Anti-orthopoxvirus recombinant polyclonal antibody

Abstract

Disclosed is an anti-orthopoxvirus recombinant polyclonal antibody comprising distinct members which in union are capable of binding at least three orthopoxvirus related antigens, a pharmaceutical composition comprising the antibody, and a method for its production. Also disclosed is a polyclonal cell line capable of producing the recombinant polyclonal antibody as therapeutic methods utilizing the polyclonal antibody. Finally, the invention also pertains to a method for screening for useful V H and V L pairs useful when preparing the polyclonal antibody.

1 . An anti-orthopoxvirus recombinant polyclonal antibody comprising distinct members which in union are capable of binding at least three orthopoxvirus related antigens. 
     
     
         2 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 1 , wherein at least two distinct epitopes on the same orthopoxvirus related antigen are bound by said polyclonal antibody. 
     
     
         3 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 1 , wherein the distinct antibody members mirror the humeral immune response with respect to diversity, affinity and specificity against antigens associated with one or more orthopoxviruses. 
     
     
         4 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 1 , wherein the distinct antibodies are encoded by nucleic acid sequences obtained from one or more donors who have raised a humeral immune response against an orthopoxvirus. 
     
     
         5 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 4 , wherein the donors have been vaccinated with a vaccinia virus strain or are recovering from an orthopoxvirus infection. 
     
     
         6 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 4 , wherein the one or more donors are human, and the polyclonal antibody is a fully human antibody. 
     
     
         7 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 3 , wherein the distinct antibody members comprise V H  and V L  pairs originally present in the one or more donors. 
     
     
         8 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 3 , wherein the specificity of the individual members of the anti-orthopoxvirus recombinant polyclonal antibody are selected such that the antibody composition collectively binds antigens that elicit significant antibody responses in mammals. 
     
     
         9 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 1 , which comprises binding reactivity against Intracellular Mature Virions (IMV) as well as Extracellular Enveloped Virions (EEV) specific antigens, where the binding reactivity is characterized by distinct members capable of binding either an IMV or an EEV specific antigen. 
     
     
         10 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 9 , which comprises binding reactivity against antigens selected among the IMV viral proteins A27L, A17L, D8L and H3L and antigens selected among the EEV viral proteins A33R and B5R. 
     
     
         11 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 10 , where the group of antigens from IMV additionally comprises the viral protein L1R and the group of antigens from EEV additionally comprises the viral protein A56R. 
     
     
         12 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 1 , where said polyclonal antibody comprises at least one individual antibody member with binding reactivity against an orthopoxvirus related regulators of complement activation (RCA) protein encoded by an orthopoxvirus. 
     
     
         13 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 12 , wherein the RCA binding reactivity is directed to a protein selected from the group consisting of VCP, SPICE, IMP, MPXV-VCP and CMLV-VCP. 
     
     
         14 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 12 , which comprises binding reactivity to at least two of the RCA domains selected from the group consisting of SCR2, SCR4 and the junction between the SCR3 and 4 domains. 
     
     
         15 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 3 , which additionally contains one or more distinct antibodies encoded from V H  and V L  pairs selected from one or more donors which have been immunized or vaccinated with a particular orthopoxvirus related antigen. 
     
     
         16 - 18 . (canceled) 
     
     
         19 . A method for treatment or prevention of adverse side effects of vaccination with vaccinia virus in a human or an animal, wherein an effective amount of the anti-orthopoxvirus recombinant polyclonal antibody according to  claim 1  is administered to said human or animal. 
     
     
         20 . A method for treatment or prophylaxis of an orthopoxvirus infection in a human or animal, wherein an effective amount of the anti-orthopoxvirus recombinant polyclonal according to  claim 1  is administered to said human or animal. 
     
     
         21 . (canceled) 
     
     
         22 . A pharmaceutical composition comprising as an active ingredient, an anti-orthopoxvirus recombinant polyclonal antibody according to  claim 1  and a pharmaceutically acceptable excipient. 
     
