Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
  • C07K16/1036—Retroviridae, e.g. leukemia viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
  • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the present invention relates to polypeptides comprising an amino acid residue sequence substantially identical to that of a viral envelope N-linked glycoprotein, and more particularly to polypeptides and nor-viruses that are substantially free of the N-linked glycosylation of a viral envelope N-linked glycoprotein and are capable of inducing the production of antibody-containing serum that (i) provides improved cross-reactivity among related viral strains and/or (ii) neutralizes the virus of said viral glycoprotein at a greater serum dilution than does antibody-containing serum induced by the viral envelope N-linked glycoprotein.
• Background of the Invention The pathways and mechanisms by which carbohydrate moieties (glycosyl groups) are added to glycoproteins are now relatively well defined, Hubbard et al., Ann. Rev. Biochem., 50, 555 (1981). However, heretofore little has been known about the role carbohydrates play in the function of this class of proteins.
• glycosylation Possible roles posed for glycosylation include increasing hydrophilicity of*certain regions of a given molecule, protection of the molecule from proteolytic attack, facilitation of mobilization to the cell surface and dictation of secretion of certain proteins. See, for example, Gibson et al., Trends Biochem.' Sci., 5_, 290 (1980); Schwarz et al., Trends Biochem. Sci., 5_, 65 (1980); Heifetz et al., Biochemistry, 18, 2186 (1979). Although examples of each of .the above suggestions are evident, generalizations regarding the role of the carbohydrate on glycoproteins as a class are not apparent. See, Sharon et al.. The Proteins, Volume V, 3d ed., Neurath et al., eds.. Academic Press, New York (1982).
• glycosidase preparations have typically been contaminated with proteases that cleave the glycoproteins into non-immunogenic fragments.
• the immunogenic contributions of the carbohydrate and polypeptide portions of the major envelope glycoprotein (gp90) of the equine infectious anemia virus called EIAV have been analyzed by Montelaro et al.. Virology, 136, 368 (1984). ' The analysis was made by measuring the effects of specific glycosidase and protease digestions on the reactivity of the glycoprotein with immune sera from infected horses. The results of both direct and competitive radioimmunoassays indicated that immune sera contained antibodies reactive with both carbohydrate and protein moieties of EIAV gp90, with the predominant reactivity apparently being against the gp90 polypeptide epitopes.
• the present invention contemplates a polypeptide free of N-linked carbohydrate having an amino acid residue sequence substantially identical to that of a viral envelope N-linked glycoprotein, as well as methods of producing and utilizing the polypeptide.
• a polypeptide having an amino acid residue sequence substantially identical to that of a viral envelope N-linked glycoprotein is contemplated.
• the polypeptide is substantially free of the N-linked glycosylation of the glycoprotein and induces the production of antibody-containing serum that (i) exhibits improved cross-reactivity among related viral strains and/or (ii) neutralizes the virus of said viral glycoprotein at a greater serum dilution than does antibody-containing serum induced by the viral envelope N-linked glycoprotein when the polypeptide and the viral envelope N-linked glycoprotein are individually utilized to induce antibody production in separate host mammals of the same strain using substantially identical immunization regimens.
• Another aspect of the present invention contemplates a modified virus or virion described herein as a nor-virus or nor-virion whose envelope protein has an amino acid residue sequence substantially identical to that of the unmodified virus envelope protein, the latter envelope protein containing N-linked glycosylation in the unmodified state.
• the envelope protein of the nor-virus is substantially free of carbohydrate moieties covalently linked to asparagine residues (N-links) and induces the production of antibody-containing serum that (i) exhibits improved cross-reactivity among related viral strains and/or (ii) neutralizes the virus of said viral glycoprotein at a greater serum dilution than does antibody-containing serum induced by the virus when the polypeptide and the virus are individually utilized to induce antibody production in separate host mammals of the same strain using substantially identical immunization regimens.
• an inoculum against infection by a retrovirus or a myxovirus is contemplated.
• the inoculum includes an effective amount of the above-described polypeptide or nor-virus of the invention in a physiologically tolerable diluent.
• the inoculum when introduced into a host, is capable of inducing the production of antibodies in the host that immunoreact with the nor-virus, the virus, and with a related viral strain, and neutralize the virus in vitro.
• such inocula are utilized to protect the host from _in vivo viral infection.
• antibodies to a retrovirus or a myxovirus are contemplated. The antibodies are raised in an animal host to the above-described polypeptide or nor-virus of the invention and have the capacity to immunoreact with the virus, the nor-virus, and a related viral strain as well as neutralize the virus ⁇ in vitro.
• a method for the production of antibodies to a virus comprises (i) introducing into an animal host an effective amount of the above-described polypeptide or nor-virus of the invention that is capable of inducing the production of antibodies in the serum of the host; and (ii) collecting induced antibody-containing serum from the host.
• the antibodies may be recovered in purified form from the induced antibody-containing serum by well known techniques, such as affinity purification.
• the antibodies and antibody-containing serum so prepared immunoreact with the virus, the nor-virus, a related viral strain and also neutralize the virus i ⁇ vitro.
• a method of improving the immunogenicity of a viral envelope N-linked glycoprotein comprises (i) providing a viral N-linked envelope glycoprotein or virus having an N-linked envelope glycoprotein; (ii) reacting the glycoprotein with a glycosidase such as an endoglycosidase or exoglycosidase to remove glycosyl groups from the glycoprotein to form the above-described polypeptide or virus, respectively.
