WO1999001153A1 - Hantavirus du rio mamore - Google Patents

Hantavirus du rio mamore Download PDF

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Publication number
WO1999001153A1
WO1999001153A1 PCT/US1997/009695 US9709695W WO9901153A1 WO 1999001153 A1 WO1999001153 A1 WO 1999001153A1 US 9709695 W US9709695 W US 9709695W WO 9901153 A1 WO9901153 A1 WO 9901153A1
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WO
WIPO (PCT)
Prior art keywords
rmv
hantavirus
virus
antigen
protein
Prior art date
Application number
PCT/US1997/009695
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English (en)
Inventor
Brian L. Hjelle
Norah Torrez-Martinez
Original Assignee
The University Of New Mexico
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The University Of New Mexico filed Critical The University Of New Mexico
Priority to PCT/US1997/009695 priority Critical patent/WO1999001153A1/fr
Publication of WO1999001153A1 publication Critical patent/WO1999001153A1/fr

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    • 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
    • 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
    • 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
    • C12N2760/12122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Hantaviruses are a group of at least 19 diverse agents. They occur worldwide in rodent hosts and cause either (1) no known human disease; (2) hemorrhagic fever with renal syndrome, HFRS, or (3) Hantavirus pulmonary syndrome (HPS) when transmitted to man.
  • HPS Hantavirus pulmonary syndrome
  • N protein is the portion of each hantavirus that is most strongly immunogenic, and the standard for diagnosis of Hantaviruses has increasingly been to rely upon the expression of homologous N protein in bacteria or other microbial expression system to generate high concentrations of recombinant-expressed antigen.
  • Classical methods of viral antibody detection have depended upon the growth of the virus in culture, with use of the viral antigens from infected cultures in immunologic detection, but these methods are increasingly falling out of favor for a variety of technical and practical reasons.
  • Hantaviruses Specific diagnostic tests are available for several previously-described Hantaviruses. For Hantaviruses in general, antibody tests are much preferred over direct detection of infectious viral particles, viral genomic RNA, or viral antigens because of the inherently superior stability, sensitivity, specificity, and ease of transfer of antibody assay technologies.
  • the following modalities are in common use (C) or are under development or research use (D) for the following Hantaviruses:
  • the invention provides a molecular clone encoding and expressing the complete nucleotide protein of Rio Mamore virus.
  • the RMV Nprotein inlcudes antigenically active domains useful in immunoassays for detecting South American Hantavirus infection, and in vaccines.
  • the far right panel is a dried polyacrylamide gel containing 4 different protein preparations.
  • the first lane (“C”) is a crude lysate of E. coli JM101 cells after induction of expression from the pET23b vector that lacks a viral genetic insert.
  • the next 3 lanes contain the pET23b-expressed, affinity-purified viral N proteins from Bayou (BAY), Rio Mamore (RM) or Sin Nombre (SN)Hantaviruses.4 western blot membranes containing the same purified proteins are at left.
  • Panels (clockwise from upper left)were probed with serum of (1) an RMV-infected Oligoryzomys microtus mouse; (2) a BAYV-infected rice rat (Oryzomys palust s) from Texas; (3) an uninfected deer mouse (Peromyscus maniculatus); (4) an SNV- infected deer mouse (Peromyscus maniculatus) from Texas.
  • Antibodies were detected with an alkaline phosphatase-conjugated goat anti P. leucopus reagent, followed by exposure to alkaline phosphatase substrate as described (Jenison et al., 1994).
  • the dark bands in the BAY, RMV, and SNV lanes indicate the presence of antigenically-active N protein that reacts with rodent serum. All N proteins are about 55 kD in apparent molecular weight. Differential reactivity is evident in each panel, in each case stronger reactivity is evident against the homologous viral antigen.
  • Figure 2 The complete sequence of the S genomic segment and associated N protein of Rio Mamore virus, specimen OM NK 13556.
  • FIG. 3 Maximum-parsimony phylogenetic tree showing position of RMV (RM-OM556) relative to prototypical Hantaviruses BAY (Bayou), SNV (SN), Hantaan (HTN, Puumala (PUU), Isla Vista (ILV), Tula (TUL), Bloodland Lake (BLLL), New York (NY), El Moro Canyon (ELMC), Black Creek Canal (BCC), Rio Segundo (RIOS), Prospect Hill (PH) and Muleshoe (MULE).
  • T e tree is based upon the entire N gene sequences (1287-nt). Detailed Description of the Invention
