WO2012083475A1 - Anticorps polyclonal neutralisant dirigé contre l'hantavirus andes, procédé de production et compositions pharmaceutiques - Google Patents

Anticorps polyclonal neutralisant dirigé contre l'hantavirus andes, procédé de production et compositions pharmaceutiques Download PDF

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Publication number
WO2012083475A1
WO2012083475A1 PCT/CL2010/000052 CL2010000052W WO2012083475A1 WO 2012083475 A1 WO2012083475 A1 WO 2012083475A1 CL 2010000052 W CL2010000052 W CL 2010000052W WO 2012083475 A1 WO2012083475 A1 WO 2012083475A1
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hantavirus
antibody
andv
serves
patients
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PCT/CL2010/000052
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English (en)
Spanish (es)
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Tobías MANIGOLD
Pablo Agustiín VIAL CLARO
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Universidad De Desarollo
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Priority to PCT/CL2010/000052 priority Critical patent/WO2012083475A1/fr
Publication of WO2012083475A1 publication Critical patent/WO2012083475A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA 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
    • 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
    • C12N2760/12011—Bunyaviridae
    • C12N2760/12111—Hantavirus, e.g. Hantaan virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/175—Bunyaviridae, e.g. California encephalitis virus, Rift valley fever virus, Hantaan virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • Viruses of the genus Hantavirus belong to the family Bunyaviridae.
  • the genome consists of a single negative strand of RNA that is divided into three segments. Segments S, M and L encode for the nucleocapsid (N protein), two glycoproteins of the Gl and G2 envelope and a viral transcriptase, respectively.
  • the genus Hantavirus is the only one of the Bunyaviridae family not transmitted by arthropods. In contrast, the natural Hantavirus reservoir are rodents of the Muridae family.
  • the different Hantavirus species have a close association with a specific host species and their circulation depends on the geographical distribution of the different species of the Muridae family.
  • ANDV is the only Hantavirus described to date. Its reservoir is the rodent Oligoryzomys longicaudatus (long-tailed mouse) present between the II and XII region. Since 1995, 395 patients with SCPH have been confirmed in Chile, of which 66.5% have been diagnosed during the last four years. While the lethality of the infection in 1997 was 60%, it has fallen to 31% in 2004, possibly due to greater knowledge of both the population and the health staff, which allow a more early and effective intervention. Regionally, the seropositivity rate for ANDV in Chilean populations can reach up to 7.5% (6). However, to date no specific vaccine or treatment has proven effective in humans.
  • Cardiopulmonary Syndrome (SCPH) due to ANDV is an acute and endemic acute viral disease in Chile that annually affects 60-70 patients between the III-XII regions of Chile, with a mortality of 37%.
  • SCPH Cardiopulmonary Syndrome
  • patients who survive the infection maintain immunity from re-infection for life. It has been shown that this immunity is due, at least in part, to the persistence of neutralizing antibodies, which prevent the virus from binding to its receptors on the surface of white cells.
  • the natural reservoir of ANDV is the rodent Oligoryzomys longicaudatus, which are infected by horizontal transmission but do not develop the disease, being chronic carriers of the virus.
  • the ANDV replicative cycle begins when it enters the airway and binds by some domain of its Gl and G2 proteins to avb3 integrins and other receptors still unknown in the cell membrane of lung endothelial cells, peripheral blood mononuclear cells (PBMC) and dendritic cells of the interstitium.
  • PBMC peripheral blood mononuclear cells
  • Hantaviruses mostly infect endothelial cells, peripheral blood mononuclear cells (PBMC) and dendritic cells; however, neither Hantavirus infection nor replication is cytopathic for these cells (7-10).
  • PBMC peripheral blood mononuclear cells
  • the immune response to the virus participates in the pathogenesis of Hantavirus disease. Specifically, the cellular response by T lymphocytes against antigens presented in infected endothelial cells is considered as causing the increased vascular permeability and pulmonary edema of SCPH (1 1-14).
  • T lymphocytes against antigens presented in infected endothelial cells is considered as causing the increased vascular permeability and pulmonary edema of SCPH (1 1-14).
  • in vivo studies it has been suggested that immunity to virus pathology is directly associated with immunity to Hantavirus (15-18). However, to date it is unknown which epitopes are recognized by these neutralizing antibodies that would mediate permanent immunity. This information is crucial both
  • Macaques vaccinated with DNA encoding the M segment developed high antibody titers against G1 / G2.
  • SCPH the only existing animal model for SCPH
  • US Patent 5614193 (US ARMY) describes vaccines with a peptide selected from SEQ ID NO: 1, 647 amino acids, which codes for Hantavirus Gl protein and which contains, at its 427-441 position, the DMDIWYCNGQRKVI peptide with 80% homology with the immunogenic peptide of the invention.
  • peptide 427-441 could have immunogenicity by itself, which describes fundamental differences in its effectiveness.
  • Japanese patent JP 8 325 291 (Yoshimatsu et al) discloses a Hantavirus antigenic protein and a monoclonal antibody, where the protein does not contain the peptide of the invention.
  • the peptides investigated and used in this patent application for the identification of the relevant epitopes consisted of 15 amino acids each with a linear conformation. Because it was possible that this methodology did not detect all specific antibodies effective in recognizing conformational epitopes present during viral replication, this was resolved using overlapping peptides of 14-21 amino acids in size. In addition, since recombinant proteins also show a linear structure and, in the case of Gl and G2, are extremely unstable, the use of overlapping peptides does not represent disadvantages with respect to other possible approaches.
  • the methodology proposed in this application represents an adequate and globally applied technique to detect specific antibodies with or without neutralizing function.
