NO. 81, and a CDRL3 denoted by SEQ ID NO. 82 (designated MV31); e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 83, CDRH2 denoted by SEQ ID NO. 84, CDRH3 denoted by SEQ ID NO. 85, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 86, a CDRL2 denoted by SEQ ID NO. 87, and a CDRL3 denoted by SEQ ID NO. 88 (designated MV32); f. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 101, CDRH2 denoted by SEQ ID NO. 102, CDRH3 denoted by SEQ ID NO. 103, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 104, a CDRL2 denoted by SEQ ID NO. 105, and a CDRL3 denoted by SEQ ID NO. 106 (designated MV49); g. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 119, CDRH2 denoted by SEQ ID NO. 120, CDRH3 denoted by SEQ ID NO. 121, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 122, a CDRL2 denoted by SEQ ID NO. 123, and a CDRL3 denoted by SEQ ID NO. 124 (designated MV57); h. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 125, CDRH2 denoted by SEQ ID NO. 126, CDRH3 denoted by SEQ ID NO. 127, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 128, a CDRL2 denoted by SEQ ID NO. 129, and a CDRL3 denoted by SEQ ID NO. 130 (designated MV60); and a second of said at least two isolated monoclonal antibodies comprises complementarity determining regions selected from a group consisting of: i. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 95, CDRH2 denoted by SEQ ID NO. 96, CDRH3 denoted by SEQ ID NO. 97, and a light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 98, a CDRL2 denoted by SEQ ID NO. 99, and a CDRL3 denoted by SEQ ID NO. 100 (designated EV42); j . a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 53, CDRH2 denoted by SEQ ID NO. 54, CDRH3 denoted by SEQ ID NO. 55, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 56, a CDRL2 denoted by SEQ ID NO. 57, and a CDRL3 denoted by SEQ ID NO. 58 (designated EV4); k. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 65, CDRH2 denoted by SEQ ID NO. 66, CDRH3 denoted by SEQ ID NO. 67, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 68, a CDRL2 denoted by SEQ ID NO. 69, and a CDRL3 denoted by SEQ ID NO. 70 (designated EVI 5); l. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 107, CDRH2 denoted by SEQ ID NO. 108, CDRH3 denoted by SEQ ID NO. 109, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 110, a CDRL2 denoted by SEQ ID NO. 11, and a CDRL3 denoted by SEQ ID NO. 112 (designated EV55); and m. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 113, CDRH2 denoted by SEQ ID NO. 114, CDRH3 denoted by SEQ ID NO. 115, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 116, a CDRL2 denoted by SEQ ID NO. 117, and a CDRL3 denoted by SEQ ID NO. 118 (designated EV56).

[0085] In a specific embodiment of the invention the pharmaceutical composition comprises at least two isolated monoclonal antibodies or antigen-binding fragments thereof wherein one of said at least two isolated monoclonal antibodies comprises: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 89, CDRH2 denoted by SEQ ID NO. 90, CDRH3 denoted by SEQ ID NO. 91, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 92, a CDRL2 denoted by SEQ ID NO. 93, and a CDRL3 denoted by SEQ ID NO. 94; and a second of said at least two isolated monoclonal antibodies comprises: b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 95, CDRH2 denoted by SEQ ID NO. 96, CDRH3 denoted by SEQ ID NO. 97, and a light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 98, a CDRL2 denoted by SEQ ID NO. 99, and a CDRL3 denoted by SEQ ID NO. 100.

[0086] The KD values of the anti-pox antibodies were calculated using biolayer interferometry (BLI). The KD values as measured by BLI are presented in Table 4. Affinities ranged from <0.001nM to 7.4nM, MV33 showing the highest affinity.

[0087] Therefore, in some embodiments the isolated monoclonal antibody or antigenbinding fragment thereof according to the present invention is wherein said antibody or antigen-binding fragment thereof binds VACV strain IHD-J, or VACV H3, D8 or A33 proteins with a KD of about 1 pM to about 10 nM.

[0088] Determination of the dissociation constant KD is well known to those of skill in the art and may be performed for example as described in the Examples below.

[0089] In another aspect the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof which binds to VACV, wherein said antibody comprises: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 89, CDRH2 denoted by SEQ ID NO. 90, CDRH3 denoted by SEQ ID NO. 91, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 92, a CDRL2 denoted by SEQ ID NO. 93, and a CDRL3 denoted by SEQ ID NO. 94 (designated MV33); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 59, CDRH2 denoted by SEQ ID NO. 60, CDRH3 denoted by SEQ ID NO. 61, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 62, a CDRL2 denoted by SEQ ID NO. 63, and a CDRL3 denoted by SEQ ID NO. 64 (designated MV7); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 71, CDRH2 denoted by SEQ ID NO. 72, CDRH3 denoted by SEQ ID NO. 73, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 74, a CDRL2 denoted by SEQ ID NO. 75, and a CDRL3 denoted by SEQ ID NO. 76 (designated MV26); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 77, CDRH2 denoted by SEQ ID NO. 78, CDRH3 denoted by SEQ ID NO. 79, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 80, a CDRL2 denoted by SEQ ID NO. 81, and a CDRL3 denoted by SEQ ID NO. 82 (designated MV31); e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 83, CDRH2 denoted by SEQ ID NO. 84, CDRH3 denoted by SEQ ID NO. 85, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 86, a CDRL2 denoted by SEQ ID NO. 87, and a CDRL3 denoted by SEQ ID NO. 88 (designated MV32); f. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 101, CDRH2 denoted by SEQ ID NO. 102, CDRH3 denoted by SEQ ID NO. 103, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 104, a CDRL2 denoted by SEQ ID NO. 105, and a CDRL3 denoted by SEQ ID NO. 106 (designated MV49); g. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 119, CDRH2 denoted by SEQ ID NO. 120, CDRH3 denoted by SEQ ID NO. 121, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 122, a CDRL2 denoted by SEQ ID NO. 123, and a CDRL3 denoted by SEQ ID NO. 124 (designated MV57); h. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 125, CDRH2 denoted by SEQ ID NO. 126, CDRH3 denoted by SEQ ID NO. 127, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 128, a CDRL2 denoted by SEQ ID NO. 129, and a CDRL3 denoted by SEQ ID NO. 130 (designated MV60); i. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 95, CDRH2 denoted by SEQ ID NO. 96, CDRH3 denoted by SEQ ID NO. 97, and a light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 98, a CDRL2 denoted by SEQ ID NO. 99, and a CDRL3 denoted by SEQ ID NO. 100 (designated EV42); j . a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 53, CDRH2 denoted by SEQ ID NO. 54, CDRH3 denoted by SEQ ID NO. 55, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 56, a CDRL2 denoted by SEQ ID NO. 57, and a CDRL3 denoted by SEQ ID NO. 58 (designated EV4); k. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 65, CDRH2 denoted by SEQ ID NO. 66, CDRH3 denoted by SEQ ID NO. 67, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 68, a CDRL2 denoted by SEQ ID NO. 69, and a CDRL3 denoted by SEQ ID NO. 70 (designated EVI 5); l. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 107, CDRH2 denoted by SEQ ID NO. 108, CDRH3 denoted by SEQ ID NO. 109, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 110, a CDRL2 denoted by SEQ ID NO. 11, and a CDRL3 denoted by SEQ ID NO. 112 (designated EV55); and m. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 113, CDRH2 denoted by SEQ ID NO. 114, CDRH3 denoted by SEQ ID NO. 115, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 116, a CDRL2 denoted by SEQ ID NO. 117, and a CDRL3 denoted by SEQ ID NO. 118 (designated EV56).

[0090] Any of the isolated antibodies of the invention may be administered as a single agent or in combination with another antibody of the invention. The antibodies that are given in combination, may be administered in a single composition or as separate compositions.

[0091] As used herein, the term “antibody” refers to a polypeptide encoded by an immunoglobulin gene that specifically binds and recognizes an antigen, in the present case a VACV protein.

[0092] The term “monoclonal antibody”, “monoclonal antibodies” or “mAb” as herein defined refers to a population of substantially homogenous antibodies, i.e., the individual antibodies comprising the population are identical except for possibly naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are directed against a single antigenic site (epitope).

[0093] Monoclonal antibodies may be prepared and purified by any method known in the art. For example, monoclonal antibodies may be prepared from B cells taken from the spleen, bone marrow, peripheral blood, or lymph nodes of immunized animals (e.g., rabbits, rats, mice, or monkeys), or isolated from the blood of patients vaccinated with vaccinia virus. mRNA is extracted from these cells, reverse-transcribed into cDNA and the heavy and light chain variable regions are amplified and a phage display library of combinatorial single chain (scFv) antibodies is constructed.

[0094] Full length antibodies are then produced from selected scFv, as known in the art, and as described below. Purification of monoclonal antibodies may be performed using any method known in the art, for example by affinity chromatography, namely, by using an affinity column to which a specific epitope (or antigen) is conjugated. Alternatively, purification of antibodies may be based on using protein A and protein G column chromatography.

[0095] An exemplary antibody structural unit comprises a tetramer, as known in the art. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light chain" and one “heavy chain The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen (or epitope) recognition.

[0096] Thus, the terms “heavy chain variable region (VH) and “light chain variable region (VL) refer to these heavy and light chains, respectively. More specifically, the variable region is subdivided into hypervariable and framework (FR) regions. Hypervariable regions have a high ratio of different amino acids in each position, relative to the most common amino acid in that position. Four FR regions which have more stable amino acids sequences separate the hypervariable regions. The hypervariable regions directly contact a portion of the antigen's surface. For this reason, hypervariable regions are referred to as "complementarity determining regions", or "CDRs", the CDRs are positioned either at the heavy chain of the antibody (a "heavy chain complementarity determining region") or at the light chain of the antibody (a "light chain complementarity determining region").

[0097] From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the CDR is located.

[0098] Thus, the complementarity determining regions CDRH1, CDRH2 and CDRH3 refer to the three complementarity determining regions starting from the N-terminus of the antibody’s heavy chain (also referred to herein as heavy chain complementarity determining region) and the complementarity determining regions CDRL1, CDRL2 and CDRL3 refer to the three complementarity determining regions starting from the N- terminus of the antibody’s light chain (also referred to herein as light chain complementarity determining region). The present invention encompasses antigen-binding fragments of the isolated anti VACV monoclonal antibodies of the invention.

[0099] As used herein the term "antigen binding fragment" relates to a fragment of the full length antibody which retains the antibody's specificity of binding to VACV. An antigen binding fragment encompasses but is not limited to Fv, single chain Fv (scFv), single chain Fv-Fc (scFv-Fc), Fab’, Fab, F(ab’)2 and F(ab)2.

[0100] Such fragments may be produced by any method known in the art, for example by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).