• a glycosidase such as an endoglycosidase or exoglycosidase
• the deglycosylated polypeptide or virus (the nor-virus) so formed when utilized in an effective amount in an inoculum, is capable of inducing the production of antibody-containing serum that (i) exhibits improved cross-reactivity among related viral strains and/or (ii) neutralizes the virus of said viral glycoprotein at a greater serum dilution than does antibody-containing serum induced by the corresponding glycoprotein or virus, respectively, when the polypeptide or nor-virus and the respective glycoprotein or virus are individually utilized to induce antibody production in separate host mammals of the same strain using substantially identical immunization regimens.
• Still another aspect of the present invention contemplates a method of immunizing an animal against a virus.
• the method comprises (i) providing a unit dose of inoculum comprising the above-describe polypeptide or nor-virus of the present invention dispersed in an effective amount in a physiologically tolerable diluent, the polypeptide or nor-virus having the capacity to induce the production of antibodies in the animal that immunoreact with the virus, nor-virus, a related viral strain as well as neutralize the virus in vitro, and preferably protect the animal from the virus; and (ii) introducing the unit dose of the inpculum into the blood stream of the animal to be immunized.
• the present invention provides several benefits and advantages.
• One benefit of the present invention is that the polypeptide or nor-virus of the invention provides improved immune system susceptibility to viral immunogens.
• antibodies raised against the polypeptide or nor-virus of the invention display a greater neutralizing titer than do antibodies raised against a corresponding glycoprotein or a fully glycosylated virus, respectively, when used as immunogens in separate animals of the same strain under similar immunization conditions, and thus provide an improved method of immunizing animals against viruses.
• a further advantage of the present invention is that antibodies raised against the polypeptide or nor-virus of the invention provide improved cross-reactivity among related viral strains, thereby providing the ability to use a nor-virus or carbohydrate-free viral envelope polypeptide of this invention in an inoculum against another, related virus.
• FIGURE 1 is a photograph of a Western blot analysis visualized by autoradiography showing the reactivity of heteroantisera to Rauscher murine leukemia virus (R-MuLV) glycoprotein gp70 before and after deglycosylation with Endoglycosidase F (Endo F) , Elder and Alexander, Proc. Natl. Acad. Sci. (USA) , 79, 4540 (1982).
• R-MuLV Rauscher murine leukemia virus
• Endo F Endoglycosidase F
• Immune reactivity was assessed against R-MuLV either untreated (native) or deglycosylated with Endo F to form a nor-virus as described in Elder and Alexander, supra, followed by separation on SDS-PAGE, as in Laemmli, Nature, 227, 680 (1970) , and transfer to nitrocellulose [Western blotting as described in Towbin et al., Proc. Natl. Acad. Sci. (USA) , 76, 4350 (1979) and modified in Johnson et al.. Gene Anal. Techn. , 1 , 3 (1984)].
• Lane B shows reactivity of goat heteroserum made against intact virus (sera obtained from the Resources Branch of the National Cancer Institute) with R-MuLV proteins before (-) and after (+) treatment of those proteins with Endo F.
• Lane C shows reactivity of a goat heteroserum made against TWEEN-ether disrupted virus (sera obtained from the Resources Branch of the National Cancer Institute) before (-) and after (+) treatment with Endo F.
• TWEEN is used to indicate polyoxyethylene (20) sorbitan monooleate. Arrows denote bands corresponding to gp70 before and after Endo F treatment, as is appropriate for the lanes.
• Radioactive bands were removed and counted to quantitate changes in reactivity following deglycosylation. Reaction with a monoclonal antibody that recognized both untreated and deglycosylated gp ' 70 verified that the relative amounts of gp70 remained unchanged after deglycosylation.
• FIGURE 2 is a photograph of a Western blot analysis visualized by autoradiography showing the effect of deglycosylation of Rauscher murine leukemia virus (R-MuLV) glycoprotein gp70 on reactivity with monoclonal antibodies. Immunoprecipitations were performed using a panel of 78 monoclonal antibodies made against purified R-MuLV gp70, as described in Niman and Elder, Virology, 123, 189 (1982). R-MuLV disrupted with Nonidet P-40 [polyoxyethylene (9) octyl phenyl ether; Sigma Chemical Co., St. Louis, MO] and radiolabeled with 125I (A ersham, Arlington Heights, IL) was used as antigen.
• Nonidet P-40 polyoxyethylene (9) octyl phenyl ether
• radiolabeled with 125I A ersham, Arlington Heights, IL
• FIGURE 3 is a photograph of a Western blot analysis visualized using peroxidase-coupled goat anti-rabbit IgG, hydrogen peroxide and o-dianisidine as colorant showing the reactivity of anti- synthetic peptide antisera to the X47 influenza h ' emagglutinin (HA) before and after deglycosylation with Endo F.
• X47 influenza virus (H 3 subtype) was grown in embryonated eggs. The virus was concentrated from allantoic fluid by centrifugation.
• Strips containing a lane of each of the glycosylated and deglycosylated samples were incubated with the various rabbit anti-synthetic peptide antisera made against synthetic peptides corresponding to regions of the X47 influenza HA molecule, as in Green et al., Cell, 28, 477 (1982) and Min Jou et al., Cell, 19_, 683 (1980). After washing, the blots were incubated with peroxidase-coupled goat anti-rabbit IgG. The blots were developed with hydrogen peroxide and o-dianisidine. Reactivities before (-) and after (+) deglycosylation with Endo F are shown.
• Lane A shows that reactivity of rabbit heteroserum against intact X47 influenza virus decreases dramatically with the deglycosylated HA molecule.