  • This methodology allows the detection of G1 / G2 epitopes that are recognized by the plasma antibodies of convalescent patients.
  • the present invention contributes to the development of passive and / or active immunotherapy.
  • the present application shows the identification of the anti-Gl_2210 antibody of Hantavirus Andes, of high prevalence among patients convalescent to the infection, being detectable with a high sensitivity (100%) by ELISA with linear peptides.
  • This antibody can be efficiently purified by affinity chromatography from the patient plasma, while maintaining its functional capacity, being able to recognize in situ the conformational structure of the epitope previously identified by ELISA with linear peptides.
  • EXAMPLE 1 Identification and quantification of epitopes of the glycoproproteins Gl and G2 of the Hantavirus Andes (ANDV).
  • Group A individuals in whom Hantavirus infection (anti-N IgG is documented
  • Group B individuals in whom Hantavirus infection (anti-N IgG positive) is documented with moderate or severe symptoms and who required mechanical ventilation and / or hemodynamic support (VM / SH) or ECMO. In addition, healthy individuals were included.
  • Hantavirus infection anti-N IgG positive
  • VM / SH hemodynamic support
  • ECMO hemodynamic support
  • VM / SH mechanical ventilation and / or hemodynamic support or ECMO
  • Antibody detection by ELISA Enzyme Immunoemayo: An ELISA based on overlapping peptides was performed encoding the ANDV Gl and G2 proteins and detecting the antibodies present in the patients' plasma (IgG) that specifically bind said peptides. The establishment of this methodology was based on the ELISA protocol by detecting specific IgG antibodies for the recombinant N protein published by the Centers of Disease Control (CDC). Triplicates of 10 mixtures of overlapping and continuous peptides (mix 1 to mix 10: concentration 20 nmol / ml per peptide) were incubated overnight at 4 ° C in a 96-pocket plate. The ANDV recombinant N protein was used as a positive control.
  • EXAMPLE 2 Identification of immuno-dominant epitopes of protein N.
  • EXAMPLE 3 Identification of immunodominant epitopes of the G2 protein. COMPARATIVE EXAMPLE WITH ART (Tischler et al).
  • EXAMPLE 4 Identification of immunodominant epitopes of the Gl protein.
  • serial dilutions of anti-Gl_2210 IgG Figure 5 we determine the dilution at which the anti-Gl_2210 antibody signal disappeared. On average, patients with mild SCPH showed an average titer of 1/6933 of anti-Gl_2210 while patients with SCPH severe showed an average grade of 1/3167, without achieving a significant difference analyzed by the student t test.
  • EXAMPLE 7 Indirect Immunofluorescence Study (IFI) to study in vitro recognition of the conformational epitope of the anti-Gl_2210 antibody.
  • Indirect Immunoflourescence was performed.
  • Vero E6 endothelial cells were cultured on 8-well Lab-Tek Chamber Slide plates (Nalge Nunc International) and incubated overnight in a culture chamber at 37 ° C until confluence. They were then infected with ANDV at an approximate concentration of 100 PFU (Plaque Forming Units) / lOOul.
  • Figure 2 Peptide and nucleotide sequences of consensus objects of the present invention.
  • Figure 3 Hanta virus infection cycle and viral structural composition.
  • Figure 4 Anti-Gl_2210 antibody isolation protocol isolated from patients' plasma.
  • Figure 5 Working protocol for identification of immunogenic peptides and specific epitopes in patients convalescent to ANDV infection
  • Figure 7 Recognition of individual peptides of mix 6 by specific IgGs, determined in 12 patients. The results indicate the percentage of patients whose samples (applied in triplicates) showed equal or more absorbance than the average of the healthy controls + 2xSD controls. Each patient's plasma was exposed in a 1: 50 dilution to each of the indicated peptides (10 nmol / ml each) in an ELISA.
  • Figure 8 Specific IgGs for individual peptides of mix 32 and 33, determined in 8 patients. The results indicate the percentage of patients whose samples (applied in triplicates) showed equal or more absorbance than the average of the healthy controls + 2 standard deviations. Each patient's plasma was exposed in a 1: 50 dilution to each of the indicated peptides (10mg / ml c / u) in an ELISA.
  • Figure 9 IgG specific for individual peptides of mix 22, determined in 27 patients. The results indicate the percentage of patients whose samples (applied in triplicates) showed equal or more absorbance than the average of the healthy controls + 2xSD control. Each patient's plasma was exposed in a 1: 50 dilution to each of the indicated peptides (10nmol / ml c / u) in an ELISA.
  • Figure 10 Specific IgG titration for Gl_2210, determined in 12 patients. The results indicate the absorbance obtained by the different serial serial dilutions of each of the patients. Results represent the average of triplicates in each patient. Plasma of each patient was exposed in different dilutions (1: 50-1: 409600) to 10nmol / ml of Gl_2210 in an ELISA.
  • Figure 11 Comparison between the anti-Gl_2210 titer in the plasma prior to chromatography and the eluate titer.
  • Figure 12 (63x) The images on the left side correspond to anti-Gl_2210 in dilutions 1: 200, 1: 400 and 1: 800 and the images on the right side correspond to anti-Gl_2210 previously incubated with recombinant N protein (* N). DAPI-stained nuclei (in blue) allow the identification of the N protein, which
  • Image 8-b shows, in the same way, the same rabbit plasma but previously incubated for 4 hours at room temperature, with 2 ⁇ g of recombinant N protein. In this, the absence of mareaje is observed, which proves the high effectiveness of this pre-incubation as a method to decrease the binding of antibodies to the viral N protein.
  • Plasma rabbit anti-N 13-a Plasma rabbit anti-N 13-b. Plasma rabbit anti-N / * N
  • Figure 14 (63x) Images 9-a and 9-b correspond to the negative controls performed with papillomavirus.
  • BPV Bovine Papiloma Virus
  • HPV Human Papiloma Virus
  • Absence of mareaje can be observed, which accounts for a high specificity for the epitope identified in the ANDV Gl protein.
  • images 9-c and 9-d 2 plasma samples of patients convalescent to ANDV infection are observed, performed as positive controls. In these, a diffuse pattern of very intense pattern is observed in the perinuclear areas, which would correspond to the extensive screening of all the antibodies developed by these patients.
  • Anti-BPV Negative Control
  • Anti-HPV Negative Control