[0101] Thus, in some embodiments the antibody according to the invention is wherein said antibody is an antibody fragment selected from the group consisting of a single-chain Fv-Fc (scFv-Fc) molecule, single chain Fv (scFv), Fv, Fab’, Fab, F(ab’)2, F(ab)2 and any combination thereof.

[0102] In some specific embodiments, the antibody or antigen-binding fragment thereof is a single chain variable fragment-Fc fragment (scFv-Fc) molecule. In other specific embodiments, the antibody or antigen-binding fragment thereof is a single chain variable fragment (scFv) molecule.

[0103] In some specific embodiments, scFv-Fc molecule is converted to a full-length IgG antibody using methods known in the art.

[0104] As demonstrated in Example 4 below, binding of the antibodies of the invention inhibited the ability of VACV to infect VERO cells or BSC-1 cells. Furthermore, as demonstrated in Example 5 the antibodies of the invention effectively increased animal survival in an animal model of Ectromelia virus (a virus of the pox-virus genus). Therefore, in certain embodiments the isolated monoclonal antibodies of the invention or the antigen-binding fragment thereof inhibits the ability of a pox virus to infect cells.

[0105] By the term “inhibi in the context of the present invention it is meant that the isolated monoclonal antibody of the invention or the antigen-binding fragment thereof impedes, hinders, prohibits or suppresses by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,

[0106] 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,

[0107] 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,

[0108] 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% of the ability of a pox virus to infect cells as compared to a pox virus infection in the absence of the antibody of the invention or the antigen-binding fragment thereof.

[0109] The term “celV or cells as referred to herein is used in its broadest sense.

[0110] In some embodiments the isolated anti VACV monoclonal antibody is a chimeric antibody, a human antibody, or a humanized antibody.

[0111] The term "human antibody" as used herein refers to an antibody that possesses an amino acid sequence corresponding to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies known in the art. This definition specifically excludes a humanized antibody that comprises non-human antigen-binding residues.

[0112] The term “chimeric antibody” as used herein refers to an antibody whose amino acid sequence is partly human and partly of animal (non-human) source. Namely, a portion of the antibody (e.g., the Fc portion) has an amino acid sequence corresponding to that of an antibody produced by a human and another portion of the antibody (e.g., the heavy and light chains variable regions) has an amino acid sequence corresponding to that of an antibody produced by a non-human animal (e.g., a monkey).

[0113] The construction of chimeric antibodies may be performed using any method known in the art, for example as described below.

[0114] For preparing large quantities of the antibody (either chimeric, humanized, or human), a stable cell line expressing the antibody can be prepared, by transfecting cells (e.g., CHO cells) with the Ig expression vector containing both heavy and light chains of the antibody. The antibodies may then be manufactured for example in a state of the art single-use bioreactor system. The antibodies may be purified to clinical grade using well established monoclonal antibody purification methods. Highly anti-VACV antibody producing clones may then be selected and expanded based on antibody levels in the supernatant, as tested by any method known in the art, for example, a VACV-specific ELISA assay, as detailed herein below. A master cell bank, developed for the specific clone, may serve as the starting growing material for all clinical grade batches.

[0115] In some embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 89, CDRH2 denoted by SEQ ID NO. 90, CDRH3 denoted by SEQ ID NO. 91, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 92, a CDRL2 denoted by SEQ ID NO. 93, and a CDRL3 denoted by SEQ ID NO. 94 (designated MV33).

[0116] In some embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 95, CDRH2 denoted by SEQ ID NO. 96, CDRH3 denoted by SEQ ID NO. 97, and a light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 98, a CDRL2 denoted by SEQ ID NO. 99, and a CDRL3 denoted by SEQ ID NO. 100 (designated EV42).

[0117] In some embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 59, CDRH2 denoted by SEQ ID NO. 60, CDRH3 denoted by SEQ ID NO. 61, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 62, a CDRL2 denoted by SEQ ID NO. 63, and a CDRL3 denoted by SEQ ID NO. 64 (designated MV7).

[0118] In various embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 71, CDRH2 denoted by SEQ ID NO. 72, CDRH3 denoted by SEQ ID NO. 73, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 74, a CDRL2 denoted by SEQ ID NO. 75, and a CDRL3 denoted by SEQ ID NO. 76 (designated MV26).

[0119] In certain embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 77, CDRH2 denoted by SEQ ID NO. 78, CDRH3 denoted by SEQ ID NO. 79, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 80, a CDRL2 denoted by SEQ ID NO. 81, and a CDRL3 denoted by SEQ ID NO. 82 (designated MV31). In other embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 83, CDRH2 denoted by SEQ ID NO. 84, CDRH3 denoted by SEQ ID NO. 85, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 86, a CDRL2 denoted by SEQ ID NO. 87, and a CDRL3 denoted by SEQ ID NO. 88 (designated MV32).

[0120] In other embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 101, CDRH2 denoted by SEQ ID NO. 102, CDRH3 denoted by SEQ ID NO. 103, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 104, a CDRL2 denoted by SEQ ID NO. 105, and a CDRL3 denoted by SEQ ID NO. 106 (designated MV49).

[0121] In other embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 119, CDRH2 denoted by SEQ ID NO. 120, CDRH3 denoted by SEQ ID NO. 121, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 122, a CDRL2 denoted by SEQ ID NO. 123, and a CDRL3 denoted by SEQ ID NO. 124 (designated MV57).

[0122] In other embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 125, CDRH2 denoted by SEQ ID NO. 126, CDRH3 denoted by SEQ ID NO. 127, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 128, a CDRL2 denoted by SEQ ID NO. 129, and a CDRL3 denoted by SEQ ID NO. 130 (designated MV60).

[0123] In other embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 53, CDRH2 denoted by SEQ ID NO. 54, CDRH3 denoted by SEQ ID NO. 55, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 56, a CDRL2 denoted by SEQ ID NO. 57, and a CDRL3 denoted by SEQ ID NO. 58 (designated EV4).

[0124] In other embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 65, CDRH2 denoted by SEQ ID NO. 66, CDRH3 denoted by SEQ ID NO. 67, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 68, a CDRL2 denoted by SEQ ID NO. 69, and a CDRL3 denoted by SEQ ID NO. 70 (designated EVI 5).

[0125] In other embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 107, CDRH2 denoted by SEQ ID NO. 108, CDRH3 denoted by SEQ ID NO. 109, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 110, a CDRL2 denoted by SEQ ID NO. 11, and a CDRL3 denoted by SEQ ID NO. 112 (designated EV55).

[0126] In other embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 113, CDRH2 denoted by SEQ ID NO. 114, CDRH3 denoted by SEQ ID NO. 115, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 116, a CDRL2 denoted by SEQ ID NO. 117, and a CDRL3 denoted by SEQ ID NO. 118 (designated EV56).

[0127] In other embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 13 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 14. In certain embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 15 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 16.

[0128] In other embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 3 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 4.

[0129] In some embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 7 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 8.

[0130] In some embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 9 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 10.

[0131] In some embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 11 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 12.

[0132] In certain embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 17 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 18.

[0133] In certain embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 23 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 24.

[0134] In certain embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 25 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 26.

[0135] In some embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 1 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 2.

[0136] In some further embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 5 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 6.

[0137] In certain embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 19 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 20. In certain embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 21 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 22.

[0138] In one embodiment, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 39 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 40, or a variant thereof.

[0139] In one embodiment, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 41 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 42, or a variant thereof.

[0140] In other embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 29 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 30, or a variant thereof.

[0141] In one embodiment, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 33 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 34, or a variant thereof. In one embodiment, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 35 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 36, or a variant thereof.

[0142] In one embodiment, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 43 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 44, or a variant thereof.

[0143] In one embodiment, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 49 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 50, or a variant thereof.

[0144] In one embodiment, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 51 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 52, or a variant thereof.

[0145] In some embodiments, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 37 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 38, or a variant thereof.

[0146] In one embodiment, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 27 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 28, or a variant thereof.

[0147] In one embodiment, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 31 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 32, or a variant thereof.

[0148] In one embodiment, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 45 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 46, or a variant thereof.

[0149] In one embodiment, the present invention provides an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 47 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 48, or a variant thereof.

[0150] In other embodiments the isolated antibody according to the invention is wherein said antibody is an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos 89-94, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 39 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 40.

[0151] In various other embodiments the isolated antibody according to the invention is wherein said antibody is an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 95-100, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 41 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 42.

[0152] In other embodiments the isolated antibody according to the invention is wherein said antibody is an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 59-64, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:29 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 30.

[0153] In various embodiments the isolated antibody according to the invention is wherein said antibody is an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 71-76, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 33 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 34.

[0154] In other embodiments the isolated antibody according to the invention is wherein said antibody is an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 77-82, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 35 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 36.

[0155] In further embodiments the isolated antibody according to the invention is wherein said antibody is an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 83-88, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 37 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 38.

[0156] In various other embodiments the isolated antibody according to the invention is wherein said antibody is an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 101-106, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 43 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 44.

[0157] In various other embodiments the isolated antibody according to the invention is wherein said antibody is an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 119-124, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 49 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 50.

[0158] In various other embodiments the isolated antibody according to the invention is wherein said antibody is an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 125-130, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 51 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 52.

[0159] In other embodiments the isolated antibody according to the invention is wherein said antibody is an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 53-58, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 27 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 28.

[0160] In further embodiments the isolated antibody according to the invention is wherein said antibody is an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 65-70, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 31 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 32. In various other embodiments the isolated antibody according to the invention is wherein said antibody is an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 107-112, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 45 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 46.

[0161] In various other embodiments the isolated antibody according to the invention is wherein said antibody is an anti VACV isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 113-118, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 47 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 48.

[0162] As detailed above, in specific embodiments the isolated antibody according to the invention is an scFv-Fc antibody, namely an antibody comprising a single chain variable fragment comprising both the heavy and light chain CDRs fused to an Fc fragment.

[0163] In another specific embodiment the isolated antibody according to the invention is an scFv-Fc antibody comprising six CDR sequences as denoted by SEQ ID Nos 89-94, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 39 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 40.

[0164] In another specific embodiment the isolated antibody according to the invention is an scFv-Fc antibody comprising six CDR sequences as denoted by SEQ ID Nos. 95- 100, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 41 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 42.

[0165] In another specific embodiment the isolated antibody according to the invention is an scFv-Fc antibody comprising six CDR sequences as denoted by SEQ ID Nos. 59- 64, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 29 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 30. In another specific embodiment the isolated antibody according to the invention is an scFv-Fc antibody comprising six CDR sequences as denoted by SEQ ID Nos. 71- 76, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 33 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 34.

[0166] In another specific embodiment the isolated antibody according to the invention is an scFv-Fc antibody comprising six CDR sequences as denoted by SEQ ID Nos. 77- 82, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 35 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 36.