  • Oryzomine rodents were selected as potentially important vectors of human Hantavirus disease in South America because (1) they are abundant, widespread, and occur in high density; (2) they favor disturbed habitat such as houses and other human habitations; (3) was recently identified as a North American oryzomine rodent, Oryzomys palusths is the host for an etiologic agent of HPS (Bayou virus) in Louisiana and Texas. Accordingly, tissue specimens of candidate oryzomine rodent hosts were obtained from the Museum of Southwestern Biology (University of New Mexico) and from the Museum of Vertebrate Zoology (University of California, Berkeley). These included:
  • RNA samples were positive for Hantavirus antibodies. These tissue samples were used to prepare RNA, and the RNA was then subjected to reverse transcription-polymerase chain reaction (RT- PCR) analysis to identify the virus and to prepare recombinant antigens through expression of the PCR-amplified DNA in molecular clones.
  • RT- PCR reverse transcription-polymerase chain reaction
  • RMV Zika virus
  • the high-level expression of the N protein allows incorporation into a variety of antibody testing formats to produce the most efficient and accurate diagnostic tests.
  • the RMV N protein is antigenically active in western blot and ELISA formats. Additional conventional formats such as immunofluorescence assay, particle agglutination and radioimmunoprecipitation assays are contemplated.
  • RMVN protein whether in humans, rodents, or other animals are described herein.
  • the western blot and ELISA assays have been reduced to practice, and other methods are readily adapted from these procedures (given the purified antigen) by trivial manipulations.
  • an ELISA system in which a microtiter well is first coated with goat IgG directed against human IgM is of particular interest. The well is then treated with the serum of a patient with suspected RMV infection, washed, then treated with purified recombinant antigen. After washing, a biotin-labeled rabbit antibody directed against the recombinant antigen of RMV (see below) is applied. Finally a streptavidin-conjugated alkaline phosphatase is used to detect the bound biotin. A chromogenic alkaline phosphatase substrate is used to detect bound alkaline phosphatase.
  • rDNA-derived antigens will continue to be useful diagnostic tools.
  • rDNA antigens provide valuable supplementary information to that provided by ELISAs using cultured virus as antigen; in some cases, rDNA antigens have superior sensitivity or superior ability to differentiate among antibodies directed against different related viruses, or ability to diagnose infection by viruses for which there is no method of culture.
  • virtually all North American HPS virus infections are diagnosed with recombinant antigen-based systems.
  • the initial reaction mixes were as follows. All mixtures contained 10 pmol of each primer; 1.7 mM 2- mercaptoethanol; 1.5mM MgC 10 mM Tris-HCI (pH 8.3); 50mM KCI; 200uM each of dATP, dTTP, dGTP, and dCTP; 10 units of AMV reverse transcriptase (Boehringer-Mannheim), and 2.5 units of AmpliTaqTM DNA polymerase (Perkin-Elmer), in a final volume of 100 ul.
  • the "second round” PCR product was prepared by thermal cycling at 94-40-72 (1 minute, 1 minute, and 3 minutes, respectively) for 8 cycles, followed by 29 more cycles at 94-42-72 (1 minute, 1 minute, and 3 minutes, respectively). The reaction was then subjected to an elongation step of 70°for 10 minutes. The DNA product was then loaded on an 1.2%-1.6% agarose gel, electrophoresed for 1 h at 80V, and the band of the appropriate molecular weight was then excised with a razor blade. The DNA was extracted from the gel with a glass-milk resin (Qiaex resin, Qiagen Inc.) after melting the gel in a sodium iodide solution.
  • a glass-milk resin Qiaex resin, Qiagen Inc.
  • the PCR product was taken up in 10 ul of (10 mM Tris-HCI, pH 8/1 mM EDTA), and 5-10 ul was ligated to the pCRII vector according to the manufacturer's instructions (Invitrogen Corp.).
  • 10 ul ligation mix was used to transform E. coli cells according to the manufacturer's instructions (Invitrogen), and the transformed cells plated onto LB media containing 50 ug/ml ampicillin and 0.005% X-Gal; plates were incubated at 37° overnight. Clear colonies were selected from the plate the following morning and expanded in 4 ml of LB media containing 50 ug/ml ampicillin.
  • PCR primers were designed either as consensus primers (conserved portions of other Hantavirus S segments that were predicted to be conserved in RMV) or by directly reading the sequences of RMV clones and designing primers from those sequences.
  • Bolded sequences in primers 4 and 5 represent restriction endonuclease sites that were introduced as the primers were designed. All coordinates refer the location of the 5'-most residue of the primer; each primer is written in 5'-3' direction.
  • RNAs from five seropositive O. microtus were used in this round, and all were positive. Since specimen OM NK 13556 (from Beni Department, Peru) produced the strongest amplification signal, it was used exclusively in further amplification reactions, and represents the prototype RMV S segment sequence.
