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A Human Monoclonal Antibody against Hepatitis B Surface Antigen with Potent Neutralizing Activity.

Cerino A, Bremer CM, Glebe D, Mondelli MU - PLoS ONE (2015)

Bottom Line: We describe the production and characterization of human monoclonal antibodies (mAb) specific for the major hepatitis B virus (HBV) S protein.The former recognized a denaturation-insensitive epitope within the p24 protein whereas the latter recognized a denaturation-sensitive, conformational epitope located within the HBsAg common "a" determinant.Recombinant variable heavy and light domain sequences derived from mAb ADRI-2F3 were cloned into eukaryotic expression vectors and showed identical fine specificity and 1 log10 higher titer than the original IgG1λ.

View Article: PubMed Central - PubMed

Affiliation: Research Laboratories, Department of Infectious Diseases, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.

ABSTRACT
We describe the production and characterization of human monoclonal antibodies (mAb) specific for the major hepatitis B virus (HBV) S protein. The mAbs, two IgG1κ and one IgG1λ, were secreted by B-cell clones obtained from peripheral blood mononuclear cells (PBMC) of one person convalescent from acute hepatitis B and one vaccinated individual. The former recognized a denaturation-insensitive epitope within the p24 protein whereas the latter recognized a denaturation-sensitive, conformational epitope located within the HBsAg common "a" determinant. This mAb, denominated ADRI-2F3, displayed a very high protective titer of over 43,000 IU/mg mAb and showed an extremely potent neutralizing activity in the in vitro model of HBV infection using primary hepatocytes from Tupaia belangeri as target. Recombinant variable heavy and light domain sequences derived from mAb ADRI-2F3 were cloned into eukaryotic expression vectors and showed identical fine specificity and 1 log10 higher titer than the original IgG1λ. It is envisaged that such mAb will be able to efficiently prevent HBV reinfection after liver transplantation for end-stage chronic HBV infection or infection after needle-stick exposure, providing an unlimited source of valuable protective anti-HBs antibody.

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A) Titration curve of humAb binding to HBsAg in an ELISA.Diamonds: ADRI-2F3, Squares: PK-3D1, Triangles PK-10C7. B) Western blot showing reactivity of humAb ADRI-2F3 (lanes 1 and 2), PK-10C7 (lane 3) and PK-3D1 (lane 4). Lane 5; positive control serum, lane 6; negative control. MW indicates a molecular weight marker. Note that in lanes 3, 4 and 5 a 24 kDa band is recognized, indicating binding to the HBsAg polypeptide. C) Competitive inhibition of serial dilutions (0 ng/ml—400 ng/ml) of humAb ADRI-2F3, and its corresponding recombinant recADR12F3, binding to HBsAg by 200 ng of murine mAb Hyb-824 specific for the HBsAg common “a” determinant. D) Competitive inhibition of humAb ADRI-2F3, recADRI2F3, PK-10C7, PK-3D1 by murine mAb Hyb-824. CM3B6, a HCV-NS3 specific humAb, served as negative control. Co20-F10 is an isotype control.
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pone.0125704.g001: A) Titration curve of humAb binding to HBsAg in an ELISA.Diamonds: ADRI-2F3, Squares: PK-3D1, Triangles PK-10C7. B) Western blot showing reactivity of humAb ADRI-2F3 (lanes 1 and 2), PK-10C7 (lane 3) and PK-3D1 (lane 4). Lane 5; positive control serum, lane 6; negative control. MW indicates a molecular weight marker. Note that in lanes 3, 4 and 5 a 24 kDa band is recognized, indicating binding to the HBsAg polypeptide. C) Competitive inhibition of serial dilutions (0 ng/ml—400 ng/ml) of humAb ADRI-2F3, and its corresponding recombinant recADR12F3, binding to HBsAg by 200 ng of murine mAb Hyb-824 specific for the HBsAg common “a” determinant. D) Competitive inhibition of humAb ADRI-2F3, recADRI2F3, PK-10C7, PK-3D1 by murine mAb Hyb-824. CM3B6, a HCV-NS3 specific humAb, served as negative control. Co20-F10 is an isotype control.

Mentions: Three human B-cell clones (ADRI-2F3, PK10C7, PK3D1) and several subclones were derived from the two subjects mentioned in Materials and Methods. All humAb IgG showed strong binding to HBsAg in ELISA (Fig 1A). ADRI-2F3 secreted IgG1λ, whereas PK10C7 and PK3D1 secreted IgG1κ. Western blot analysis showed that PK10C7 and PK3D1 recognized a sequential, denaturation-insensitive epitope located within a band corresponding to the expected molecular weight (24 kDa) of the major S envelope protein [17], whereas ADRI-2F3 did not bind to any protein band, indicating that it recognized a denaturation-sensitive conformational epitope (Fig 1B). Competitive ELISA showed that ADRI-2F3 and its recombinant recADRI2F3 binding to HBsAg was almost completely inhibited by addition of murine mAb specific for the HBsAg common “a” determinant (Fig 1C & 1D) while PK10C7 and PK3D1 binding to HBsAg was not significantly affected by the same mAb (Fig 1D)


A Human Monoclonal Antibody against Hepatitis B Surface Antigen with Potent Neutralizing Activity.