[0167] In another specific embodiment the isolated antibody according to the invention is an scFv-Fc antibody comprising six CDR sequences as denoted by SEQ ID Nos. 83- 88, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 37 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 38.

[0168] In another specific embodiment the isolated antibody according to the invention is an scFv-Fc antibody comprising six CDR sequences as denoted by SEQ ID Nos. 101- 106, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 43 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 44.

[0169] In another specific embodiment the isolated antibody according to the invention is an scFv-Fc antibody comprising six CDR sequences as denoted by SEQ ID Nos. 119- 124, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 49 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 50.

[0170] In another specific embodiment the isolated antibody according to the invention is an scFv-Fc antibody comprising six CDR sequences as denoted by SEQ ID Nos. 125- 130, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 51 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 52.

[0171] In one specific embodiment the isolated antibody according to the invention is an scFv-Fc antibody comprising six CDR sequences as denoted by SEQ ID Nos. 53-58, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 27 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 28.

[0172] In another specific embodiment the isolated antibody according to the invention is an scFv-Fc antibody comprising six CDR sequences as denoted by SEQ ID Nos. 65- 70, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 31 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 32.

[0173] In another specific embodiment the isolated antibody according to the invention is an scFv-Fc antibody comprising six CDR sequences as denoted by SEQ ID Nos. 107- 112, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 45 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 46.

[0174] In another specific embodiment the isolated antibody according to the invention is an scFv-Fc antibody comprising six CDR sequences as denoted by SEQ ID Nos. 113- 118, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 47 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 48.

[0175] The nucleic acid sequences encoding the heavy and light chains of the antibodies termed herein EV4, MV7, EV15, MV26, MV31, MV32, MV33, EV42, MV49, EV55, EV56, MV57, and MV60 are detailed in Table 1 below. In addition, the amino acid sequences of the heavy and light chains of the antibodies described herein are detailed in Table 2 below. Furthermore, the sequences of the CDRs of the above antibodies are displayed in Table 3 below.

[0176] Table 1: Nucleic acid sequences of the light and heavy chains of the antibodies EV4, MV7, EV15, MV26, MV31, MV32, MV33, EV42, MV49, EV55, EV56, MV57, and MV60 Table 2: Amino acid sequences of the light and heavy chains of the antibodies EV4, MV7, EV15, MV26, MV31, MV32, MV33, EV42, MV49, EV55, EV56, MV57, and MV60

[0177] The CDR sequences are highlighted within the heavy and the light chain amino acid sequences. Table 3: CDR amino acid sequences of the antibodies EV4, MV7, EV15, MV26, MV31, MV32, MV33, EV42, MV49, EV55, EV56, MV57, and MV60

[0178] The present invention also encompasses variants of the heavy and light chain variable regions. The variants may include mutations in the complementarity determining regions of the heavy and light chains which do not alter the activity of the antibodies herein described, or in the framework region.

[0179] By the term “variant” it is meant sequences of amino acids or nucleotides different from the sequences specifically identified herein, in which one or more amino acid residues or nucleotides are deleted, substituted, or added.

[0180] It should be appreciated that by the term "added ", as used herein it is meant any addition of amino acid residues to the sequences described herein.

[0181] Variants encompass various amino acid substitutions. An amino acid “substitution” is the result of replacing one amino acid with another amino acid which has similar or different structural and / or chemical properties. Amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0182] Typically, variants encompass conservative amino acid substitutions. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0183] Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another:

[0184] 1) Alanine (A), Glycine (G);

[0185] 2) Aspartic acid (D), Glutamic acid (E);

[0186] 3) Asparagine (N), Glutamine (Q);

[0187] 4) Arginine (R), Lysine (K);

[0188] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0189] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0190] 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).

[0191] Conservative nucleic acid substitutions are nucleic acid substitutions resulting in conservative amino acid substitutions as defined above.

[0192] Variants in accordance with the invention also encompass non-polar to polar amino acid substitutions and vice-versa.

[0193] As used herein, the term “ amino acid" or “ amino acid residue refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function like the naturally occurring amino acids.

[0194] Variant sequences refer to amino acid or nucleic acids sequences that may be characterized by the percentage of the identity of their amino acid or nucleotide sequences with the amino acid or nucleotide sequences described herein (for example, the amino acid or nucleotide sequences of the heavy and light chains of the antibodies herein described).

[0195] In some embodiments, variant sequences as herein defined refer to nucleic acid sequences that encode the heavy and light chain variable regions, each having a sequence of nucleotides with at least 70% or 75% of sequence identity, around 80% or 85% of sequence identity, around 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity when compared to the sequences of the heavy and light chain variable regions described herein.

[0196] By the term “activity of the antibodies it is meant the ability of the antibodies to bind a VACV protein, and preferably to inhibit the ability of VAC V to infect mammalian cells. The activity of the antibodies can be measured in vivo or in vitro using methods well known in the art, e.g., as described in the Examples below.

[0197] The binding of the antibody of the invention to its target protein may be measured for example using ELISA, biolayer interferometry (BLI), Western blot or immunofluorescence assays.

[0198] The biological activity of the antibodies can be measured for example in a functional assay testing the infectivity of VACV in cultured mammalian cells or testing the antibodies’ ability to protect animals in in vivo models of infection, for example as detailed in the Examples below.

[0199] In another one of its aspects the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof according to the invention. The term nucleic acid” or nucleic acid molecule” as herein defined refers to a polymer of nucleotides, which may be either single- or double-stranded, which is a polynucleotide such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides. The term DNA used herein also encompasses cDNA, i.e., complementary DNA produced from an RNA template by the action of reverse transcriptase (RNA-dependent DNA polymerase).

[0200] The invention further provides an expression vector comprising the isolated nucleic acid molecule as herein defined.

[0201] “Expression vector” sometimes referred to as “expression vehicle” or “expression construct”, as used herein, encompasses vectors such as plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles, which enable the integration of DNA fragments into the genome of the host. Expression vectors are typically self-replicating DNA or RNA constructs containing the desired gene or its fragments, and operably linked genetic control elements that are recognized in a suitable host cell and effect expression of the desired genes. These control elements are capable of effecting expression within a suitable host. The expression vector in accordance with the invention may be competent with expression in bacterial, yeast, or mammalian host cells, to name but few.

[0202] In yet another one of its aspects the present invention provides a host cell transfected with the isolated nucleic acid molecule according to the invention or with the expression vector according to the invention.

[0203] The term "host cells” as used herein refers to cells which are susceptible to the introduction of the isolated nucleic acid molecule according to the invention or with the expression vector according to the invention. Preferably, said cells are mammalian cells, for example CHO cells. Transfection of the isolated nucleic acid molecule or the expression vector according to the invention to the host cell may be performed by any method known in the art.

[0204] The anti VACV antibodies of the invention may be administered in combination with at least one additional therapeutic agent. The term “additional therapeutic agent used herein refers to any agent that may be used for treating pox virus infections or symptoms associated with such infections. In accordance with certain embodiments said at least one additional therapeutic agent is selected from a group consisting of anti VACV monoclonal antibodies, anti VACV VIG, antiviral agents, or anti-inflammatory agents.

[0205] In certain embodiments the additional therapeutic agent is an additional antibody. The term “additional antibody as herein defined refers to antibodies of the invention as well as to an antibody, which is not the antibody according to the invention, which may be used in combination with the antibody of the invention for treating VACV.

[0206] In a specific embodiment, the pharmaceutical composition of the invention is for use in the treatment of a pox-virus infection or for use in reducing undesired side effects of vaccinia-based vaccination.

[0207] The “pharmaceutical composition'’’’ of the invention generally comprises one, but preferably at least two isolated monoclonal antibodies or antigen-binding fragments thereof which bind to vaccinia virus (VACV) as herein defined and one or more pharmaceutically acceptable carriers, excipients and / or diluents as known in the art.

[0208] As used herein the term “pharmaceutically acceptable carrier, excipient or diluent includes any solvents, dispersion media, coatings, antibacterial and antifungal agents and the like, as known in the art. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.

[0209] In other embodiments the pharmaceutical composition according to the invention further comprises an additional therapeutic agent. Non-limiting examples of additional therapeutic agents include additional anti VACV monoclonal antibodies or VIG and antiviral agents. Thus, in specific embodiments the pharmaceutical composition according to the invention further comprises an additional therapeutic agent which is an antiviral agent.

[0210] The antibodies of the invention (e.g., MV33 and EV42 as shown in Example 5) provide protection against a lethal Ectromelia (a poxvirus) infection of BALB / c mice. The data demonstrate the therapeutic value of the chimeric monoclonal antibodies of the invention as a life-saving treatment for pox infection.

[0211] Accordingly, the present invention also provides methods of prophylaxis, treatment or amelioration of pox infection or of symptoms associated with vaccinia-based vaccinations comprising administering to a subject in need thereof a therapeutically effective amount of at least two isolated monoclonal antibodies or antigen-binding fragments thereof which bind to VACV of the invention, or a pharmaceutical composition comprising at least two isolated monoclonal antibodies or antigen-binding fragments thereof which bind to VACV of the invention.

[0212] The term “pox infection is used herein at its broadest sense and refers to any infection caused by an Orthopoxvirus (OPV). Including, for example, Variola virus (VARV), the causative agent of smallpox, monkeypox virus (MPXV), the causative agent of monkeypox, cowpox virus (CPXV), Vaccinia virus (VACV), and ectromelia virus (ECTV).

[0213] The terms "subject" or "patient" are used interchangeably and refer to a subject that may benefit from the present invention such as a mammal (e.g., canine, feline, ovine, porcine, equine, bovine, nonhuman primate, or human). In one specific embodiment the patient is human. Diagnosis of a poxvirus infection may be performed by a skilled physician by methods known in the art. The term “subject in need thereof" in the context of the present invention inter alia refers to mammals and particularly to human subjects suffering from a poxvirus infection or from undesirable symptoms and / or side effects of vaccinia-based vaccination, as defined herein. The term “subject in need thereof" further encompasses mammals and in particular human subjects at risk of being infected by a poxvirus (including but not limited to health care practitioners, subjects having a compromised immune system, elderly people and subjects suffering from other diseases, for example chronic diseases).

[0214] By the term “ prophylaxis"" as herein defined it is meant to provide a "preventive treatment" or "prophylactic treatment", namely acting in a protective manner, to defend against or prevent the appearance of a symptom of a poxvirus infection, vaccination adverse reactions or poxvirus-related onset or progression of disease.

[0215] It is to be understood that the terms "treat", “treating"", “treatment" or forms thereof, as used herein, mean reducing, preventing, curing, reversing, ameliorating, attenuating, alleviating, minimizing, suppressing, or halting the deleterious effects of a disease or a condition or delaying the onset of one or more clinical indications of a poxvirus infection, or poxvirus-related disease, as defined herein. In some embodiments the methods according to the invention are wherein said methods further comprise administering to a subject in need thereof an additional therapeutic agent as herein defined.