• Lane B shows that anti-synthetic peptide serum 15 (HA sequence positions 140-156 that contain no site of glycosylation) reacts with neither glycosylated nor deglycosylated HA.
• Lane C shows that anti-synthetic peptide serum 2 (HA sequence positions 1-36 that contain 2 sites of glycosylation at positions 8 and 23) improves in reactivity when the HA is deglycosylated.
• Lane D shows that anti-synthetic peptide 17 (HA sequence positions 174-196 with no site of glycosylation) reacts equally well with either glycosylated or deglycosylated HA.
• the arrows denote bands corresponding to the influenza HA molecule before and after Endo F treatment, as is appropriate.
• FIGURE 4 is a graph showing the adsorption of neutralizing antibodies by deglycosylated Rauscher murine leukemia virus (R-MuLV). The ordinate shows the percent of neutralization and the abscissa shows antibody dilution over an average of five adsorption studies.
• Antibodies raised to non-deglycosylated R-MuLV ( ⁇ - ⁇ ) were used as a control and adsorption of neutralizing antibodies by untreated R-MuLV (0-0) and by R-MuLV deglycosylated with Endo F (•-•) were compared.
• FIGURE 5 is a photograph of a Western blot analysis visualized using peroxidase-coupled goat anti-rabbit antibodies, hydrogen peroxide and 4-chloro-2-naphthol as described in Section III C, and shows the reactivity of antisera raised against R-MuLV or deglycosylated, nor-R-MuLV.
• the R-MuLV gp70 was deglycosylated (treated) with Endo F thereby providing the deglycosylated protein called as p49.
• the left panel of lanes shows the reactivity of antisera raised against deglycosylated R-MuLV to native and deglycosylated R-MuLV.
• the right panel of lanes shows the reactivity of antisera raised against native R-MuLV to native and deglycosylated R-MuLV.
• FIGURE 6 is a graph showing the neutralization of R-MuLV by antisera raised .against a control and against deglycosylated R-MuLV. The ordinate shows the percent of neutralization and the abscissa shows the antibody dilution. The neutralization of untreated R-MuLV (0-0) was compared to that of R-MuLV treated with Endo F (nor-R-MuLV) (•-•).
• FIGURE 7 is a graph showing the neutralization of feline leukemia virus (FeLV) by antisera raised against untreated FeLV (0-0) and deglycosylated FeLV (nor-FeLV) (•-•). As above for FIGURE 6, the ordinate shows the percent of neutralization and the abscissa shows the antibody dilution.
• FIGURE 8 is a photograph of a Western blot analysis visualized as described in FIGURE 5 showing the relative lack of cross-reactivity of antisera raised against native FeLV with R-MuLV.
• the left lane shows the reactivity of the antisera to native and deglycosylated R-MuLV.
• the right lane shows the reactivity of the antisera to native and deglycosylated FeLV.
• FIGURE 9 is a photograph of a Western blot analysis visualized as described in FIGURE 5 showing cross-reactivity of antisera raised against deglycosylated, nor-FeLV.
• the left lane shows the reactivity of the antisera to native and to deglycosylated R-MuLV.
• the right lane shows the reactivity of the antisera to native and to deglycosylated, nor-FeLV.
• the present invention is directed to a polypeptide that is free of N-linked carbohydrate that has an amino acid residue sequence substantially identical to that of a viral envelope N-linked glycoprotein, its related nor-virus whose envelope protein is free of N-linked carbohydrate, and to methods of producing and utilizing same.
• the polypeptide is substantially free of glycosylation of the N-linked glycoprotein and induces the production of antibody-containing serum that (i) provides improved cross-reactivity among related viral strains and/or (ii) neutralizes the virus of said viral glycoprotein at a greater serum dilution than does antibody-containing serum induced by the viral envelope N-linked glycoprotein when the polypeptide and the viral envelope N-linked glycoprotein are individually utilized to induce antibody production in separate host mammals of the same strain using substantially identical immunization regimens.
• a polypeptide of this invention is a nor- or deglycosylated form of a native viral envelope glycoprotein that contains a plurality of asparagine-linked (N-linked) carbohydrate moieties in the native form.
• a polypeptide is substantially the same length and has substantially the identical amino acid residue sequence as does the native envelope glycoprotein.
• An alternative description of such a polypeptide is that it is a deglycosylation reaction product of an N-linked viral envelope glycoprotein.
• Particularly preferred polypeptides are reaction products of an endoglycosidase or ' exoglycosidase, such as neuraminidase, and an N-linked viral envelope glycoprotein.
• the endoglycosidase is the enzyme referred to herein as Endo F and the viral envelope glycoprotein is that of a retrovirus such as a leukemia-inducing retrovirus or a myxovirus such as an influenza virus.
• the polypeptide reaction product has substantially the same amino acid residue length and substantially identical amino acid residue sequence as does the native viral envelope glycoprotein.
• the polypeptide may differ by a few residues, e.g., about 5 to about 20 residues, from the native glycoprotein.
• the polypeptide is relatively free from proteolytic cleavage that reduces its amino acid residue sequence to less than about 90 percent, or more preferably, less than about 95 percent, of the number of residues present in the native glycoprotein. Most preferably, the polypeptide has the same number of amino acid residues as does the native glycoprotein. Thus, deglycosylation of the N-linked carbohydrate moieties does not substantially reduce the length of the native viral envelope protein.
• polypeptide as having a substantially identical amino acid residue sequence to that of the native glycoprotein, it is meant that the amino acid residues present in the polypeptide are also present in the glycoprotein. Thus, for example, if a few amino acid residues have been lost by proteolysis during the deglycosylation reaction, those remaining in the polypeptide are also present in the glycoprotein.
