WO2013097838A1

WO2013097838A1 - Live attenuated salmonella enterica capm 6449 strain for oral vaccination of farm animal

Info

Publication number: WO2013097838A1
Application number: PCT/CZ2012/000138
Authority: WO
Inventors: Daniela KARASOVÁ; Alena ŠEBKOVÁ; Hana HAVLĺČKOVÁ; Marta MATULOVÁ; Magdaléna CRHÁNOVÁ; Jíří VOLF; Martin FALDYNA; František ŠIŠÁK; Ivan RYCHLÍK
Original assignee: VÝZKUMNÝ ÚSTAV VETERINÁRNÍHO LÉKAŘSTVÍ, v.v.i.
Priority date: 2011-12-27
Filing date: 2012-12-19
Publication date: 2013-07-04
Also published as: EP2841094A1; CZ2011887A3; RU2014125037A; CZ307299B6

Abstract

The solution refers to a live attenuated strain Salmonella enterica CAPM 6449 which is deposited in the Collection of Zoopathogenic Microorganisms, Brno, Czech Republic and is intended as a live attenuated vaccine for oral vaccination of farm animals.

Description

Live attenuated Salmonella enterica CAPM 6449 strain for oral vaccination of farm animals

Technology Area

The patent claims a protection of a live attenuated strain of Salmonella enterica CAPM 6449 with specific deletions used for its attenuation, and which is suitable for oral immunisation and protection of farm animals against Salmonella infection.

The Current State of Technology

Protection of farm animals against Salmonella infections by vacciantion can be achieved by two types of vaccines - inactivated or live attenuated vaccines. Due to the course of infection, the live attenuated vaccines are more protective than the inactivated ones (Papezova et al. 2008, Vet. Med. Czech 53, 315; Robertsson et al. 1983, Infect. Immun. 41 , 742; Silva et al., 1981 , Avian Dis. 25, 38-52).

The first attenuated vaccines were obtained by prolonged subcultures in vitro or random mutagenesis induced by physical or chemical agents. Such experimental approaches resulted in a selection of strains with reduced virulence but retained immunogenicity. Although such vaccines are fully functional and have been extensively characterised, the real reason of their reduced virulence and attenuation is unknown (Guzman et al., 2006, Vaccine 24, 3804-11 ; Smith 956, J Hyg.(Lond);54, 419-32).

This is a reason why from the 80th of last century, new genetically modified and defined vaccines are being tested. Great advantage of such vaccines is their clear definition of their attenuation. Those tested the most frequently include aroA, aroD or phoP mutants and mutations (Cooper et al. 1990, Microb. Pathog. 9, 255; Hormaeche et al. 1996, Vaccine 14, 251 ; Groisman et al. 1992, Proc. Natl. Acad. Sci. USA 89, 1 1939). Approximately 10 years later, genes directly involved in Salmonella virulence have been identified. Some of the mutations in such genes, e.g. those localised at Salmonella pathogenicity island 2 (SPI2) lead to an over-attenuation and loss of immunogenicity of such mutants. On the other hand, inactivation of genes encoded by Salmonella pathogenicity island 3 (SPI-3) leads to only a minor decrease in virulence of such a mutant which need not be suitable enough for the vaccination of immunocompromised individuals. This is why new ways of attenuation of Salmonella strains are being sought.

To both increase attenuation and safety, vaccine strains attenuated by two independent mutations have been tested. These include vaccines with inactivated htrA and aroA genes (Tacket a Levine 2007, Clin. Infect. Dis. 45, S20), or phoP and rpoS genes (Methner et al. 2004, Vet. Microbiol. 98, 37-43).

The Essence of the Invention

All of the above mentioned limitations are absent in bacterium Salmonella enterica CAPM 6449 in which is attenuated by two particular mutations. Combination of two specific mutations is the subject of this application. Resulting live attenuated Salmonella strain is intended for a vaccination of farm animals and is deposited in the Collection of Zoopathogenic Microorganisms in Brno, CZ.

To increase safety of live attenuated Salmonella vaccine for farm animals we constructed a strain of Salmonella enterica from which we removed both the whole pathogenicity island 1 (SPI1 ) and a gene coding for Lon protease. Salmonella attenuation by inactivation of biological functions encoded by SPI1 was originally overlooked, since functions encoded by SPI1 are disposable for Salmonella virulence in Balb/C mice, in which SPI1 mutants cause lethal infection (Karasova et al. 2010, BMC Microbiology 10, 75). However, inactivation of SPI1 encoded functions results in decrease in virulence of such mutant for chickens and pigs (Rychlik et al. 2009, BMC Microbiology 9, 268). lon gene encodes Lon protease, inactivation of which leads to Salmonella attenuation both in mice and poultry. Moreover, the Ion mutant retains its immunogenicity, both in mice and poultry. In this patent application we therefore combined two modes of attenuation which is a subject of the patent claims.