  • the intact N gene open reading frame was then cloned by amplifying with primers 2 and 3, followed by nesting with primers 4 and 5, yielding a 1287-nt product.
  • Plasmid DNA was prepared from the cultures according to standard methods, and then digested with various restriction enzymes to verify that the correct insert had been obtained. Clones that appeared to have the correct restriction enzyme digestion pattern were subjected to DNA sequencing (according to the manufacturer of the SequenaseTM sequencing system, US Biochemicals) to verify that DNA with characteristics appropriate for a novel Hantavirus had been amplified and cloned. The DNA was examined for strong homology to (but not identity to) previously-described Hantaviruses SNV and Bayou virus, and relative conservation of the protein product that would be predicted from the nucleotide sequence.
  • the complete N gene open reading frame was excised from the cloning vector pCRII, and subcloned at the Hind III and Xho I restriction sites (which were incorporated into the PCR primers 2 and 3).
  • the gene was transferred into the pET 23b vector (Novagen Inc.) at the Hind III and Xho I sites and expanded in E. co// strain BL21 , wherein the gene is not expressed.
  • This vector places the N gene in fusion with a small leader genetic element derived from the T7 bacteriophage. Correct clones were identified by restriction endonuclease digestion and DNA sequencing.
  • the clone was then transfected into the expression-competent cell, BL21 (DE3), where N gene expression was induced with IPTG according to the manufacturer's instructions.
  • the use of the pET 23b expression system is designed to allow the ready purification of recombinant antigens for preparation of ELISAs and "slot-blot" format assays such as the RIBATM (Chiron Corporation). These assays require that the antigen be extremely pure, because they are subject to false reactivities that can be associated with even small amounts of contaminating E. co// ' antigens.
  • the purification of the recombinant proteins is facilitated by the presence in the pET 23b of a genetic segment that encodes a "histidine tag", ie, a series of 6 histidine residues in tandem.
  • This peptide tag has high affinity for certain metal ions (Cesium, Nickel) that can be incorporated into an affinity column.
  • the Novagen Corporation sells such columns as kits for purification of recombinant proteins. These columns were purchased and found to work very well for purification of the RMV N protein.
  • the purified N protein was placed on western blots and in microtiter wells, and probed with serum from animals and humans with and without Hantavirus infection ( Figure 1). Strong immunoreactivity of the RMV N protein was evident in these studies, indicating that it is useful in detection of Hantavirus antibodies.
  • the proteins After expressing recombinant proteins in E. coli or bacuiovirus systems from our rDNA RMV clones, the proteins are purified and used to immunize rabbits, mice and/or other animals by injection.
  • the resultant antibodies (both polyclonal and monoclonal) are useful in diagnosis, treatment, or prophylaxis of RMV infection in the following ways.
  • the RMV N antigen is expressed in cultured cells under the control of a vaccinia or other heteroiogous virus' replication machinery, and used to prepare live or killed-virus vaccinia antigens.
  • the RMV DNA is used as a substrate for "naked DNA vaccines", i.e., immunization by injection of purified RMV DNA intramuscularly into humans or animals.
  • Purified RMV N protein, expressed in bacuiovirus, yeast, or E. coli is injected into humans or animals in combination with a customary pharmacological carrier to allow development of an immune response and protective immunity.
  • RMV infection will result in a rapid drop in the level of circulating virus during the course of a patient's treatment.
  • RMV Clones for In Situ Hybridization.
  • the availability of the RMV S genome sequence allows the preparation of specific probes for detection of RMV RNA in cell culture, in human and animal tissues, and in human blood cells. Specific probes can be made to either strand of the virus. Plus strand probes, corresponding to the mRNA of the virus, should be useful in detecting RMV (a minus-strand virus), wherever it occurs. Minus strand probes should be useful in detecting the antigenomic strand and mRNA of RMV, which would occur only in tissues in which the virus is replicating.
  • In situ hybridization has great potential as a research tool for understanding the replication of the virus, and some potential as a diagnostic, prognostic, or therapeutic tool if it proves able to detect replicating and non- replicating RMV in patient blood specimens, human or animal tissues, or viral cultures.
  • This method is developed by expressing radio labeled RNA from DNA clones of the RMV in vitro, and using the RNA probes as reagents for in situ hybridization studies of infected tissues.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

