Her Majesty the Queen in Right of Canada, As Represented by the Minister of National Defence

National Research Council of Canada

Her Majesty the Queen in Right of Canada, As Represented by the Minister of National Defence of Her Majesty's Canadian Government

Other

Canadian Patents & Development Ltd.

Her Majesty the Queen As Represented by the Minister of National Defence

Her Majesty the Queen in Right of Canada, As Represented by the Minister of National Defence in Her Brittanic Majesty's Government of the United Kingdom of Great Britain and Northern Ireland

Her Majesty the Queen in Right of Canada Represented by the Minister of National Defence of Her Majesty's Canadian Government

John Walter Cherwonogrodzky

Scott James Jager

Wei-Gang Hu

Christopher Sikora

Junfei Yin

Bradley Berger

Laurel M Negrych

Damon Chau

Jonathan P Wong

David R Bundle

Malcolm B Perry

Kamil Lotfali

J Robert Duncan

Klaus Nielsen

William Edward Lee

Peter F Wright

Vincent L Di Ninno

Maureen L Simpson

Martine G Caroff

Bill Kournikakis

Robert Toussaint Poirier

Vincent L Dininno

The present invention relates to anti-ricin antibodies and uses thereof. More specifically, the invention relates to anti-ricin antibodies and fragments thereof as well as their use in therapy or prophylaxis.

Anti-ricin antibodies and uses thereof

Low molecular weight serum components (less than 10,000 m.w.), in vaccinated animals and a human subject who has been exposed to a threat agent inadvertently, bound to purified O-polysaccharide (OPS, a polymer of formamido-mannose) and a candidate of a threat agent, such as145 vaccine is disclosed. These components formed a loose reversible precipitin with OPS in a high-salt borate-buffered agarose gel and bound to the candidate vaccine as observed by capillary electrophoresis. By using modified capillary electrophoresis, the invention also discloses the presence of two larger serum components, one similar in size to that of serum albumin and one resembles that of mannan-binding lectin, that bound to the vaccine. An indirect method for identifying vaccination is the presence of antibodies against-OPS-antibodies. ELISA, capillary electrophoresis and animal challenge studies showed that as high as 30% of the control animals did not require vaccination. These animals could have been exposed to cross-reactive cross-protective antigens naturally.

Serum components that bind to threat agents

This invention discloses methods for identifyingvaccine candidates. It enables identification of novel vaccine candidates and quality assurance for vaccine batches, assessment of protection in vaccinates and identification of the infecting agent in vaccinates. Mice were first vaccinated withO-polysaccharide (OPS) vaccine. These animals were then given 10 LDs oflive vaccine strain (LVS). Sixty percent (60%) of the vaccinated mice survived the multiple lethal doses. Sera were collected from these surviving mice and the antibodies were used to probe supernatant and cell lysates of liveLVS cultures. Severalcomponents were identified only by the noted 'survivor' antisera. Of these identified proteins, enzyme digestions and chemical oxidation suggest post-translational modifications of some proteins e.g. a 52 kDa glycoprotein, a 45 kDa lipoprotein and a 19 kDa nucleoprotein. The 52 kDa component caused nitrous oxide induction in tissue cultures at low concentrations, cell death at high concentrations. Vaccination with this gave partial protection while addition of other components acted synergistically to give enhanced protection from 250 LDs ofLVS.

Use of cross-protection to identify novel vaccine candidates for infectious agents

A vaccine comprising a combination of Brucella &ldquo;A&rdquo; and &ldquo;M&rdquo; outer-polysaccharides (OPSs) and &ldquo;R&rdquo; protein antigens for enhancing immunity against brucellosis is disclosed. The OPS may be obtained from different strains or species of Brucellae (i.e. combining OPS extracted from different bacteria expressing &ldquo;A&rdquo; or &ldquo;M&rdquo; OPS, or combining OPS and OPS-protein complexes extracted from different bacteria). The OPS or OPS-protein complexes may also be obtained from a single strain expressing more than one OPS (e.g. from strain 145 which expresses &ldquo;A&rdquo;, &ldquo;M&rdquo; and possibly other OPSs). The vaccine according to the present invention overcomes the limitation of previously discovered &ldquo;A&rdquo; OPS which only protects against species and strains of Brucella that had &ldquo;A&rdquo; OPS but not against others with different OPS.