[0216] Administration according to the present invention may be performed by any of the following administration routes: oral, intravenous, intramuscular, intraperitoneal, intrathecal, subcutaneous, intra-rectal, intranasal, ocular, intracranial, or topical administration.

[0217] In specific embodiments administration according to the present invention may be performed intravenously. In other specific embodiments administration may be performed intraperitoneally. In other specific embodiments administration may be performed by inhalation.

[0218] The antibodies or antibody fragments as herein defined, or any pharmaceutical compositions comprising the same may be administered to a subject prior to or post disease onset, in a single dose or in multiple doses.

[0219] Thus, in some embodiments the method of prophylaxis, treatment, or amelioration of poxvirus infection according to the invention is where said isolated anti VACV antibodies or any antigen-binding fragments thereof according to the invention, or pharmaceutical composition according to the invention is administered to said subject prior to or after disease onset.

[0220] A therapeutically effective amount of the isolated monoclonal antibody or any antigen-binding fragment thereof according to the invention, or the pharmaceutical composition according to the invention for purposes herein defined is determined by such considerations as are known in the art to cure, arrest or at least alleviate or ameliorate the medical condition. For any preparation used in the methods of the invention, the dosage or the therapeutically effective amount can be estimated initially from in vitro cell culture assays or based on suitable animal models.

[0221] In some embodiments the therapeutically effective amount in accordance with the invention is in the range of 10 pg / kg to about 50 mg / kg.

[0222] In other embodiments the therapeutically effective amount in accordance with the invention is in the range of 0.1 mg / kg to 40 mg / kg, 1 mg / kg to 10 mg / kg, or 5 mg / kg to 10 mg / kg. Specific exemplary doses include but are not limited to 0.5mg / kg, or 0.75 mg / kg, or 2.5 mg / kg, or 5 mg / kg, or 10 mg / kg, or 50mg / kg each given as a single dose.

[0223] The present invention further provides the isolated anti VACV antibodies or any antigen-binding fragment thereof according to the invention or the pharmaceutical composition according to the invention for use in a method of prophylaxis, treatment, or amelioration of a poxvirus infection or poxvirus-related disease as defined herein.

[0224] Still further the present invention provides use of the isolated monoclonal antibodies or antigen-binding fragments thereof of the invention in the preparation of a medicament for the prophylaxis, treatment or amelioration of a poxvirus infection or poxvirus-related disease in a patient.

[0225] In specific embodiments the invention provides an isolated scFv-Fc anti VACV antibody fragment, wherein the antibody fragment comprises: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 89, CDRH2 denoted by SEQ ID NO. 90, CDRH3 denoted by SEQ ID NO. 91, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 92, a CDRL2 denoted by SEQ ID NO. 93, and a CDRL3 denoted by SEQ ID NO. 94 (designated MV33); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 95, CDRH2 denoted by SEQ ID NO. 96, CDRH3 denoted by SEQ ID NO. 97, and a light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 98, a CDRL2 denoted by SEQ ID NO. 99, and a CDRL3 denoted by SEQ ID NO. 100 (designated EV42); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 59, CDRH2 denoted by SEQ ID NO. 60, CDRH3 denoted by SEQ ID NO. 61, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 62, a CDRL2 denoted by SEQ ID NO. 63, and a CDRL3 denoted by SEQ ID NO. 64 (designated MV7); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 71, CDRH2 denoted by SEQ ID NO. 72, CDRH3 denoted by SEQ ID NO. 73, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 74, a CDRL2 denoted by SEQ ID NO. 75, and a CDRL3 denoted by SEQ ID NO. 76 (designated MV26); e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 77, CDRH2 denoted by SEQ ID NO. 78, CDRH3 denoted by SEQ ID NO. 79, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 80, a CDRL2 denoted by SEQ ID NO. 81, and a CDRL3 denoted by SEQ ID NO. 82 (designated MV31); f. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 83, CDRH2 denoted by SEQ ID NO. 84, CDRH3 denoted by SEQ ID NO. 85, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 86, a CDRL2 denoted by SEQ ID NO. 87, and a CDRL3 denoted by SEQ ID NO. 88 (designated MV32); g. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 101, CDRH2 denoted by SEQ ID NO. 102, CDRH3 denoted by SEQ ID NO. 103, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 104, a CDRL2 denoted by SEQ ID NO. 105, and a CDRL3 denoted by SEQ ID NO. 106 (designated MV49); h. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 119, CDRH2 denoted by SEQ ID NO. 120, CDRH3 denoted by SEQ ID NO. 121, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 122, a CDRL2 denoted by SEQ ID NO. 123, and a CDRL3 denoted by SEQ ID NO. 124 (designated MV57); i. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 125, CDRH2 denoted by SEQ ID NO. 126, CDRH3 denoted by SEQ ID NO. 127, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 128, a CDRL2 denoted by SEQ ID NO. 129, and a CDRL3 denoted by SEQ ID NO. 130 (designated MV60); j . a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 53, CDRH2 denoted by SEQ ID NO. 54, CDRH3 denoted by SEQ ID NO. 55, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 56, a CDRL2 denoted by SEQ ID NO. 57, and a CDRL3 denoted by SEQ ID NO. 58 (designated EV4); k. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 65, CDRH2 denoted by SEQ ID NO. 66, CDRH3 denoted by SEQ ID NO. 67, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 68, a CDRL2 denoted by SEQ ID NO. 69, and a CDRL3 denoted by SEQ ID NO. 70 (designated EVI 5); l. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 107, CDRH2 denoted by SEQ ID NO. 108, CDRH3 denoted by SEQ ID NO. 109, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 110, a CDRL2 denoted by SEQ ID NO. 11, and a CDRL3 denoted by SEQ ID NO. 112 (designated EV55); and m. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 113, CDRH2 denoted by SEQ ID NO. 114, CDRH3 denoted by SEQ ID NO. 115, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 116, a CDRL2 denoted by SEQ ID NO. 117, and a CDRL3 denoted by SEQ ID NO. 118 (designated EV56) for use in a method of prophylaxis, treatment, or amelioration of poxvirus infection or poxvirus-related disease.

[0226] In other embodiments the invention provides an isolated scFv-Fc anti VACV antibody fragment, wherein the antibody fragment comprises a heavy chain variable region and a light chain variable region as disclosed in Table 2, or a variant thereof, for use in a method of prophylaxis, treatment, or amelioration of poxvirus infection or poxvirus-related disease.

[0227] It is appreciated that the term purified or "isolated" refers to molecules, such as amino acid or nucleic acid sequences, peptides, polypeptides, or antibodies that are removed from their natural environment, isolated, or separated. An “isolated antibody” is therefore a purified antibody. As used herein, the term purified or purify also refers to the removal of contaminants from a sample.

[0228] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. Disclosed and described, it is to be understood that this invention is not limited to the examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing specific embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

[0229] It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[0230] Throughout this specification and the Examples and claims which follow, 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 integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0231] EXAMPLES

[0232] Materials and Methods

[0233] Viruses and proteins

[0234] VACV Lister (Israeli Ministry of Health) was grown on the chorioallantoic membranes (CAMs) of 12 d SPF embryonated eggs according to the protocol for vaccine production of the Israeli Ministry of Health (Gerichter, 1966). The virus was titrated on Vero (ATCC-CCL-81) cells. The CAMs were ground in glycerol buffer, filtered through a stainless-steel filter, and frozen. This strain was used for non-human primate (NHP) immunization as well as neutralization assays.

[0235] VACV-WR used for immunization was propagated in HeLa (ATCC-CCL-2) cells, prepared as crude, and titrated on BSC-1 cells (ATCC-CCL-26). VACV IHD-J was grown in HeLa cells and titer determined in BSC 1 cells. For commet-inhibition assays, crude viruses were used. For ELISA and panning, the virus was purified by centrifugation in sucrose density gradient (using sucrose cushion) and inactivated using PpL.

[0236] Vaccinia viruses: VACV-WR-vFire, VACV-IHDJ-vFire, VACV-Wyeth-vFire, and VACV-Copenhagen-vFire, expressing both GFP and Firefly luciferase under the control of an early-late promotor (Townsley et al., 2006). Viruses were grown in HeLa cells and titer determined in Vero cells. VACV AH3 was grown in BSC1 cells and inactivated using PpL.

[0237] MV preparations-VACV strains were propagated in HeLa cells in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 1% glutamine, 1% non-essential amino acids (NEAAs), and 0.5% Pen Strep Nistatin in T175 flasks. Cells were infected at a multiplicity of infection (MOI) of 0.5 and harvested 3 d post infection. The virus was recovered by rapidly freeze-thawing the cell pellet three times and sonicating for 1.5 min on ice. Cell debris was removed by centrifugation (at 1,200 rpm for 10 min), and the virus was aliquoted and stored at -80°C.

[0238] Monkeypox viruses 2018 (Mpox2018; accession no. MN648051) and 2022 (Mpox2022; accession no. ON649879) were obtained from clinical samples monitored during the mentioned outbreaks. The surveillance of monkeypox cases, collection, and analysis of the data were executed by health authorities and the Israel Institute for Biological Research as a national reference laboratory for identification and characterization of Mpox according to their mandate provided by the Israeli Ministry of Health. All samples were processed in an anonymized fashion. Viruses were grown in HeLa cells and prepared as described above (“MV preparations”). Titer was determined in BSC1 cells (Erez rt al, 2019; Paran et al, 2022). The final preparation was not further purified as previously described.

[0239] Expression of VACV A33 recombinant protein - A33R gene sequence, corresponding to amino acids 89-185 of A33 protein: SSTTQYDHKESCNGLYYQGSCYILHSDYQLFSDAKANCTAESSTLPNKSDVLIT WLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCVKTMN (SEQ ID NO: 131), was cloned to pRSET vector and expressed in Escherichia coli. N- terminal his-tag was included in the construct to facilitate protein purification. Recombinant proteins: H3, A27, LI, A33 and B5 were purchased from Sino Biological (Beijing, China). D8 protein was purchased from Cusabio Technology (Houston, TX, USA).

[0240] Immunization

[0241] Two non-human female primates (Macaca mulatta Macaques) were immunized with live vaccinia virus (VACV) Lister, IxlO6plaque forming units (pfu) per animal, followed by four monthly booster injections of IxlO9pfu. The first animal received a fifth booster injection of VACV Lister, 7 months after the last boost (48 weeks from the onset of immunization) and the second animal received three additional booster injections of VACV Western Reserve (WR) strain: 33, 53 and 63 weeks from the onset of immunization. Seven days after the last boost, the animals were sacrificed, and samples were taken from the blood, spleen, bone marrow and lymphatic nodes, to isolate mRNA that was subsequently used for amplification of the Heavy and Light chains variable regions (VH and VL, respectively).