     
         23 . A screening procedure for selecting V 14  and V L  sequence pairs capable of encoding a broad diversity of anti-orthopoxvirus antibodies comprising:
 (a) expressing an antibody or antibody fragment from a host cell transfected with a screening vector comprising a distinct member of the repertoire of V H  and V L  coding pairs,   (b) contacting said antibody or antibody fragment with at least two different vaccinia virus strains and one or more antigens selected from the group consisting of A27L, A17L, D8L, H3L, L1R, A33R, B5R and VCP in parallel,   (c) repeating step a) and b) for each V H  and V L  sequence pair in the repertoire of sequence pairs, and   (d) selecting the V H  and V L  sequence pairs encoding an antibody or antibody fragment which bind to at least one of the vaccinia virus strains or one of the antigens.   
     
     
         24 . The screening method according to  claim 23 , wherein the V H  and V L  coding pairs are cognate pairs. 
     
     
         25 . The screening method according to  claim 23 , wherein the screening procedure does not utilize phage display. 
     
     
         26 . A method for generating a repertoire of V H  and V L  coding pairs, where the members mirror the gene pairs responsible for the humeral immune response upon challenge with an orthopoxvirus, comprising:
 (a) providing a lymphocyte-containing cell fraction from a donor vaccinated with an orthopoxvirus or recovering from an orthopoxvirus infection;   (b) optionally enriching B cells or plasma cells from said cell fraction;   (c) obtaining a population of isolated single cells, comprising distributing cells from said cell fraction individually into a plurality of vessels;   (d) amplifying and effecting linkage of the V H  and V L  coding pairs, in a multiplex overlap extension RT-PCR procedure, using a template derived from said isolated single cells; and   (e) optionally performing a nested PCR of the linked V H  and V L  coding pairs.   
     
     
         27 . The method according to  claim 26 , wherein the linked V H  and V L  coding pairs are subjected to a screening procedure comprising:
 (a) expressing an antibody or antibody fragment from a host cell transfected with a screening vector comprising a distinct member of the repertoire of V H  and V L  coding pairs,   (b) contacting said antibody or antibody fragment with at least two different vaccinia virus strains and one or more antigens selected from the group consisting of A27L, A17L, D8L, H3L, L1R, A33R, B5R and VCP in parallel,   (c) repeating step a) and b) for each V H  and V L  sequence pair in the repertoire of sequence pairs, and   (d) selecting the V H  and V L  sequence pairs encoding an antibody or antibody fragment which bind to at least one of the vaccinia virus strains or one of the antigens.   
     
     
         28 . A polyclonal cell line capable of expressing a recombinant polyclonal anti-orthopoxvirus antibody according to  claim 1 . 
     
     
         29 . A polyclonal cell line wherein each individual cell is capable of expressing a single V H  and V L  coding pair and the polyclonal cell line as a whole is capable of expressing a collection of V H  and V L  coding pairs, where each V H  and V L  coding pair encode an anti-orthopoxvirus antibody. 
     
     
         30 . The polyclonal cell line according to  claim 29 , wherein said collection of V H  and V L  coding pairs are generated according to the method comprising:
 (a) providing a lymphocyte-containing cell fraction from a donor vaccinated with an orthopoxvirus or recovering from an orthopoxvirus infection;   (b) optionally enriching B cells or plasma cells from said cell fraction;   (c) obtaining a population of isolated single cells, comprising distributing cells from said cell fraction individually into a plurality of vessels;   (d) amplifying and effecting linkage of the V H  and V L  coding pairs, in a multiplex overlap extension RT-PCR procedure, using a template derived from said isolated single cells; and   (e) optionally performing a nested PCR of the linked V H  and V L  coding pairs.   
     
     
         31 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 1 , wherein said anti-orthopoxvirus recombinant polyclonal antibody is essentially free from immunoglobulin molecules that do not bind to orthopoxvirus antigens. 
     
     
         32 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 1 , wherein the constant region of said anti-orthopoxvirus recombinant polyclonal antibody belongs to the isotype of IgG, IgA, IgE, IgM, or IgD. 
     
     
         33 . The anti-orthopoxvirus recombinant polyclonal antibody according to  claim 1 , wherein the constant region of said anti-orthopoxvirus recombinant polyclonal antibody belongs to one or two particular immunoglobulin subtypes of IgG or IgA.