1. Methodology for Development of Defined Deletion Mutant Vaccine for Salmonellosis
Dr. Bhoj R singh, Principal Scientist (VM)
I/C Epidemiology; Centre for Animal Disease Research and Diagnosis
Indian Veterinary Research Institute, Izatnagar , Bareilly, UP, India.  TeleFax 

2. Importance Live Salmonella vaccines are required:
For long lasting and cross protection
Due to their potential as mucosal vaccines.
To induce cell-mediated, humoral and secretory antibody responses.
Types of Live Vaccines
Attenuated homologous vaccines
i. Non defined attenuated (9-R; S-51)
ii. Defined attenuated
a. Insertions to inactivate target genes
(phage transduction, Transposon mutagenesis)
b. Deletions in target genes to attenuate
2. Vectored vaccines
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

3. Defined deletions help us in:
1. Maintaining desired level of virulence in vaccine strains so that it must maintain enough antigenic concentration and antigenicity. 2. Identifying the strain using site specific PCR, probes or even ELISA and simpler diagnostic tools. 3. Devising the DIVA targets 4. Development of marker vaccines 5. Vectored vaccine
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

4. Common targets gene of Salmonella for deletion mutagenesis
galE aroA cya
crp PhoP phoQ
vPla nuoG secC
htrA dam phoA
sodCl spvBC sopE
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

5. General methods to produce genetically defined deletion vaccines
Development of deletion mutant of desired strain (Phage transduction, Suicide plasmid).
Confirmation of deletion
Permission from Biosafety and Animal Ethics committee
In-vitro and in-vivo evaluation for attenuation
Stability of mutation (testing for reversion (in-vitro and in-vivo)
Safety, immunogenicity and efficacy testing in experimental model.
Safety, efficacy and Vertical/ horizontal transmissibility studies
in target animal
DIVA designing, posology and excipient selection.
Self life and stability testing and Scaling the production
Approval from Animal Ethics committee, Biosafety Committee, Drug Comptroller etc.
Evaluation in field
Validation
Patent/ Release/ transfer of technology
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

6. Conventional defined deletion method
PCR amplification of target gene with flanking ( bp on each side).
TA cloning or cloning at specific RE site in cloning vector
RE Digestion of cloning vector to delete the target sequence from the gene and ligation to recircularize the vector. Confirmation of deletion by PCR or probes through southern blotting.
Cutting of the truncated gene with flanking regions and cloning into suicide vector.
Transfer of suicide vector into target strain (conjugation/ electroporation/ chemical transformation)
Removal of Suicide vector (sacB)
Confirmation of deletion in Salmonella strain.
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

7. A simple gene disruption strategy
(Kirill A. Datsenko and Barry L. Wanner, 2000).
H1 and H2 refer to the homology extensions or regions. P1 and P2 refer to priming sites. FRT=FLP recognition target for FLP recombinase. Step one- PCR using plasmid with special gene.
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

8. In-frame deletion mutagenesis Method
Beliaev et al., 2001.
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

9. Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

10. Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

11. One step site directed deletion/ insertion
(Salerno et al., 2005; Liu and Naismith, 2008; Qi and Scholthop, 2008)
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

12. Overlap extension protocol
(Lee et al., 2004)
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

13. Example 1
aroA mutation
1.676 kB segment having full length aroA was amplified from S. Typhimurium by PCR and TOPO-TA cloned.
A 392 bp fragment was deleted from ORF of aroA by Hinc II digestion
The aroA gene harbouring the deletion was then subcloned into a positive-selection suicide vector, pCVD442 which carries sacB and ampR genes .
Construct was introduced into S. Abortus-equi under antibiotic selection through conjugation with E. coli S 17.1pir
Integrated vector was removed from Salmonella under sucrose selection (sacB) .
Mutant strains were confirmed by PCR and southern blotting .
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

14. Example 2
htrA mutation
1.758 kB segment having full length htrA was amplified from S. Typhimurium by PCR and cloned into pUC19 digested with Sph I and Sac I.
A 608 bp fragment was deleted from ORF of htrA by Hinc II digestion.
htrA gene harbouring the deletion was then subcloned into pCVD442.
Construct was introduced in to S. Abortus-equi under antibiotic selection through conjugation with E. coli S 17.1 pir.
pCVD442 was removed under sucrose selection (sacB) .
Mutant strains were confirmed by PCR and southern blotting .
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

15. Testing for Attenuation
In-vivo
In experimental models for virulence and pathogenicity.
In-vitro
Conditioned bacteriological culture(s)
Toxigenicity assay (serological, chemical, physical)
Pathogenicity assay in cell lines, mononuclear cells, macrophages.
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

16. Stability testing
Reversion rate for deletion mutants is low (10-12 to-13)
To prove that mutant is reversion free test should be done in vitro and in vivo in co-culture condition in back passage mode.
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

17. Safety, immunogenity, efficacy and Vertical/ horizontal transmissibility
Permission from Animal ethics and biosafety committee is essential.
Test in different dosages to determine the minimum dose required to induce protective immune response against challenge with wild type strain(s).
Test for safety using at least 100 times of the immunogenic dose giving desired protection.
Safety should be tested in animals of different age, under different physiological conditions, through different routes of inoculation.
Environmental safety must be tested through determining survivability of the mutant under prevailing environmental conditions in soil, water and other biotic and abiotic components of the environment.
Vertical and horizontal transmission studies should be done under different settings as in companion animals, direct contacts, mating mates.
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

18. DIVA designing, posology and excipient selection
For DIVA designing you need to develop suitable and simple tests for differentiating vaccinated animals from infected.
You need to define the simple test to differentiate vaccine strain from field strains.
Although mucosal route is the best and most warranted, one studies are required to prove that which of the route gives best protection against natural way of infection.
Suitable dosages defined in terms of colony forming units (cfu) per dose have to be determined.
Live vaccines require a bit of nutrient to support the life and side by side there is need to keep the cfu at constant level therefore live vaccines need a suitable excipient when the vaccine is not a freeze dried one and when it is freeze dried it need a suitable freeze drying and re-suspension medium to be choosen after experimentation.
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

19. Self life and stability testing and Scaling the production
To define transport and storage conditions:
Self life and stability of the final preparations is needed to be tested at different storage and transport conditions under probable environmental exposures to clearly detail the survival of effective dose in the vaccine.
Scaling the production is integral for any commercial product, for vaccine fermenter technology has be metered for the developed vaccine.
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar

20. In field evaluation of vaccine
Before going to field trials of a live, genetically engineered vaccine you need approval from Animal Ethics committee, Biosafety Committee, Drug Comptroller etc., for starting the work.
Evaluation in field involve all getting fully informed consent from the live stock owner, always keep some exigency plan to face adversity while evaluating your vaccine in field.
Validation: Testing/ evaluation of vaccine by third party.
Patent/ Release/ transfer of technology
Defined deletion vaccines
BR Singh, CADRAD, IVRI, Izatnagar