[0242] Phage display scFv library construction

[0243] Total RNA was extracted from lymph nodes, spleen, bone marrow and peripheral blood of the immunized Macaque using the RNeasy mini kit (Qiagen GmbH, Germany), and was reverse transcribed into cDNA using the Verso cDNA synthesis kit (Thermo Scientific, USA) and used as a template for Abs variable region coding fragment amplification. Briefly, the VH and VL were amplified from the cDNA, using a set of primers designed to cover all known antibody families of Monkeys. VH and VL were randomly assembled by PCR to construct a combinatorial single-chain (scFv) library (Noy-Porat T. et al. 2016), which was then cloned into phagmid pCC16 plasmid (Rosenfeld R. et al. 2009). A total of 2 * 109independent clones were obtained, representing the library’s complexity.

[0244] The plasmids were transformed into E. coli TGI electro-competent cells (Lucigen, Middleton, WI, USA). The transformed bacteria, containing the final scFv library, were plated on YPD agar (BD, Franklin Lakes, NJ, USA) supplemented with 100 pg / mL ampicillin and 100 mM glucose and, after an overnight culture at 30 °C, were harvested, aliquoted and stored at -80 °C.

[0245] Library Screening

[0246] For phage production 25 mL of logarithmic bacteria culture (OD600 = 0.6) in YPD supplemented with 100 pg / mL ampicillin and 1% glucose (YPD-Amp-Glu) was infected with M13KO7 helper phage (New England Biolabs, USA) at 7 * 109 plaqueforming unit (PFU) per mL (~1 :20 multiplicity of infection) by incubating at 37°C for 30 min without shaking, followed by 30 min at 120 rpm. Infected cells were then harvested (5 min, 4000 rpm) and re-suspended in 100 mL YPD with 100 pg / mL ampicillin and 50 pg / mL kanamycin. After an overnight culture at 30 °C at 200 rpm, cells were pelleted by centrifugation for 10 min, 4000 rpm at 4 °C, and the supernatant containing the phages was filtered through a 0.45-pm filter and then precipitated with 1 / 5 volume of 20% PEG6000 (polyethylene glycol) / 2.5 M NaCl solution for 2 hours on ice. The phages were pelleted by centrifugation of 1 hour at 9000 x g, 4 °C, and re-suspended in 5 mL PBS.

[0247] Library panning was performed against inactivated VACV, H4D-J or AH3 strains, or against recombinant A33 protein, directly absorbed to polystyrene plates (Maxisorb 96-well microtiter plates; Nunc, Denmark) and against biotinylated-VACV H4D-J attached to streptavidin coated magnetic beads (Dynabeads M-280, Invitrogen #11206D). Blocking of plates, beads, and phages was conducted for 60 min using two blocking solutions: 3% BSA (in PBS) or 2% skimmed milk and 0.05% Tween 20 in PBS. The blocking solutions were alternated between panning cycles.

[0248] Phages (approximately 1 x 1011) were incubated with the antigen coated plates for 60 min or with the blocked beads for 90 min, followed by 6-10 washings with phosphate buffered saline Tween-20 (PBST) for both plates and beads. Phages were eluted by incubation with 1ml of 100 mM Triethylamine (Sigma- Aldrich, St. Louis, MO, USA) for 30 min following neutralization (in 200 pl IM Tris-HCl, pH 7.4). The resulting phages were used to infect TGI cells and grown over night for phage enrichment. The culture was collected and lOOpl were used for phage rescue, to prepare the input for the next round of panning. Two additional panning cycles were conducted essentially similarly, with the following modifications: phage production conducted in 10 mL of medium, as described above, 1010and 109phages were used as input (for the second and third cycles, respectively). In total, three rounds of selection were used to isolate the anti-vaccinia antibodies.

[0249] Single colonies were randomly picked from the third panning output, and phages were rescued and tested for their ability to bind VACV.

[0250] ELISA

[0251] Maxisorp 96-well microtiter plates (Nunc, Sigma-Aldrich, St. Louis, MO, USA) were coated overnight with IxlO7pfu / ml of VACV H4D-J (50pl / well) in NaHCO3 buffer (50mM, pH 9.6), washed and blocked with PBST buffer (0.05% Tween 20, 2% BSA in PBS) at room temperature for one hour. Individual phage clones or antibodies were added to the plates for a one-hour incubation; the plates were then washed with PBST and incubated with the detecting antibody: horseradish peroxidase (HRP)-conjugated anti- M13 antibody (GE healthcare, Little Chalfont, UK) for phage clones or anti -human IgG conjugated to alkaline phosphatase (Jackson immunoresearch, West Grove, PA, USA) for full antibodies. Detection of HRP conjugates was achieved with 3, 3', 5,5'- tetramethybenzidine (TMBZE, Millipore, - 89 - Billerica, MA, USA) while detection of alkaline phosphatase conjugates was achieved with SIGMAFAST p-nitrophenyl phosphate tablets (Sigma-Aldrich, St. Louis, MO, USA).

[0252] Nucleic Acid Analysis

[0253] Phagemid DNA was isolated using the QIAprep spin Miniprep kit (Qiagen, GmbH, Hilden, Germany), and scFvs were sequenced by the SeqStudio Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using primers TAB-RI and CBD-AS (NoyPorat T. et al. 2016).

[0254] Production of full-length antibodies

[0255] Phagemid DNA of the antibody clones were isolated using QIAprep spin Miniprep kit and scFv sequences were cloned into a mammalian scFv-Fc expression vector (Noy-Porat et al. 2020). Antibodies were expressed using ExpiCHO™ Expression system (Thermo Scientific, Waltham, MA, USA) and purified on HiTrap Protein-A column (GE healthcare, Little Chalfont, UK).

[0256] Biolayer interferometry for affinity measurements

[0257] Binding studies were carried out using the Octet system (ForteBio, USA, Version 8.1, 2015) that measures biolayer interferometry (BLI). All steps were performed at 30°C with shaking at 1500 rpm in a black 96-well plate containing 200 pl solution in each well. Nickel-coated biosensors (Ni-NTA) were loaded with his-taged proteins (50nM) to reach 0.7-1 nm wavelength shift followed by a wash. The sensors were then reacted for 300 s with increasing concentrations of Antibody (association phase) and then transferred to buffer-containing wells for another 600 s (dissociation phase). Binding and dissociation were measured as changes over time in light interference after subtraction of parallel measurements from unloaded biosensors. Sensorgrams were fitted with a 1 : 1 binding model using the Octet data analysis software 8.1 (Fortebio, USA, 2015), and the presented values are an average of several repeated measurements. In vitro MV neutralization assay

[0258] Vero cells were seeded at 3xl04cells in 1 OO .1 of DMEM supplemented with 10% FBS, 1% Glutamin, 1% non-essential amino acids (NEAA) and 0.5% Pen Step Nistatin in 96 well plates and incubated overnight at 37°C for 1 hour in 5%CO2. Antibodies were serially diluted 2-fold or 10-fold starting from 20 pg / ml, added to 0.1 MOI vaccinia WR- vFIRE virus and 1% guinea pig complement sera and incubated at 37°C for 1 hour in 5%CO2. The antibody-virus mixtures were then transferred to 96-well plates containing the cell monolayers and plates were incubated for 24 hours at 37°C and 5%CO2. Following incubation, the cells were washed once with PBS before lysis with 25pl / well of cell culture 5xlysis buffer reagent (Promega) and incubated for 15 minutes at room temperature. Luciferase assay substrate (25 pl per well) was added, and the luminescence was immediately measured using infinite 200pro reader (Tecan). The neutralization value was calculated using the following equation:

[0259] % Neutralization = 100 100

[0260] Each antibody dilution was run in duplicate, with appropriate positive and negative controls.

[0261] In vitro Comet inhibition assay

[0262] BSC-1 cells were seeded in 12-well plates (3xl05cells / well) and incubated overnight at 37°C. Cells were infected with 10 pfu / well vaccinia H4DJ and incubated at 37°C for 1.5 hours. After washing with MEM containing 2% FBS, the cells were overlaid with Iml / well of the same medium supplemented with 1.5% carbonate bicarbonate (overlay). Antibodies were then added to the overlay at concentrations ranging from 1.25- lOpg / ml in triplicates. The cells were incubated for 2 days at 37°C and then fixed and stained with crystal violet.

[0263] Plaque reduction neutralization test (PRNT)

[0264] PRNT50 was conducted as previously described (Eyal et al. 2005). Briefly, all serum samples were heat inactivated (HI) (at 56 °C for 30 min), then diluted in twofold serial dilutions (between 1 : 100 and 1 :25,600). Monoclonal antibodies were serially diluted twofold starting from 20 pg / ml. Diluted sera were incubated with 500 pfu / mL of the following VACV strains: Lister, Copenhagen-vFIRE, WR-vFIRE, IHD-J-vFIRE and Wyeth-vFIRE or Mpox 2018 / 2022 (1 h at 37 °C). Diluted antibodies were incubated with 500 pfu / m of either Mpox 2018 or Mpox 2022 (1 h at 37 °C). Vero cells were infected with the virus-serum or virus-antibodies mixtures and incubated at 37°C and 5% CO2 for Ih. Overlay was then applied, and the plates were incubated for additional 72 hours. Following incubation, overlay was aspirated, and the cells were fixed and stained with 1 ml / well of crystal violet solution. Plaques were counted and NT50 was calculated using GraphPad Prism 6 software (GraphPad Software Inc., San Diego, CA, USA).

[0265] In vivo protection experiments

[0266] Ectromelia experiment - Female BALB / c mice between 5-8 weeks of age were purchased from Charles River laboratories, UK. Mice were anesthetized (Ketamine 75mg / kg, Xylazine 7.5mg / kg in PBS) and infected by intranasal route (i.n.) with a lethal dose of 50pfu (50LD50) of Ectromelia. Treatment was performed intraperitoneally (i.p.) on different days post exposure with a single dose of the following monoclonal antibodies: MV33, EV42, MV49, EV55, EV56, MV57, MV60 and VIG (Omrix) or with a combination of MV33 and EV42. Animals were monitored daily for morbidity (assessed by weight loss) and mortality for 21 days. Infected untreated mice and mice treated with an irrelevant antibody (BLN12), were used as control.

[0267] MPXV experiment - Female Cast / EiJ mice (8-10 weeks, 10-15 gr.) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and acclimatized under supervision for a week prior to the experiment. Mice were challenged (i.n.) with 3.5xl06PFU MPXV (1-2 LD50) (2018; clade lib), treated with MV33+EV42 (100+100 pg) at 2dpi or left untreated.