Clone moléculaire codant et exprimant la protéine nucléocapsidique du segment S du hantavirus du Rio Mamoré.
PCT/US1997/009695 1997-07-03 1997-07-03 Hantavirus du rio mamore WO1999001153A1 (fr)

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Application Number Priority Date Filing Date Title
PCT/US1997/009695 WO1999001153A1 (fr) 1997-07-03 1997-07-03 Hantavirus du rio mamore

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PCT/US1997/009695 WO1999001153A1 (fr) 1997-07-03 1997-07-03 Hantavirus du rio mamore

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002038174A2 (fr) * 2000-11-08 2002-05-16 Humboldt-Universität Zu Berlin Universitätsklinikum Charite Vaccins contenant des proteines hantavirus recombinantes, leur procede de production et leur utilisation

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
HJELLE et al., "Hantavirus Pulmonary Syndrome-Related Virus from Bolivia", LANCET, 06 January 1996, Vol. 347, page 57. *
LEDUC et al., "Isolation of a Hantaan-Related Virus from Brazilian Rats and Serologic Evidence of its Widespread Distribution in South America", AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 1985, Vol. 34, No. 4, pages 810-815. *
LEVIS et al., "New Hantaviruses Causing Hantavirus Pulmonary Syndrome in Central Argentina", THE LANCET, 05 April 1997, Vol. 349, pages 998-999. *
LOPEZ et al., "Genetic Identification of a New Hantavirus Causing Severe Pulmonary Syndrome in Argentina", VIROLOGY, 1996, Vol. 220, pages 223-226. *
WELLS et al., "An Unusual Hantavirus Outbreak in Southern Argentina: Person-to-Person Transmission", EMERGING INFECTIOUS DISEASES, April-June 1997, Vol. 3, No. 2, pages 171-174. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002038174A2 (fr) * 2000-11-08 2002-05-16 Humboldt-Universität Zu Berlin Universitätsklinikum Charite Vaccins contenant des proteines hantavirus recombinantes, leur procede de production et leur utilisation
WO2002038174A3 (fr) * 2000-11-08 2003-08-28 Humboldt Uni Zu Berlin Univers Vaccins contenant des proteines hantavirus recombinantes, leur procede de production et leur utilisation

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