Cerino A, Bremer CM, Glebe D, Mondelli MU - PLoS ONE (2015)

A) Titration curve of humAb binding to HBsAg in an ELISA.Diamonds: ADRI-2F3, Squares: PK-3D1, Triangles PK-10C7. B) Western blot showing reactivity of humAb ADRI-2F3 (lanes 1 and 2), PK-10C7 (lane 3) and PK-3D1 (lane 4). Lane 5; positive control serum, lane 6; negative control. MW indicates a molecular weight marker. Note that in lanes 3, 4 and 5 a 24 kDa band is recognized, indicating binding to the HBsAg polypeptide. C) Competitive inhibition of serial dilutions (0 ng/ml—400 ng/ml) of humAb ADRI-2F3, and its corresponding recombinant recADR12F3, binding to HBsAg by 200 ng of murine mAb Hyb-824 specific for the HBsAg common “a” determinant. D) Competitive inhibition of humAb ADRI-2F3, recADRI2F3, PK-10C7, PK-3D1 by murine mAb Hyb-824. CM3B6, a HCV-NS3 specific humAb, served as negative control. Co20-F10 is an isotype control.
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Related In: Results  -  Collection

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pone.0125704.g001: A) Titration curve of humAb binding to HBsAg in an ELISA.Diamonds: ADRI-2F3, Squares: PK-3D1, Triangles PK-10C7. B) Western blot showing reactivity of humAb ADRI-2F3 (lanes 1 and 2), PK-10C7 (lane 3) and PK-3D1 (lane 4). Lane 5; positive control serum, lane 6; negative control. MW indicates a molecular weight marker. Note that in lanes 3, 4 and 5 a 24 kDa band is recognized, indicating binding to the HBsAg polypeptide. C) Competitive inhibition of serial dilutions (0 ng/ml—400 ng/ml) of humAb ADRI-2F3, and its corresponding recombinant recADR12F3, binding to HBsAg by 200 ng of murine mAb Hyb-824 specific for the HBsAg common “a” determinant. D) Competitive inhibition of humAb ADRI-2F3, recADRI2F3, PK-10C7, PK-3D1 by murine mAb Hyb-824. CM3B6, a HCV-NS3 specific humAb, served as negative control. Co20-F10 is an isotype control.
Mentions: Three human B-cell clones (ADRI-2F3, PK10C7, PK3D1) and several subclones were derived from the two subjects mentioned in Materials and Methods. All humAb IgG showed strong binding to HBsAg in ELISA (Fig 1A). ADRI-2F3 secreted IgG1λ, whereas PK10C7 and PK3D1 secreted IgG1κ. Western blot analysis showed that PK10C7 and PK3D1 recognized a sequential, denaturation-insensitive epitope located within a band corresponding to the expected molecular weight (24 kDa) of the major S envelope protein [17], whereas ADRI-2F3 did not bind to any protein band, indicating that it recognized a denaturation-sensitive conformational epitope (Fig 1B). Competitive ELISA showed that ADRI-2F3 and its recombinant recADRI2F3 binding to HBsAg was almost completely inhibited by addition of murine mAb specific for the HBsAg common “a” determinant (Fig 1C & 1D) while PK10C7 and PK3D1 binding to HBsAg was not significantly affected by the same mAb (Fig 1D)

Bottom Line: We describe the production and characterization of human monoclonal antibodies (mAb) specific for the major hepatitis B virus (HBV) S protein.The former recognized a denaturation-insensitive epitope within the p24 protein whereas the latter recognized a denaturation-sensitive, conformational epitope located within the HBsAg common "a" determinant.Recombinant variable heavy and light domain sequences derived from mAb ADRI-2F3 were cloned into eukaryotic expression vectors and showed identical fine specificity and 1 log10 higher titer than the original IgG1λ.

View Article: PubMed Central - PubMed

Affiliation: Research Laboratories, Department of Infectious Diseases, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.

ABSTRACT
We describe the production and characterization of human monoclonal antibodies (mAb) specific for the major hepatitis B virus (HBV) S protein. The mAbs, two IgG1κ and one IgG1λ, were secreted by B-cell clones obtained from peripheral blood mononuclear cells (PBMC) of one person convalescent from acute hepatitis B and one vaccinated individual. The former recognized a denaturation-insensitive epitope within the p24 protein whereas the latter recognized a denaturation-sensitive, conformational epitope located within the HBsAg common "a" determinant. This mAb, denominated ADRI-2F3, displayed a very high protective titer of over 43,000 IU/mg mAb and showed an extremely potent neutralizing activity in the in vitro model of HBV infection using primary hepatocytes from Tupaia belangeri as target. Recombinant variable heavy and light domain sequences derived from mAb ADRI-2F3 were cloned into eukaryotic expression vectors and showed identical fine specificity and 1 log10 higher titer than the original IgG1λ. It is envisaged that such mAb will be able to efficiently prevent HBV reinfection after liver transplantation for end-stage chronic HBV infection or infection after needle-stick exposure, providing an unlimited source of valuable protective anti-HBs antibody.

Show MeSH
Related in: MedlinePlus