[0268] Example 1

[0269] Isolation of antibodies from immunized non-human primates

[0270] Tissue samples were taken from two non-human primates, immunized with VACV, as described above. Total RNA was extracted, reverse-transcribed into cDNA and further used for construction of a phage-display library, as detailed above. Individual phage clones were selected as described above. After panning and screening of the phage display library, 13 different antibodies, termed EV4, MV7, EV15, MV26, MV31, MV32, MV33, EV42, MV49, EV55, EV56, MV57 and MV60 were isolated and sequenced, as described above. The nucleic acid sequences of the heavy and the light chains of the antibodies are shown in Table 1 above. The amino acid sequences of the heavy and the light chains of the antibodies are shown in Table 2 above and the amino acid sequences of the heavy chain CDRs and light chains CDRs of the antibodies are shown in Table 3 above.

[0271] Example 2

[0272] Characterization of anti-vaccinia antibodies specificity

[0273] The specific binding of the various antibodies to VACV and to its major surface proteins was determined by ELISA assay. Proteins H3, D8, A27, and LI are the major surface proteins of the MV form, while A33 and B5 are known as the major surface proteins of the EV form of the virus. Based on this assay (Fig. 1), four mAbs were found to bind H3 protein: MV7, MV26, MV31, and MV32; two antibodies were found to bind D8 protein: MV33 and MV49; and additional three antibodies were characterized as anti- A33 protein: EVI 5, EV42, and EV56. Antibodies EV4 and EV55 could not bind any of the proteins examined and therefore probably recognize a different epitope on the EV form (Fig. 1). The set of the selected antibodies can therefore recognize both forms of the virus (MV and EV) and bind variable epitopes of the virus.

[0274] Example 3

[0275] Affinity Characterization of the anti-vaccinia antibodies

[0276] The affinity of the mAbs toward the whole virus was assessed next. The binding profile of all mAbs was obtained by ELISA against VACV H4D-J. Based on their binding profiles, the mAbs could be divided into two groups, with one group, consisting of mabs EV4, EVI 5, EV55, and EV56, exhibiting a distinct binding profile, reflecting relatively low binding affinity towards the virus compared to the second group. Interestingly, all these mAbs recognize the EV form of the virus. Nevertheless, EV42, which also binds the EV form, displayed substantially stronger binding to the virus, although it demonstrated a lower Bmax value than the other mAbs in this group, which probably reflects fewer available epitopes. The remaining mAbs showed similar binding profiles towards the virus.

[0277] Those antibodies to which a specific target protein could be assigned, were analyzed by Biolayer interferometry (BLI) to determine their affinity (KD values) towards the specific protein. Antigens, carrying his tag, were immobilized to nickel sensors, and interacted with increasing amounts of the relevant antibody. H3 protein was used for affinity measurements of MV7, MV26, MV31 and MV32. D8 protein was used for affinity measurements of MV33 and MV49. A33 protein was used for affinity measurements of EV42. Binding kinetics were fitted using the 1: 1 binding model. The antibodies exhibited good to excellent affinities, with MV33 showing extremely high affinity. The association rate of this antibody was fast, 2.1 x 106, while the dissociation rate was extremely slow (below the OctetReddevice detection limit of 1 x IO-7s-1) and hence could not be measured. This puts the affinity of the mAb in the sub-pM range (koff <1 x 10’7s ’1(Table 4).

[0278] Table 4: Binding characteristics of the antibodies determined using biolayer interferometry. MV49 also exhibited an extremely high affinity towards D8 (0.011 nM) despite its inferior neutralization efficiency compared to MV33 (see Table 5 below). EV42 was found to recognize A33 protein with a very high affinity (0.095nM), while all four mAbs recognizing the H3 protein exhibited similar affinities towards the protein, in the low nano molar range (2.7-7.4nM).

[0279] Example 4

[0280] Antibody mediated in vitro neutralization of VACV

[0281] The mAbs were subjected to an in vitro neutralization assay to characterize their neutralization potential and identify the form of the virus they recognize (MV or EV). All antibodies were first tested for their ability to neutralize MV virions in a high-throughput MV-neutralization test. Those antibodies that did not show any neutralization potential in this test, were further examined in an EV-neutralization test (comet assay). All neutralization assays included VIG samples for qualitative comparison. All antibodies were found to be neutralizing, showing diverse neutralization potential. The capability of each antibody to neutralize Vaccinia is shown as the concentration of antibody needed to neutralize 50% of the virus (for MV-neutralizing antibodies (NT50)) or as a positive result in an EV-neutralization comet assay (Table 5).

[0282] Six of the anti -MV mAbs exhibited effective MV neutralization capabilities that were superior to those exhibited by VIG (NT50 of VIG in this assay was 2-5 pg / ml), of which MV33 was found to be highly potent.

[0283] Five mAbs were able to neutralize the EV form of the virus, with EV42 exhibiting the most effective neutralization (Fig. 2). This mAb was able to block virus spread and comet formation even at a low antibody concentration (full inhibition at 5 pg / mL and partial but substantial inhibition at 2.5 pg / mL) and was more effective than VIG.

[0284] Despite their comparable affinities, the anti-H3 mAbs differed in their ability to neutralize the virus, with MV31 and MV32 showing strong neutralization, MV26 exhibiting an order of magnitude lower neutralization potency and MV7 showing even lower neutralization capabilities (Table 5). These differences may suggest that the mAbs bind different epitopes on the H3 protein, with some epitopes allowing more efficient neutralization than others. Table 5: in vitro neutralization assay.

[0285] Given the notable binding and neutralization capabilities of the isolated mAbs against VACV, it was next examined whether these mAbs can also neutralize Mpox virus. MV33 mAb was selected as a representative of the antibodies recognizing D8 protein and MV32 as a representative of the mAbs binding H3. Both were tested in an in vitro MV neutralization assay against both Mpox 2018 and Mpox 2022 strains (Fig. 3). Both antibodies exhibited very efficient neutralization of the two Mpox strains with similar NT50 values (0.5-0.99 and 0.03-0.06 pg / ml forMV32 and MV33, respectively). However, the maximum neutralization percentage exhibited by both mAbs reached only around 60%. This may suggest a slightly reduced neutralization efficiency, compared to VACV, or can suggest the presence of EV particles in the virus samples. Nevertheless, both mAbs neutralized Mpox better than VIG (Fig. 3), with MV33 showing significantly better neutralization than VIG of both strains (p=0.01 and p=0.0003 for 2018 and 2022 strain, respectively) and MV32 exhibiting significantly better neutralization of 2022 strain (p=0.005). The Mpox neutralization potential of EV42, which binds the A33 protein and neutralizes the EV form of the virus, could not be accurately assessed in vitro since the comet-inhibition assay is not currently adapted for Mpox.

[0286] Example 5

[0287] In vivo therapeutic potential

[0288] The ability of selected antibodies to protect mice from morbidity and mortality following infection, was examined by in vivo experiments. Mice were infected with SOLDso of Ectromelia virus (ECTV), closely related family member of the VACV which produces, in mice, a similar disease manifestation as seen in humans following VACV infection.

[0289] A single dose treatment (200pg) with the following antibodies: MV33, EV42, MV49, EV55, EV56, MV57, MV60 or 4mg of VIG (Omrix), was given 24 hours post infection. Infected untreated mice or mice infected and treated with irrelevant antibody served as control. Morbidity and mortality were monitored for 21 days. As can be seen in Figure 4A, all untreated mice succumbed to infection by day 10. Mice treated with either MV33, MV49 or VIG resulted in partial protection of 65, 15 or 50%, respectively, while treatment with EV42, resulted in 100% survival. The four additional antibodies EV55, EV56, MV57 or MV60 (Figure 5) didn't show any improvement of body weight (not shown) nor survival rate as compared to infected untreated mice.

[0290] Next, the putative positive effect of a mixture of antibodies was evaluated. To that aim, mice were infected, and treated 24 hours later with either a half dose (lOO+lOOpg) or double dose (200+200pg) of MV33 and EV42. Both concentrations of the antibody combinations resulted in full protection (Figure 4B).

[0291] Based on these results, the therapeutic window of both MV33 and EV42 alone or in combination, was further tested at days 2-5 post infection. VIG (Omrix) served as a gold standard control. As seen in Figures 4C-F, mice infected and treated with irrelevant antibody resulted in 0-15% survival rate. VIG treatment resulted in partial protection of 85%, 35% and 35% on days 2, 3 and 4 respectively, and no protection at day 5. However, a dramatic improvement resulting in full protection was observed when mice treated with either 200 pg of either MV33, EV42 or a combination of both (half dose of each) at day 2 or 3 post infection (Figures 4C and 4D, respectively). On day 4 post infection (Figure 4E), mice treated with EV42 or MV33 alone resulted in partial but significant protection of 65% and 80%, respectively, while treatment with a mixture of MV33 and EV42 (with half doses of each) resulted in 100% survival. Similar results were observed at day 5 (Figure 4F), where treatment of either MV33 or EV42 had a partial protection effect but the combination of both resulted in full protection. These results suggest a potential synergistic effect of these two antibodies which are directed against both viral infectivity forms (MV and EV), when acting at the same time.

[0292] Example 6

[0293] Treatment with MV33 and EV42 antibodies inhibits ECTV replication and dissemination.

[0294] To further investigate the impact of these two mAbs on virus spread throughout the disease progression, whole-body bioluminescent imaging (BLI) was conducted using a recombinant ECTV expressing firefly Luciferase (ECTV-Luc). The generation of ECTV-Luc led to lethal dose differences. This was adjusted by using 140 PFU (20 LD50) for ECTV-Luc in comparison to 50 PFU (50 LD50) for the parental virus used in Example 5. Mice were infected and treated with a single dose of 200 pg of either MV33, EV42, a combination of the two (100+100 pg), VIGIV (4 mg) or with an isotype control (BLN12; 200 pg) at 3- dpi. Whole body images were performed, starting 24 hours after treatment namely, on days 4, 6, 8, 10, 12 and 14 post infection. A similar experiment took place in which the treatment commenced on day 5 post infection (5 -dpi). Whole body images were performed, starting 24 hours after treatment namely, on days 6, 8, 10, 12 and 14 post infection. The total photon flux values were quantified, and the signal / noise intensity is presented in Figures 6 and 7.

[0295] At 4 dpi, the luciferase signal in the isotype control antibody (BLN12) treated mice was evident mainly in the nasal cavity and the spleen. With time, the signal intensity increased mainly at the thoracic and abdominal regions encompassing the spleen, liver, and lungs (day 8 post infection), along with body weight loss followed by death 2-3 days later. In contrast, all MV33, EV42 or MV33+EV42 mAbs treated mice showed a reduction in bioluminescence signal (Figure 6). This reduced signal correlates with body weight gain followed by complete recovery. VIGIV treatment resulted in reduced luciferase signal, yet 3 out of 4 mice exhibited delayed clearance (day 12; Figure 6), a phenomenon not observed with the humanized mAbs. It is noted that the apparent enhancement of signal in VIGIV treated group on day 12 is due to a lower presented dynamic range (5xl06- 5xl07photon / second / cm2 / steradian). As mAbs treatment ameliorated the disease and prevented mortality even when provided at later time points post infection, the effect of these antibodies on viral dissemination was also examined in mice treated at 5 dpi. As seen in Figure 7, mice treated with a single dose of either MV33 or EV42 exhibited a partial signal reduction as compared with the isotype control, which corresponded with their partial mortality rate. However, the combined therapy effect was evident, and luciferase signal was reduced with time.