Combination vaccine for enhancing immunity against brucellosis

The present invention relates to the effects of bacterial polysaccharides on cell mediated immunity in animals. Polysaccharides of the present invention comprise the outer polysaccharides (OPS) located on bacterial cell membranes and other polysaccharides (e.g. &ldquo;Poly B&rdquo;) either secreted or contained within the periplasmic space. These polysaccharides have been found to enhance the general or cell mediated immunity of animals to various diseases. The invention provides for the use of such polysaccharides in preventing and treating various infections as well as in treating carcinomas. The invention also provides for synthetic polysaccharides having the same immuno-modulating effect as the bacterial polysaccharides.

Bacterial and synthetic polysaccharide immunomodulators that enhance general immunity

A bacteriophage linked to an enzyme can replace an antibody in a system for detecting the presence of a bacteria in a sample. Specifically (a pathogen which causes brucellosis in cattle) can be detected using Brucella bacteriophage for the virus, urease for the enzyme linked to the bacteriophage, m-maleimidobenzoyl-N-hydrosysuccimide ester as a coupling reagent, sera from mice immunized with Brucella bacteriophage for a detector antibody, urease conjugated to anti-mouse sheep antibody for an indicator, and urea with bromcresol purple as the substrate. The materials can be used in indirect (sandwich) or direct enzyme-linked viral assays (ELVirA).

Method of detecting a pathogen using a virus

Liposome encapsulated antibiotic therapy has limited application against infectious organisms, which can sequester in non-phagocytic cells. Virulence factors of these infectious organisms, for example bacterial components, when used in the formulation of liposomes can enhance the effectiveness of liposomes as delivery systems in the treatment of disease. In this manner, multi-functional liposomes can be developed to treat target diseases. In addition to serving as antibiotic delivery systems, such liposomes also have an immunization effect. Thus, the liposomes can be used for both the prevention and treatment of diseases.

Use of virulence factors of pathogens to improve liposomal delivery of therapeutic agents

A vaccine comprising purified outer-polysaccharide (OPS) is effective for protection against brucellosis. The vaccine is derived from Brucella or a variety of cross reactive bacteria. The vaccine can be administered by different routes (intramuscularly, subcutaneously, intraperitoneally, orally). The vaccine is effective in protecting against other infectious bacteria, aside from Brucella. It is likely that the vaccine can be given after infection to reduce illness.

Polysaccharide vaccine to enhance immunity against brucellosis

A method is disclosed for discriminating between cattle vaccinated against and those infected with Brucella spp. The method involves immunoassay using a purified polysaccharide containing 4,6-dideoxy-4-acylamido-D-mannopyranosyl units obtained from B. abortus or from cross-reacting organisms, and results in improved differentiation between vaccinated and infected animals. Test kits are also disclosed for performing the assay and a process is disclosed for obtaining the O-chain polysaccharides in high purity and yield.

Immunoassays for discriminating between brucellosis infections and

The antigenic O-chain polysaccharides from Brucella abortus and Yersinia enterocolitica serotype 0:9 have similar structure, i.e. 1-2 linked 4,6-dideoxy-4-formamido-.alpha.-D-mannopyranosyl units. The antigen is more readily isolated from the Yersinia and may be chemically modified to have a hydrophobic moiety attached thereto. The antigen can be adsorbed or coupled to carriers suitable for immunoassay use such as ELISA and RIA. A monoclonal antibody capable of specifically binding to B. abortus and Y. enterocolitica, has been produced. Assay procedures and kits therefor, are described.

Antigenic polysaccharide specific to Brucella abortus and Yersinia

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Medicine Hat, Canada

John Walter Cherwonogrodzky