• a free, uncombined polypeptide molecule of this invention is most preferably prepared from an N-linked viral envelope glycoprotein of a native virus as contrasted to the possible preparation by recombinant DNA technology of a non-glycosylated or glycosylated protein of the same amino acid residue sequence. It is believed that the three-dimensional structure imposed upon the envelope protein during its synthesis and glycosylation is of some importance to the preparation and presentation of naturally occurring epitopes that induce production of neutralizing and/or protective antibodies.
• polypeptide used in relation to a before-described product is meant to encompass a reaction product as a free molecule as well as a reaction product molecule in combined form such as the envelope protein of an intact virus or partially disrupted virus.
• an intact virus having an envelope glycoprotein containing N-linked carbohydrate moieties may be deglycosylated by reaction with a deglycosylase to form a nor-virus that previously contained N-linked carbohydrate moieties and is substantially free of N-linked carbohydrate moieties.
• nor-virus includes the before-described polypeptide in a combined form as the viral envelope "protein", that nor-virus is considered herein to be within those materials encompassed ⁇ by the before-described term "polypeptide". Contrarily, the term “nor-virus” is used more narrowly herein, and excludes the before-described polypeptide as a free molecule. When the phrase “polypeptide or nor-virus” appears herein, it is meant to encompass the free, uncombined polypeptide molecule and the combined viral or disrupted viral form of that molecule, respectively. Definitions
• Virus or virion An infectious agent that lacks independent metabolism and is able to replicate only within a living host cell.
• the individual virus particle (virion) consists of DNA or RNA and a protein shell or envelope.
• Nor-virus or nor-virion A virus particle or virion that normally contains asparagine-linked (N-linked) carbohydrate moieties from which such N-linked carbohydrates have been removed.
• Native The word "native" used with respect to a virus or protein refers to the usual chemical state of that virus or protein even though the virus or protein may be made by man's intervention through mutation. Thus, a native virus or protein as used herein contains N-linked carbohydrate moieties.
• Heteroserum or Heteroantiserum Antibody-containing serum that contains antibodies that immunoreact with a plurality of epitopes from a plurality of antigenic determinants at several places on the immunogenic entity, e.g., virus, protein, or glycoprotein, when that entity is used as an antigen.
• immunogenic entity e.g., virus, protein, or glycoprotein
• sequence substantially identical is used herein to mean little if any cleavage of proteins, particularly in. the middle of the sequence, although a few amide bonds at the ends of the protein may be cleaved.
• heteroantisera monoclonal antibodies and anti-synthetic peptide (oligoclonal) antibodies to immunoreact with deglycosylated viral glycoproteins has been examined.
• results discussed hereinafter indicate that the reactivities of the majority of antibodies (antibody-containing sera) raised against these glycoproteins were markedly influenced by the attached carbohydrate (glycosyl) moieties.
• the assays discussed were facilitated by the use of Endoglycosidase F, Elder and Alexander, Proc. Natl. Acad. Sci. (USA), supra, that efficiently cleaves both N-linked high mannose and complex glycans from glycoproteins, and thus permits direct screening of aspects of the carbohydrate-protein interaction.
• the polypeptides of the invention were obtained from viral envelope glycoproteins, illustratively, by immunoreaction with Endo F. However, they may also be obtained by immunoreaction with endoglycosidase? * called Endo H and Endo D, Elder and Alexander, supra, or by immunoreaction with neuraminidase, an exoglycosidase.
• R-MuLV envelope glycoprotein gp70 results in the formation of a protein called p49.
• Further assays were undertaken to characterize the relationship between those antibodies influenced by carbohydrate and the antibodies responsible for virus neutralization. The results discussed hereinafter illustrate that carbohydrate directs the immune response to determinants other than those involved in viral infectivity. Surprisingly, it was found that viral antigens exhibited a relatively stronger neutralizing response after carbohydrate removal. This result was directly opposite to those previously reported, Bruck et al.. Virology, supra; Miller et al., 5th International Symposium of Bovine Leukosis, supra; Sch err et al., Virology, supra; Portetelle et al., Virology, supra.
• the initial assay in this regard was performed based upon the observation that all heterosera or oligoclonal sera raised against retroviruses (murine, feline and primate) as well as influenza virus were virtually unreactive to their respective envelope glycoprotein antigens by immune precipitation or Western blotting after carbohydrate was removed from these antigens by treatment with Endo F. As the surface glycoproteins are the primary targets of virus neutralization, it was expected that neutralization of deglycosylated virus would be similarly diminished.
• polypeptides of the invention comprising amino acid residue sequences substantially identical to those of Rauscher murine leukemia virus, (R-MuLV), feline leukemia virus (FeLV) and influenza virus envelope N-linked glycoproteins.
• polypeptides of the invention also include polypeptides comprising amino acid residue sequences substantially identical to those of any virus generally, and particularly to those of leukemia virus and myxovirus envelope N-linked glycoproteins.
• Leukemia viruses include a group of RNA viruses causing leukemia and/or leucocyte-related tumors in animals. These viruses include avian leukosis virus, Rous sarcoma and murine leukemia viruses.
• Myxoviruses include a group of viruses, such as the viruses of influenza, parainfluenza, mumps and Newcastle disease, that characteristically cause agglutination of erythrocytes.
• FIGURE 1 shows the results of Western blot analyses of untreated (native) and deglycosylated R-MuLV using heterosera prepared to purified R-MuLV glycoprotein (gp70) , intact R-MuLV, and TWEEN-ether disrupted R-MuLV. All three antisera showed diminished reactivity to gp70 after treatment of the virus with Endo F.