[0296] Example 7

[0297] Treatment with MV33 and EV42 antibodies reduces viral loads in target organs.

[0298] To obtain a focused and quantitative evaluation of the impact of the mAbs on viral dissemination and based on the BLI data of day 8 post infection, viral loads were assessed in target organs, namely spleen, liver, and lung. Mice were infected with 50 PFU of ECTV and treated at either 3 or 5 dpi with MV33 (200 pg), EV42 (200 pg), MV33+EV42 (100+100 pg), VIGIV (4mg), or with an isotype control (BLN12; 200 pg). At 8 dpi, mice were sacrificed, and their organs were collected for viral load analysis by plaque assay. As shown in Figure 8A, C, E, in the BLN12 treated groups at 3 dpi, the spleen, liver and lung already reached very high titers of 1.9xl08, 1.6xl07and 8.2xl07PFU / gr tissue, respectively. In the spleen, treatment with MV33 or EV42 alone at 3 dpi resulted in a nearly one-log and two-log reduction in viral titers (1.9xl07PFU / gr and 2.4xl06PFU / gr, respectively) (Figure 8A). However, a significant five-log reduction (4xl02PFU / gr) to nearly undetectable levels was observed in the MV33+EV42 treated group (Figure 8A). In the liver, the effect was even more pronounced (Figure 8C). The MV33 mAbs treated group resulted in almost undetectable levels (7.6x*102PFU / gr), while virus was undetectable in either the EV42 or MV33+EV42 treated groups. Treatment at 5 dpi was less effective than treatment at 3 dpi in reducing viral loads at this time point (8 dpi). However, the combination of MV33+EV42 resulted in a significant one-log reduction in the spleen (Figure 8B) and a five-log reduction in the liver (Figure 8D), reaching undetectable levels. Surprisingly, no significant reduction in viral loads was observed in the lungs of all treated groups (Figure 8E and 8F). When comparing these results to the BLI data (Figure 6 and 7), it is important to clarify that the bioluminescence images are presented at a certain dynamic range and thus do not provide the full and precise data as observed with the viral load analysis. Next, it was determined whether the combined effect of MV33+EV42 antibodies is additive or synergistic. To that aim, the average viral load reduction of each antibody separately or in combination, was calculated as compared with the isotype control group. A synergistic effect was determined when the fold change of the reduction of both antibodies was greater than the sum of the fold-change reduction of each antibody alone. For example, when mice were treated at 3 dpi, the viral load in the spleen of MV33 or EV42 treated mice was reduced by 10 or 80-fold, respectively, while the combination treatment resulted in a 4.7xl03-fold reduction (Figure 8A). This synergistic effect was evident in the spleen and liver of mice treated at 3 or 5 dpi, respectively (Figure 8A and 8D). These results further indicate a synergistic mode of action of both MV33+EV42 mAbs.

[0299] Example 8

[0300] Treatment with MV33 and EV42 antibodies protects against MPXV challenge in mice.

[0301] Based on the observation that both MV33 and EV42_mAbs conferred protection against ECTV infection, along with their demonstrated ability to neutralize several other orthopoxviruses in vitro, their potential protective effects in vivo against MPXV, were assessed. Since commonly used laboratory mice strains like BALB / c are resistant to MPXV, CastZEiJ mice were utilized. These mice were previously shown to be sensitive to previous isolates of MPXV exhibiting weight loss and death following MPXV infections [Earl et al (2012)]. Mice were intranasally challenged with 3.5x106PFU / mouse of MPXV (clade lib) and treated at 2 dpi with MV33+EV42 (100 pg each) by intraperitoneal injection. As shown in Figures 9A, and 9B, in the untreated group, all infected mice displayed morbidity, with a mortality rate of 75%. However, in the treated group, all mice survived, and no signs of morbidity could be observed. Based on these results the therapeutic window was extended to 3-5 dpi. As seen in Figure 9C, all infected untreated mice start to lose weight at around day 5 post infection, resulting in a mortality rate of 35%. Treated mice at 3 dpi showed significantly reduced morbidity, evident in improved body weight compared to infected untreated mice (Figure 9C) and were fully protected (Figure 9D). However, when extending the therapeutic window to 4 and 5 dpi, MV33+EV42 treatment resulted in partial reduction in morbidity and mortality effect. These results underscore the therapeutic potential of both MV33+EV42 when administered up to 3 dpi.

Claims

CLAIMS:

1. A pharmaceutical composition comprising as an active ingredient at least two isolated monoclonal antibodies or antigen-binding fragments thereof which bind to VACV, and a pharmaceutically acceptable carrier, excipient, or diluent, wherein one of said at least two isolated monoclonal antibodies comprises complementarity determining regions selected from a group consisting of: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 89, CDRH2 denoted by SEQ ID NO. 90, CDRH3 denoted by SEQ ID NO. 91, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 92, a CDRL2 denoted by SEQ ID NO. 93, and a CDRL3 denoted by SEQ ID NO. 94 (designated MV33); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 59, CDRH2 denoted by SEQ ID NO. 60, CDRH3 denoted by SEQ ID NO. 61, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 62, a CDRL2 denoted by SEQ ID NO. 63, and a CDRL3 denoted by SEQ ID NO. 64 (designated MV7); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 71, CDRH2 denoted by SEQ ID NO. 72, CDRH3 denoted by SEQ ID NO. 73, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 74, a CDRL2 denoted by SEQ ID NO. 75, and a CDRL3 denoted by SEQ ID NO. 76 (designated MV26); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 77, CDRH2 denoted by SEQ ID NO. 78, CDRH3 denoted by SEQ ID NO. 79, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 80, a CDRL2 denoted by SEQ ID NO. 81, and a CDRL3 denoted by SEQ ID NO. 82 (designated MV31); e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 83, CDRH2 denoted by SEQ ID NO. 84, CDRH3 denoted by SEQ ID NO. 85, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 86, a CDRL2 denoted by SEQ ID NO. 87, and a CDRL3 denoted by SEQ ID NO. 88 (designated MV32);f. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 101, CDRH2 denoted by SEQ ID NO. 102, CDRH3 denoted by SEQ ID NO. 103, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 104, a CDRL2 denoted by SEQ ID NO. 105, and a CDRL3 denoted by SEQ ID NO. 106 (designated MV49); g. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 119, CDRH2 denoted by SEQ ID NO. 120, CDRH3 denoted by SEQ ID NO. 121, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 122, a CDRL2 denoted by SEQ ID NO. 123, and a CDRL3 denoted by SEQ ID NO. 124 (designated MV57); h. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 125, CDRH2 denoted by SEQ ID NO. 126, CDRH3 denoted by SEQ ID NO. 127, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 128, a CDRL2 denoted by SEQ ID NO. 129, and a CDRL3 denoted by SEQ ID NO. 130 (designated MV60); and a second of said at least two isolated monoclonal antibodies comprises complementarity determining regions selected from a group consisting of: i. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 95, CDRH2 denoted by SEQ ID NO. 96, CDRH3 denoted by SEQ ID NO. 97, and a light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 98, a CDRL2 denoted by SEQ ID NO. 99, and a CDRL3 denoted by SEQ ID NO. 100 (designated EV42); j . a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 53, CDRH2 denoted by SEQ ID NO. 54, CDRH3 denoted by SEQ ID NO. 55, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 56, a CDRL2 denoted by SEQ ID NO. 57, and a CDRL3 denoted by SEQ ID NO. 58 (designated EV4);k. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 65, CDRH2 denoted by SEQ ID NO. 66, CDRH3 denoted by SEQ ID NO. 67, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 68, a CDRL2 denoted by SEQ ID NO. 69, and a CDRL3 denoted by SEQ ID NO. 70 (designated EVI 5); l. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 107, CDRH2 denoted by SEQ ID NO. 108, CDRH3 denoted by SEQ ID NO. 109, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 110, a CDRL2 denoted by SEQ ID NO. 11, and a CDRL3 denoted by SEQ ID NO. 112 (designated EV55); and m. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 113, CDRH2 denoted by SEQ ID NO. 114, CDRH3 denoted by SEQ ID NO. 115, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 116, a CDRL2 denoted by SEQ ID NO. 117, and a CDRL3 denoted by SEQ ID NO. 118 (designated EV56).

2. The pharmaceutical composition of claim 1, wherein one of said at least two isolated monoclonal antibodies comprises: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 89, CDRH2 denoted by SEQ ID NO. 90, CDRH3 denoted by SEQ ID NO. 91, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 92, a CDRL2 denoted by SEQ ID NO. 93, and a CDRL3 denoted by SEQ ID NO. 94; and a second of said at least two isolated monoclonal antibodies comprises: b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 95, CDRH2 denoted by SEQ ID NO. 96, CDRH3 denoted by SEQ ID NO. 97, and a light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 98, a CDRL2 denoted by SEQ ID NO. 99, and a CDRL3 denoted by SEQ ID NO. 100.

3. The pharmaceutical composition according to claim 1, wherein one of said at least two isolated monoclonal antibodies comprises a heavy chain variable region and a light chain variable region, wherein: a. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 13 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 14; or b. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 3 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 4; or c. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 7 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 8; or d. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 9 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 10; or e. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 11 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 12; or f. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 17 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 18; org. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 23 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 24; or h. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 25 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 26; and a second of said at least two isolated monoclonal antibodies comprises a heavy chain variable region and a light chain variable region, wherein: i. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 15 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 16; or j. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 1 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 2; or k. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 5 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 6; or l. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 19 and wherein said light chain variable region is encodedby a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 20; or m. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 21 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 22.

4. The pharmaceutical composition according to any one of the preceding claims, wherein one of said at least two isolated monoclonal antibodies comprises: a. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 39 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 40, or a variant thereof; or b. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 29 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 30, or a variant thereof; or c. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 33 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 34, or a variant thereof; or d. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 35 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 36, or a variant thereof; or e. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 37 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 38, or a variant thereof; or f. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 43 or a variant thereof and a light chain variable regioncomprising the amino acid sequence denoted by SEQ ID NO. 44, or a variant thereof; or g. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 49 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 50, or a variant thereof; or h. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 51 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 52, or a variant thereof and a second of said at least two isolated monoclonal antibodies comprises: i. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 41 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 42, or a variant thereof; or j . a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 27 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 28, or a variant thereof; or k. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 31 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 32, or a variant thereof; or l. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 45 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 46, or a variant thereof; or m. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 47 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 48, or a variant thereof.