We combined the removal of whole pathogenicity island 1 (SPI1 ) and Ion mutant what leads to a construction of an ideal vaccine strain which i) is attenuated by two independent mutations what leads to its increased safety

ii) is highly immunogenic (Ion mutation results in an overproduction of capsular polysaccharides, SPI1 mutants do not induce cell death in macrophages) iii) strains with this combination of mutations can be easily differentiated from wild type Salmonella enterica strains as the strain forms mucoid colonies on common nutrient agars

Basis of the discovery lies in simultaneous removal of SPI1 and Ion in order to reduce Salmonella enterica virulence and enable its use as a live attenuated vaccine for the protection of farm animals. As SPI1 mutation it is understood any loss of function of type III secretion system encoded by SPI1 (Salmonella Pathogenicity Island 1 ). Ion mutation is understood as any inactivation of function of Lon protease. The bacterial strain according to this patent claims was successfully tested at the Veterinary Research Institute, Brno, Czech Republic. The following examples document, but do not restrict, the use of the above mentioned strain.

Operation Examples

Example 1

The attenuated strain of Salmonella enterica CAPM 6449 was constructed by lambda red recombination. Individual mutations, i.e. SPM and lon, were constructed first. After replacement of whole SPI1 with a chloramphenicol gene cassette, this mutation was transferred to a new recipient by P22 phage mediated transduction. Following the transduction, the chloramphenicol resistance gene was excised by flipase encoded by transiently introduced pCP20 plasmid. The strain with a total deletion of SPI1 was transduced with P22 phage grown on Salmonella enterica /on::Cm. Selection of chloramphenicol resistant colonies resulted into Salmonella enterica ASPI1 /on::Cm strain which was used for immunogenicity testing. The immunogenicity testing was performed with Salmonella enterica CAPM 6449 (ASPI1 /on::Cm) which was orally applied to newly hatched chickens of ISA Brown line. The chickens were revaccinated 3 weeks later and additional 3 weeks later, i.e. when aged 42 days, the chickens were challenged with the wild type Salmonella enterica serovar Enteritidis. Challenge was performed both orally and intravenously. The chickens were sacrificed 4 and 14 days after the challenge and Salmonella counts were determined in liver, spleen and caecum (Tab. 1 ). As can be seen in Tab. 1 , the vaccination with Salmonella enterica CAPM 6449 (ASPI1 /on::Cm) protected chickens against the infection with the wild type Salmonella enterica serovar Enteritidis since Salmonella counts were 10 to 100 times lower in the vaccinated than in the non-vaccinated chickens. Even in the cases when the presence of Salmonella was only qualitatively determined, the numbers of Salmonella positive chickens in the vaccinated chickens were lower than in the non-vaccinated controls.

Table 1

Salmonella detection in liver, spleen and caecum of the vaccinated and non- vaccinated chickens 4 (day 46) and 14 (day 56) days after oral or intravenous challenge, respectively. Data in the table show either log CFU/g) or number of positive out of total number of tested chickens.

liver spleen caecum

day 46 day 56 day 46 day 56 day 46 day 56

without vaccination p.o. 2/6 4/6 2/6 4/6 6/6

SPI1 Ion vaccination p.o. 1/6 1/6 2/6 0/6 5/6

without vaccination i.v. 4,71 5/5 6,68 4,16 6/6

SPI1 Ion vaccination i.v. 3,94 3/6 5,20 2,40 6/6

p.o. - oral challenge, i.v. - intravenous challenge Industrial Usability

Subject of the patent claims is the new strain of bacterium Salmonella enterica CAPM 6449 in which a specific combination of mutations which led to its attenuation was constructed. This resulted into its attenuation with preserved immunogenicity. This strain is suitable as a live attenuated vaccine for the protection of farm animals against Salmonella infection.

Claims

PATENT CLAI MS

1. Live attenuated strain of Salmonella enterica CAPM 6449 with a specific combination of mutations leading to its attenuation with preserved immunogenicity. This strain is deposited in the Collection of Zoopathogenic Microorganisms, Brno, Czech Republic and is intended as a live attenuated vaccine for oral vaccination of farm animals.