• the heteroserum prepared against purified gp70 i munore acted to only 40 of percent control levels after carbohydrate removal (calculated by removing and counting the radioactive bands from nitrocellulose strips) as shown in FIGURE 1A.
• FIGURE 2 representative immune precipitations using anti-R-MuLV gp70 monoclonal antibodies, as in Niman and Elder, Virology, supra, versus untreated and deglycosylated R-MuLV are shown.
• the results of this assay demonstrate that some monoclonal antibodies immunoprecipitated control and Endo-F treated gp70 very poorly (FIGURE 2A) , others precipitated gp70 only after carbohydrate was removed (FIGURE 2B), and another group only reacted with untreated R-MuLV gp70 (FIGURE 2C) .
• X47 influenza virus was immunoreacted with glycosylated virus and deglycosylated virus (nor-virus) (FIGURE 3A) , results strikingly similar to those obtained with heteroantisera against the retroviruses were obtained (FIGURE 2B) ; namely. deglycosylation substantially reduced to abolished the reactivity of the anti-influenza ' virus antiserum.
• the virus and nor-virus were subsequently pelleted from each of the sera, and the respective sera were then sequentially adsorbed four additional times with the remaining viral pellets of each type, following the same general procedures. A portion of the serum was saved at each step so that at the end, antisera samples adsorbed lx, 2x, 3x and 4x either with control (R-MuLV) or deglycosylated R-MuLV were obtained.
• carbohydrate moiety is not the only immunogenic part of gp70, since strong antibodies were raised in their absence. Additionally, the carbohydrate moieties masked immunogenic epitopes present in the protein backbone that were subsequently exposed upon deglycosylation.
• FIGURE 6 the results of neutralization assays are shown using sera from the before-mentioned rabbits.
• the nor-virus treated with Endo F elicited a much stronger initial neutralizing titer than did control virus when both virus and nor-virus were individually utilized to induce antibody production in separate host mammals of the same strain using substantially identical immunization regimens.
• the control virus-immunized rabbit eventually began producing more neutralizing antibodies, and its antibody-containing serum approached the titer of the Endo F-treated immunogen after five or six boosts.
• FIGURE 7 again illustrate viral neutralization at a greater serum dilution by antibody-containing serum induced by a polypeptide of this invention (nor-virus) than was obtained by serum induced by the viral envelope N-linked glycoprotein (native virus) when the polypeptide and viral envelope N-linked glycoprotein were individually utilized to induce antibody production in separate host mammals of the same strain using substantially identical immunization regimens.
• the polypeptides and nor-viruses of the invention are capable of inducing the production of antibody-containing serum that neutralizes the native virus at a greater serum dilution than does antibody-containing serum induced by the N-linked envelope glycoprotein or native virus, respectively, when the polypeptide or nor-virus and the N-linked envelope glycoprotein or virus, respectively, are individually utilized to induce antibody production in separate host mammals of the same strain using substantially identical immunization regimens.
• C. Cross-Reactivity Studies The seriological classification and subdivision of antigenically related viruses is known to result from the polymorphism of certain viral proteins. Viral polymorphism reflects the response of specific viral genes to selective pressure and influences the dynamics of the host-parasite relationship. Such is the case for the hemagglutinin molecule of the influenza viruses, Fenner et al., "The Biology of Animal Viruses", 2d ed.. Academic Press, New York (1973).
• Antigenic cross-reactivity based upon amino acid residue sequence homology is a phenomenon well known in the art.
• sequence homology does not guarantee cross-reactivity because the presentation of antigenic determinants may differ from one viral strain to another.
• R-MuLV and FeLV-B are believed to have gp70 sequence homology because of the known sequence homology between Friend murine leukemia virus (F-MuLV) and FeLV-B and the close serological relationship between F-MuLV and R-MuLV as demonstrated by Niman and Elder, supra.
• F-MuLV Friend murine leukemia virus
• FeLV-B Friend murine leukemia virus
• heteroserum made to native FeLV-B virion does not cross-react with R-MuLV gp70. This result, shown in FIGURE 8, suggests that antigenic determinants shared by R-MuLV and FeLV-B are not presented to their hosts in the same way.
• sequence homologies are known to be present among FeLV, HTLV I, HTLV II, HTLV III, MCF, and ATLV envelope glycoproteins, and cross-reactivity between serum of a patient with HTLV-II and a recombinant DNA-produced protein encoded by the env gene of HTLV-I has been reported [Samuel et al.
• polypeptides or nor-viruses of this invention when introduced into an animal host as a unit dose inoculum having an effective amount of polypeptide or nor-virus in a physiologically tolerable diluent, are capable of inducing production of antibodies in the host mammal that immunoreact with the related virus, neutralize the virus in vitro, and preferably protect the host animal from in vivo infection caused by that virus.
• the "effective amount" of polypeptide or nor-virus in a unit dose depends upon a number of factors. Included among those factors are the body weight of the animal immunized and the number of inoculations desired to be used.
• Individual unit dose inoculations typically contain about 10 micrograms to about 500 milligrams of polypeptide or nor-virus per kilogram body weight of the mammalian host. Inoculation methods and amounts in rabbits for the purpose of raising antibodies are described below.
• Useful free polypeptides of this invention may be obtained from whole viruses by well-known techniques. See, for example, Niman and Elder, “Monoclonal Antibodies and T-Cell Products", supra; Niman and Elder, Virology, 123, 187 (1982); and Niman and Elder, Proc. Natl. Acad. Sci. (USA), 77, 4524
• Influenza hemagglutinin may be obtained as described in Brown et al., J. Immunol. , 125, 1583 (1980) and Aitken et al., Eur. J. Biochem., 107, 51 (1980).