5. An isolated monoclonal antibody or antigen-binding fragments thereof which binds to the MV form of VACV, wherein said isolated monoclonal antibody comprises complementarity determining regions selected from a group consisting of: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 89, CDRH2 denoted by SEQ ID NO. 90, CDRH3 denoted by SEQ ID NO. 91, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 92, a CDRL2 denoted by SEQ ID NO. 93, and a CDRL3 denoted by SEQ ID NO. 94 (designated MV33); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 59, CDRH2 denoted by SEQ ID NO. 60, CDRH3 denoted by SEQ ID NO. 61, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 62, a CDRL2 denoted by SEQ ID NO. 63, and a CDRL3 denoted by SEQ ID NO. 64 (designated MV7); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 71, CDRH2 denoted by SEQ ID NO. 72, CDRH3 denoted by SEQ ID NO. 73, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 74, a CDRL2 denoted by SEQ ID NO. 75, and a CDRL3 denoted by SEQ ID NO. 76 (designated MV26); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 77, CDRH2 denoted by SEQ ID NO. 78, CDRH3 denoted by SEQ ID NO. 79, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 80, a CDRL2 denoted by SEQ ID NO. 81, and a CDRL3 denoted by SEQ ID NO. 82 (designated MV31); e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 83, CDRH2 denoted by SEQ ID NO. 84, CDRH3 denoted by SEQ ID NO. 85, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 86, a CDRL2 denoted by SEQ ID NO. 87, and a CDRL3 denoted by SEQ ID NO. 88 (designated MV32); f. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 101, CDRH2 denoted by SEQ ID NO. 102, CDRH3 denoted by SEQ ID NO. 103, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 104, a CDRL2 denoted by SEQID NO. 105, and a CDRL3 denoted by SEQ ID NO. 106 (designated MV49); g. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 119, CDRH2 denoted by SEQ ID NO. 120, CDRH3 denoted by SEQ ID NO. 121, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 122, a CDRL2 denoted by SEQ ID NO. 123, and a CDRL3 denoted by SEQ ID NO. 124 (designated MV57); and h. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 125, CDRH2 denoted by SEQ ID NO. 126, CDRH3 denoted by SEQ ID NO. 127, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 128, a CDRL2 denoted by SEQ ID NO. 129, and a CDRL3 denoted by SEQ ID NO. 130 (designated MV60).

6. The isolated monoclonal antibody according to claim 5, wherein said isolated monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein: a. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 13 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 14; or b. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 3 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 4; or c. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 7 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 8; ord. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 9 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 10; or e. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 11 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 12; or f. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 17 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 18; or g. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 23 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 24; or h. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 25 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 26.

7. The isolated monoclonal antibody according to any one of claims 5 or 6, wherein said isolated monoclonal antibody comprises: a. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 39 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 40, or a variant thereof; or b. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 29 or a variant thereof and a light chain variable regioncomprising the amino acid sequence denoted by SEQ ID NO. 30, or a variant thereof; or c. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 33 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 34, or a variant thereof; or d. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 35 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 36, or a variant thereof; or e. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 37 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 38, or a variant thereof; or f. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 43 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 44, or a variant thereof; or g. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 49 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 50, or a variant thereof; or h. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 51 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 52, or a variant thereof.

8. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 5 to 7, wherein said antibody or antigenbinding fragment thereof binds to at least one surface protein of the MV form of VAC V.

9. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 8, wherein said at least one surface protein of the MV form of VACV is selected from a group consisting of H3, D8, A27, and LI.

10. An isolated monoclonal antibody or antigen-binding fragments thereof which binds to the EV form of VACV, wherein said isolated monoclonal antibody comprises complementarity determining regions selected from a group consisting of: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 95, CDRH2 denoted by SEQ ID NO. 96, CDRH3 denoted by SEQ ID NO. 97, and a light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 98, a CDRL2 denoted by SEQ ID NO. 99, and a CDRL3 denoted by SEQ ID NO. 100 (designated EV42); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 53, CDRH2 denoted by SEQ ID NO. 54, CDRH3 denoted by SEQ ID NO. 55, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 56, a CDRL2 denoted by SEQ ID NO. 57, and a CDRL3 denoted by SEQ ID NO. 58 (designated EV4); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 65, CDRH2 denoted by SEQ ID NO. 66, CDRH3 denoted by SEQ ID NO. 67, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 68, a CDRL2 denoted by SEQ ID NO. 69, and a CDRL3 denoted by SEQ ID NO. 70 (designated EVI 5); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 107, CDRH2 denoted by SEQ ID NO. 108, CDRH3 denoted by SEQ ID NO. 109, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 110, a CDRL2 denoted by SEQ ID NO. 11, and a CDRL3 denoted by SEQ ID NO. 112 (designated EV55); and e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 113, CDRH2 denoted by SEQ ID NO. 114, CDRH3 denoted by SEQ ID NO. 115, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 116, a CDRL2 denoted by SEQ ID NO. 117, and a CDRL3 denoted by SEQ ID NO. 118 (designated EV56).

11. The isolated monoclonal antibody according to claim 10, wherein said isolated monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein: a. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 15 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 16; or b. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 1 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 2; or c. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 5 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 6; or d. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 19 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 20; or e. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 21 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 22.

12. The isolated monoclonal antibody according to any one of claims 10 or 11, wherein said isolated monoclonal antibody comprises: a. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 41 or a variant thereof and a light chain variable regioncomprising the amino acid sequence denoted by SEQ ID NO. 42, or a variant thereof; or b. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 27 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 28, or a variant thereof; or c. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 31 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 32, or a variant thereof; or d. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 45 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 46, or a variant thereof; or e. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 47 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 48, or a variant thereof.

13. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 10 to 12, wherein said antibody or antigenbinding fragment thereof binds to at least one surface protein of the EV form of VAC V.

14. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 13, wherein said at least one surface protein of the EV form of VACV is A33 or B5.

15. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 5 to 9, for use in combination with at least one isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 10 to 14.

16. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 10 to 14, for use in combination with at least one isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 5 to 9.

17. The pharmaceutical composition or the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein said antibody or antigen-binding fragment thereof binds VACV with a KD of about 1 pM to about 10 nM.

18. The pharmaceutical composition or the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein said antibody or antigen-binding fragment thereof inhibits the ability of a pox virus to infect mammalian cells.

19. The pharmaceutical composition or the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein said antibody or antigen-binding fragment thereof is a singlechain Fv-Fc (scFv-Fc) molecule, single-chain Fv (scFv), Fv, heavy chain variable region, light chain variable region, Fab, F(ab)2(or any combination thereof.

20. The pharmaceutical composition or the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein said antibody or antigen-binding fragment thereof is a scFv- Fc molecule.

21. The pharmaceutical composition or the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein said antibody is a neutralizing antibody.

22. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or any antigen-binding fragment thereof according to any one of the preceding claims.

23. An expression vector comprising the isolated nucleic acid molecule according to claim 22.

24. A host cell transfected with the expression vector according to claim 23.

25. The pharmaceutical composition according to any one of claims 1 to 4, wherein said pharmaceutical composition further comprises an additional therapeutic agent.

26. A method of prophylaxis, treatment, or amelioration of infections caused by pox viruses, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition accordingto any one of claims 1 to 4, at least one, or at least two of the isolated monoclonal antibodies or antigen-binding fragments thereof according to any one of claims 5 to 14.

27. A method of reducing side effects of vaccinia-based vaccines, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 4, at least one, or at least two of the isolated monoclonal antibodies or antigenbinding fragments thereof according to any one of claims 5 to 14.

28. The method according to claim 26 or 27, wherein said method further comprises administering to a subject in need thereof an additional therapeutic agent.

29. A method of inhibiting the ability of VACV to infect mammalian cells in a subject comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 4, at least one, or at least two of the isolated monoclonal antibodies or antigen-binding fragments thereof according to any one of claims 5 to 14.

30. The method according to any one of claims 26 to 29, wherein said pharmaceutical composition, said at least one, or at least two isolated monoclonal antibodies or antigen-binding fragment thereof, are administered to said subject before or after exposure to a pox virus.

31. A method of detecting VACV in a sample, said method comprising: a. contacting said sample with at least one isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 5 to 14; and b. detecting said at least one isolated monoclonal antibody or any antigenbinding fragment thereof; wherein the presence of said isolated monoclonal antibody or any antigenbinding fragment thereof indicates the presence of VACV in said biological sample.

32. A method of detecting VACV in a sample, said method comprising: a. immobilizing at least one isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 5 to 14 (the "capture antibody") onto a substrate, wherein said substrate is placed in a vessel;b. contacting said capture antibody with said sample; and c. adding to said vessel at least one isolated monoclonal antibody or antigenbinding fragment thereof of the invention (the "reporter antibody"), wherein said at least one reporter antibody is different from said at least one capture antibody, and wherein said reporter antibody is optionally detectably labelled; wherein detecting the presence of said at least one reporter antibody indicates the presence of VACV in said sample.

33. The method of claim 32 wherein said at least one reporter antibody is a biotinylated antibody or is being associated with a detectable molecule (e.g., a fluorophore or an enzyme such as horse radish peroxidase or alkaline phosphatase).

34. The method of any one of claims 31-33 wherein said sample is a biological sample obtained from a subject.

35. The pharmaceutical composition according to any one of claims 1 to 4, or the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 5 to 14, for use in a method of prophylaxis, treatment, or amelioration of infections caused by pox viruses, or for use in reducing side effects of vaccinia-based vaccines, said methods comprising administering to a subject in need thereof a therapeutically effective amount of said pharmaceutical composition, at least one, or at least two of said isolated monoclonal antibodies or antigen-binding fragments thereof.

36. The pharmaceutical composition or the isolated monoclonal antibody or antigen-binding fragment thereof, for use according to claim 35, wherein said method further comprises administering to a subject in need thereof an additional therapeutic agent.

37. The pharmaceutical composition according to any one of claims 1 to 4, or the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 5 to 14, for use in a method of inhibiting the ability of VACV to infect cells in a subject, said method comprising administering to a subject in need thereof a therapeutically effective amount of said pharmaceutical composition, at least one, or at least two of said isolated monoclonal antibodies or antigen-binding fragments thereof.

38. The pharmaceutical composition or the isolated monoclonal antibody or antigen-binding fragment thereof, for use according to any one of claims 35- 37, wherein said method comprising administering said pharmaceutical composition, said at least one, or at least two isolated monoclonal antibodies or antigen-binding fragment thereof, to said subject before or after exposure to a pox virus.

39. A kit for detecting VACV infection comprising:(a) at least one isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 5-14;(b) means for detection of said isolated monoclonal antibody; and optionally(c) instructions for use of said kit.

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