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  • Health & Medical Sciences (AREA)
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  • General Physics & Mathematics (AREA)
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Abstract

La présente invention concerne l'identification de l'anticorps polyclonal humain anti-Gl_2210, qui est spécifique contre la glycoprotéine G1 de l'Hantavirus andes (ANDV), de haute prévalence chez les patients convalescents sujets à l'infection, et détectable avec une forte sensibilité (100%) au moyen d'une technique ELISA avec des peptides linéaires. Cet anticorps peut être purifié efficacement par chromatographie d'affinité à partir du plasma de patients, conserver sa capacité fonctionnelle et être capable de reconnaître in situ la structure conformationnelle de l'épitope identifié préalablement au moyen d'une technique ELISA avec des peptides linéaires. L'anticorps anti Gl_2210 peut être utilisé en tant que neutralisant in vivo et in vitro contre l'infection par ANDV ou d'autres Hantavirus sous forme d'immunothérapie passive ou active. La présente invention concerne également le peptide immunogénique, des compositions, des vaccins et des trousses, ainsi que leur utilisation contre les infections par Hantavirus ou pour effectuer la détection d'échantillons biologiques positifs pour Hantavirus.
PCT/CL2010/000052 2010-12-20 2010-12-20 Anticorps polyclonal neutralisant dirigé contre l'hantavirus andes, procédé de production et compositions pharmaceutiques WO2012083475A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015130722A1 (fr) * 2014-02-25 2015-09-03 Albert Einstein College Of Medicine Of Yeshiva University Méthodes et dosages pour le traitement d'infections par des hantavirus

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040053216A1 (en) * 1999-01-29 2004-03-18 Hooper Jay W. DNA vaccines against hantavirus infections
WO2006088478A2 (fr) * 2004-06-18 2006-08-24 Novartis Vaccines And Diagnostics Inc. Methodes et reactifs permettant de diagnostiquer une infection a hantavirus

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040053216A1 (en) * 1999-01-29 2004-03-18 Hooper Jay W. DNA vaccines against hantavirus infections
WO2006088478A2 (fr) * 2004-06-18 2006-08-24 Novartis Vaccines And Diagnostics Inc. Methodes et reactifs permettant de diagnostiquer une infection a hantavirus

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MANIGOLD, T. ET AL.: "Highly differentiated, resting Gn-specific memory CD8+ T cells persist years after infection by Andes Hantavirus.", PLOS PATHOGENS., vol. 6, no. 2, February 2010 (2010-02-01), pages 1 - 16 *
TISCHLER, N. D. ET AL.: "Human and rodent humoral immune responses to Andes virus structural proteins.", VIROLOGY., vol. 334, no. 2, April 2005 (2005-04-01), pages 319 - 326 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015130722A1 (fr) * 2014-02-25 2015-09-03 Albert Einstein College Of Medicine Of Yeshiva University Méthodes et dosages pour le traitement d'infections par des hantavirus
US10105433B2 (en) 2014-02-25 2018-10-23 Albert Einstein College Of Medicine, Inc. Methods and assays for treating hantavirus infections

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