• the resulting polypeptides are then deglycosylated with Endo F, as described in Section III hereinafter, and may then be utilized in the inocula of the invention.
• Physiologically tolerable diluents are well known in the art, and alone are not part of the present invention. Exemplary of such diluents are distilled or deionized water, normal saline solutions and phosphate-buffered saline (PBS) solutions.
• PBS phosphate-buffered saline
• the immunizing composition or inoculum may be introduced into the host by intravenous injection, or the like, using known methods.
• Adjuvants such as complete Freund's adjuvant (CFA), incomplete Freunds's adjuvant (IFA), alum, tetanus toxoid and the like as are well known in the immunological arts may also be included in the inocula as part of the physiologically tolerable diluent.
• Booster injections may also be given, as desired, to build a desired antibody titer in the host's serum.
• Exact doses depend on the animal and polypeptide or nor-virus used, and can be determined using known challenge techniques. Additional exemplary amounts of immunogen such as the polypeptide or nor-virus, and specific reaction conditions for the inoculum preparation may be found in Bittle et al., Nature, 298, 30-33 (July, 1982).
• the term "inoculum” is used herein to mean any immunizing composition. As such, the term also embraces vaccines that are useful in man and other mammals for conferring i vivo protection against a . native virus. A given vaccine and inoculum may be identical where non-human mammalian hosts are involved, but typically differ where humans are the intended hosts. The reason for that difference is that adjuvants such as CFA are not utilized in humans, and another adjuvant must be used if any adjuvant is to be present in a human vaccine. E. Antibodies
• Antibodies to the polypeptides or nor-viruses of the present invention can be used in assays or to treat virus infections.
• the antibodies can be used directly as whole, intact antibodies or may be processed to provide Fab or F(ab * ) portions, all of which are biologically active.
• the term "antibody” indicates a whole, intact antibody or the idiotype-containing polyamide portion of the antibody that is biochemically active and is capable of immunoreacting with or binding to its antigenic ligand on the native virus, nor-virus or glycoprotein.
• an immunizing inoculum described before is introduced into the host mammal as by injection.
• the host is maintained for a time sufficient for antibodies to be induced, usually for one to about four months.
• the desired antibodies raised are thereafter harvested from host fluids.
• the whole antibodies so induced can be used directly, or they may be cleaved with pepsin or papain as is well known to provide F(ab' ) 2 or Fab portions that may be used.
• the antibodies produced may also be used as therapeutic agents for passive immunoprophylaxis.
• An animal infected by a retrovirus such as a leukemia virus or a myxovirus such as an influenza virus may be treated with antibodies preferably as whole antibodies raised to the polypeptides or nor-viruses of the present invention.
• the antibodies are administered in a unit dose having an effective amount of antibodies dispersed in a physiologically tolerable diluent such as saline or phosphate buffered saline.
• an effective amount of such antibodies varies depending on the reactivity and type of the antibodies, but generally about 1 milligram to about 50 milligrams of antibody per kilogram animal weight is considered effective. In the case of mice and native murine leukemia virus, 1.5 milligrams of IgG antibody in ascites fluid were found to be effective in prolonging survival. See "Monoclonal Antibodies", Kennett et al. ed., Plenum Press (1980).
• the antibodies may be introduced intravenously or mtraperitoneally, with several administrations given at three to seven day intervals. The antibodies may also be given in conjunction with surgical treatment.
• the antibodies may be obtained from sera of a second animal, different from the first animal to be treated, by raising antibodies to the polypeptides or nor-viruses of this invention.
• the antibodies may also be obtained from monoclonal sources such as ascites fluid by preparing a hybridoma cell line using known techniques. Whole antibodies are preferred as the antibodies since they are capable of activating the complement system when an immune complex is formed.
• unit dose refers to physically discrete units suitable as unitary dosages for animals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent or vehicle.
• the specifications for a novel unit dose of this invention are dictated by and are directly dependent on (a) the unique characteristics of the immunogen and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such active material for therapeutic use in animals.
• Feline leukemia virus B FeLV-B
• Rauscher murine leukemia virus R-MuLV
• Endo F endo-beta-N-Acetylglucosaminidase F
• Endo F Elder and Alexander, supra
• a similar preparation of Endo F is also available from New England Nuclear (Boston, MA).
• the removal of carbohydrate sidechains from R-MuLV was performed under three different virus denaturing (disrupting) conditions. Non-denaturing carbohydrate removal was accomplished by deglycosylating R-MuLV in deglycosylation buffer alone 10 millimolar (mM) sodium phosphate (pH 6.1), 5 mM EDTA and 0.15 molar (M) NaCl).
• Deglycosylation resulting in mild disruption of the nor-virus was performed using deglycosylation buffer containing 1 percent Nonidet P40.
• Carbohydrate removal was also performed under disrupting-reducing conditions using deglycosylation buffer containing 1 percent Nonidet P40 and 1 percent 2-mercaptoethanol.
• FeLV-B was deglycosylated using only the latter of the above buffer conditions.
• Virus deglycosylated using non-denaturing conditions was first inactivated by exposing 2 milliliters (ml) of virus [0.1 milligram (mg)/ml in deglycosylation buffer] in an" uncovered petri dish (Falcon model 1007; Falcon, Oxnard, CA) to ultraviolet light (uv) (Sylvania Ger icidal model G30T8, GTE-Sylvania, Stamford, CT) for 5 minutes. Complete inactivation was confirmed by the inability to induce virus producing foci when used as a control in the appropriate neutralization assay described hereinafter.
• the uv inactivated virus was then split into two 1 ml aliquots, one of which was incubated with 50 units of Endo F at 37°C for 16 hours, the control aliquot being similarly incubated without Endo F.
• Deglycosylation under 1) mild disrupting and 2) disrupting-reducing conditions was performed by incubating 10 units of Endo F with 200 micrograms of virus in 200 microliters of the appropriate deglycosylation buffer for 16 hours at 37°C. Control virus was similarly prepared without treatment with Endo F.
• Q deglycosylation was accomplished by admixing 2x10 plaque-forming units (pfu) of X47 influenza virus in 0.1M Tris HC1 (pH 7.6), 0.1 percent sodium dodecyl sulfate (SDS), 1 percent 2-mercaptoethanol, and 0.05M EDTA. The resulting admixture was then immersed in boiling water for 2 minutes. Subsequently, Nonidet P40 was added to provide a concentration of 1 percent. The admixture was cooled to 22°C and 20 units of Endo F were added thereto. The new admixture was then incubated at 22°C for 16 hours, immersed in boiling water for 2 minutes, and then cooled to 22°C. Then, an additional 20 units of Endo F were admixed therein and the resulting admixture was incubated at 22°C for 16 hours. At this point, the deglycosylated influenza virus was suitable for injection into rabbits.
• SDS sodium dodecyl sul
• Virus Neutralizing Antibody Adsorption The ability of an antigen to immunoreact with antibodies indicates that the antigen contains determinants that are the same or similar to those immunogenic determinants that induced the antibodies. Therefore, the ability of virus-neutralizing antibodies induced by native virus to immunoreact with deglycosylated virus indicates the presence of virus-neutralizing immunogenic determinants on the deglycosylated virus.
• the present assays used a virus-neutralizing antibody adsorption technique to examine whether deglycosylated virus (R-MuLV or FeLV-B) could adsorb out (immunoreact with) the neutralizing antibodies in heterosera induced by native virus (R-MuLV or FeLV-B) .
• Goat anti-TWEEN-ether disrupted virion (R-MuLV or FeLV-B) was serially adsorbed (immunoreacted) four times using 200 micrograms of deglycosylated (uv inactivated, non-denatured) or control virus per adsorption.
• 1.5 ml of goat anti-gp70 antiserum diluted 1:100 using Minimum Eagle's Medium (MEM) containing 10 percent fetal calf serum was admixed (immunoreacted) with virus for 1 hour at 20°C with agitation. The reaction mixture was centrifuged for 5 minutes in a Fisher model 235A microcentrifuge to pellet the immunoreaction products.
• MEM Minimum Eagle's Medium
• the im unoblotting neutralization assay described in Elder et al., Bio Techniques, 170-172, May-June, 1984 was used to examine, 1) the ability of deglycosylated virus to adsorb out neutralizing antibodies, and 2) the ability of antiserum induced by deglycosylated virus to neutralize native virus.
• the assay measured i ⁇ _ vitro viral neutralization (inactivation) by immunoreaction between antibodies and live (infectious) virus particles.
• the assay was reproducible and yielded a percent reduction in virus producing foci between control and experimental sera.
• the type of cell line subjected to infection in the assay depended upon the virus under investigation because most viruses have limited host specificity.
• the dog thymus cell line CF2th (ATCC CRL 1430, American Type Culture Collection, Bethesda, MD) was used as viral host.
• the murine virus R-MuLV was being investigated, the mouse embryo cell line SC-1 (ATCC CRL 1404, American Type Culture Collection, Bethesda, MD) was used as viral host.
• Both cell lines were grown in Minimum Eagle's Medium (MEM) supplemented with 10 percent fetal calf serum, penicillin (100 units/milliliter) , streptomycin (100 milligrams/milliliter) , 4 mM L-glutamine, and 1.0 mM sodium pyruvate. Following trypsinization with 0.025 percent trypsin in phosphate-buffered saline (PBS) and washing in MEM, 5 3X10 cells were added to tissue culture petri dishes (Falcon #3003, Falcon, Oxnard, CA) and were incubated overnight at 37°C in a humidified atmosphere of 5 percent CO-.
• MEM Minimum Eagle's Medium
• PBS phosphate-buffered saline
• Study and control immunoreactions were performed in separate containers by admixing 5 microliters of antiserum with 100 microliters of MEM containing 400 focus-forming units of virus. Each mixture was incubated at 37°C in a humidified 5 percent CO, atmosphere for 40 minutes. A solution of 5 milliliters of MEM containing 10 micrograms per milliliter hexadimethrine bromide (Polybrene P415, Sigma Chemical Co. St. Louis, MO) was then added. The resulting admixture was then used to inoculate the appropriate host cell culture.
• the host cell cultures were inoculated by replacing the overnight growth MEM with 5 millilite'rs of the above admixture. This mixture was replaced after 24 hours and the cultures were incubated in MEM for an additional 4 day period at 37°C in a 5 percent C0 « atmosphere. The cultures were then terminated by aspirating off the MEM, washing 3 times with 10 milliliters of PBS and allowing the monolayers to dry. Virus-producing cell foci in the monolayer were detected by two techniques: (1) a radioimmune assay (RIA) and (2) an ELISA.
• RIA radioimmune assay
• Both assays were performed by pre-wetting a nitrocellulose disc designated BA85 (Schleicher & Schuell, Inc., Keene, NH) in PBS, placing the wetted disc over the monolayer and pressing firmly so as to transfer the cells from the petri dish onto the disc.
• the cells were fixed on the disc by placing the disc in Amido Black Dye [0.045 percent Naphthol Blue Black (Sigma Chemical Co. St. Louis, MO), 45 percent methanol, 10 percent acetic acid] for 1 minute and then destained (45 percent methanol, 10 percent acetic acid) for 5 minutes. Non-specific binding sites were then blocked by incubating the discs in BLOTTO [Bovine Lacto Transfer Technique
• the discs were then washed with 5 milliliters of BLOTTO three times for 15 minutes each.
• the washed discs were placed in 5 milliliters of BLOTTO containing 5 microliters of rabbit anti-goat antisera and were incubated at 20°C for 1 hour with shaking. They were then again washed 3 times with BLOTTO.
• Goat anti-whole virus antiserum was prepared by injecting either FeLV or R-MuLV virus particles into goats and recovering the sera.
• the rabbit anti-goat antisera were prepared by injecting rabbits as described above using purified goat gamma-globulin as immogen in a physiologically tolerable diluent.
• Staphylococcus aureus Protein A was labeled with 125I, and 5 milliliters of BLOTTO containing 2.5 microliters of 1-labeled Protein
• peroxidase-coupled goat anti-rabbit IgG (Tago, Burlingame CA) was diluted 1:500 in BLOTTO.
• the BLOTTO-washed discs were then immersed in an amount of the diluted, peroxidase-coupled goat anti-rabbit IgG composition to cover the discs, and maintained for a time period of 1.5 hours to bind the peroxidase-labeled goat antibodies to the disc-bound rabbit antibodies.
• the discs were thereafter washed in a bath of BLOTTO for 10 minutes followed by a washing in a deionized water bath for 10 minutes.
• An ELISA-developing bath was prepared by admixing the following: 8 ml of a solution of
• FIGURES 5, 8 and 9 The Western blot analyses of FIGURES 5, 8 and 9 were also visualized following the above ELISA technique.
• the Western Blot technique was done according to published procedures and was used to examine the ability of heteroserum raised against the whole virus to bind deglycosylated gp70.
• the viral proteins were separated by gradient (5-17.5 percent) SDS-polyacrylamide gel electrophoresis. See Laemmli, Nature, supra, and Towbin, et al., Proc. Natl. Acad Sci. (USA), supra.
• Proteins were electrophoretically transferred to nitrocellulose (Schleicher & Schuell, Keene, NH) as described by Towbin et al., Proc. Natl. Acad. Sci. (USA), 7_6, 4350 (1976), using an electroblot apparatus, (E.C. Apparatus Corp., Jacksonville, FL) with buffer consisting of 25 millimolar Tris Base, 192 millimolar glycine, 20 percent methanol and 0.1 percent sodium dodecyl sulfate (pH 8.3). Following the transfer, the nitrocellulose was blocked in BLOTTO to reduce non-specific binding.
• electroblot apparatus E.C. Apparatus Corp., Jacksonville, FL
• the resulting blots were reacted with 100 microliters of heteroserum to R-MuLV or FeLV as appropriate in 10 milliliters of BLOTTO for 3 hours, and were then washed three times for 1 hour with 50 milliliters of fresh BLOTTO.
• Antibodies bound to specific control and deglycosylated viral proteins were detected by one of two methods.
• antibodies were detected by reacting the blots with 20 microliters of
• the antisera against deglycosylated, nor-virus used in the neutralization assays described herein were obtained from immunized rabbits. Control sera were obtained from rabbits by bleeding just prior to initial immunization.
• Antiserum to deglycosylated nor-R-MuLV was obtained by immunization with 200 micrograms nor-R-MuLV, deglycosylated under mild disrupting conditions, as described hereinabove, in complete Freund's adjuvant (CFA), in incomplete Freund's adjuvant (IFA) and on alum (5 milligrams/milliliter) on days 0, 14 and 21, respectively. Thereafter, the rabbit was boosted with nor-R-MuLV, deglyqosylated under disrupting-reducing conditions, as described hereinabove, or alum on a monthly basis.
• CFA complete Freund's adjuvant
• IFA incomplete Freund's adjuvant
• Rabbits immunized with influenza-related immunogens were injected with inocula containing 2X10 7 plaque-forming units of virus or nor-virus following the above immunization regimen. Each immunization consisted of introducing the inoculum in four subcutaneous injections of 50 micrograms of deglycosylated, nor-virus, one on each shoulder and hip. Rabbits were bled 7 days after the last immunization, and in some cases, the rabbits were further boosted, as described above, with immunogen in alum and bled as necessary. The collected blood was allowed to clot overnight and then was centrifuged to obtain the sera used in the assays discussed hereinabove.
• the inocula also contained a physiologically tolerable diluent such as water, or phosphate-buffered saline (pH 7.4) .
• a physiologically tolerable diluent such as water, or phosphate-buffered saline (pH 7.4) .
• Inocula stock solutions were prepared with CFA or IFA as follows: An amount of deglycosylated nor-virus conjugate sufficient to provide the desired amount of deglycosylated nor-virus inoculation was dissolved in phosphate-buffered saline (PBS). Equal volumes of CFA or IFA were then mixed with the deglycosylated nor-virus conjugate solution to provide an inoculum containing deglycosylated nor-virus, water and adjuvant solution in which the water to oil ratio was 1:1. The mixture was thereafter homogenized to provide the inoculum stock solution.
• PBS phosphate-buffered saline

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