The document discusses various approaches to veterinary bacterial vaccine design, detailing different types of vaccines such as inactivated, live attenuated, subunit, and gene deletion vaccines. It highlights the development of third-generation vaccines through advanced methods like reverse vaccinology and functional genomics, aiming for improved efficacy and reduced cold-storage requirements. The conclusion emphasizes the need for modern techniques to enhance vaccine stability and effectiveness in controlling animal diseases and thereby benefiting human health.

1. New generation vaccines
A
Seminar
on
DIVISION OF BACTERIOLOGY AND MYCOLOGY, IVRI
Mamta singh
Phd Scholar
Roll no. 177

3. Approaches to veterinary bacterial vaccine design
Vaccine
generation
Type Development
process
Vaccine examples
I Inactivated whole
bacteria
(bacterins) or
culture
supernatants
Chemical or physical
inactivation to
eliminate infectivity
but retain
immunogenicity
Leptospira Hardjo,
Pasteurella multocida
I Live attenuated
whole bacteria
Application of in
vitro passage or
random chemical
mutagenesis to
attenuate the strain
and lose infectivity
Brucella abortus S19,
Salmonella Pullorum
I and II Subunit or extract
vaccines
1.Purified proteins
that are chemically
Inactivated
2. Purified capsular
polysaccharide that
are conjugated with
protein
Clostridium tetani toxoid,
Clostridium perfringens type
D epsilon toxoid
Actinobacillus pleuropneumoniae
serotype 5b capsular
polysaccharide-tetanus toxoid
conjugate (Andresen et al., 1997)

4. II Gene deletion
vaccines
Rational attenuation
through mutagenesis
or gene knockout
procedures
Chlamydophila abortus
temperature sensitive live
vaccine (Chalmers et al.,
1997)
II Recombinant
component
vaccines
Cloning of appropriate
genes and
expression of their
products
Subunit vaccine
candidates
for Streptococcus equi
(Timoney et al., 2007;
Wallera et al., 2007)
III Reverse
vaccinology
In silico analysis of the
genome to
select vaccine targets
Subunit vaccine
candidates for
Dichelobacter nodosus
(Myers et al., 2007)
Approaches to veterinary bacterial vaccine
design

5. New generation vaccine-IIIrd generation
Changing
in antigen
type
Changing
in delivery
methods
Changing
in
adjuvants
type

6. New generation vaccines

7. Category –I Antigens Generated by Gene Cloning

8. Recombinant vaccines
• Recombinant vaccines commercially
available for Mannheimia haemolytica
and Actinobacillus pleuropneumoniae
based upon the leukotoxins produced
by these organisms, as well as
transferrin-binding proteins
• Recombinant cocktail vaccine
comparising rOmpA and rOmp C
provide 83.33% protection against
salmonella challenge (IVRI, annual
report,2013-14)

9. 9
• Transposon technology
• Precise genomic excision of
genes
• Used as marker vaccine
Category –II Genetically Attenuated Organisms
(Gene deleted vaccines)

10. 10

11. Gene deleted vaccines
• Gene-deleted Salmonella enterica serovar typhimurium and
serovar enteritidis vaccines have been licensed for use in poultry
(Babu et al., 2004; Meesun et al., 2007)
• AroA gene-deleted Streptococcus equi vaccine Equilis StrepE
vaccine from the S. equi TW928 deletion mutant lacking bp 46 to
978 of the aroA gene has been licensed for use in horses
(Meesun et al., 2007)
• A double gene (gE and TK) deleted Pseudorabies virus marker
vaccine licensed for use in pigs( Meesun et al., 2007)
• gE deleted a Bovine herpesvirus-1 marker vaccine has been
licensed for use in cattle (Meesun et al., 2007)
• A deletion mutant of Brucella abortus S19 (IVRI, annual
report,2013-14)

12. Category –III Recombinant vectored vaccine

14. 14
Category –III Recombinant vectored vaccine
Viral Vectored Vaccines
– Pox viruses, Adenoviruses and Herpesvirus-as
delivery systems for foreign antigens
– Virus – simply as a vector
• E.g. Vaccinia-rabies recombinant vaccine
– Virus – both as vector & vaccine against the
infection by the wild vector itself
• E.g. recombinant capripox virus expressing PPR
virus Ag

15. 15

16. 16
Advantages and disadvantages of recombinant
vectored vaccines
Advantages
– Rapid generation
– No need for protein expression and purification
– Potentially generic and low-cost manufacturing
processes
– Thermostability
– Leading technology for T cell induction
Disadvantages
– Affected by maternal antibody

17. 17
Category –IV DNA Vaccines/Polynucleotide
immunization
Gene for an antigenic determinant is inserted
into a plasmid
Genetically engineered plasmid injected into
the host
Within the host cells, the foreign gene can be
expressed from the plasmid DNA
If sufficient amounts of the foreign protein are
produced, they will elicit an immune response

18. 18
Production of DNA vaccines

19. 19

20. Delivary of DNA Vaccines

21. 21
Advantage of DNA vaccine
Only protective antigen is included like subunit
vaccine
Avoid the problem of incorrect folding and
glycosylation of antigenic protein
Easy inclusion of regulatory cytokines
Stable, less variation and cheaper to prepare
Safe and long lived immunity(HMI & CMI)
Inexpensive & Multivalency could be achieved
 Can induce immune responses in the presence of
maternal antibodies

22. 22
DNA Vaccines-Disadvantages
• Limited to protein immunogen only
• Potential integration of plasmid into host genome
leading to insertional mutagenesis
• Chromosomal integration – cell transformation
• Induction of autoimmune responses (e.g. pathogenic
anti-DNA antibodies)
• Induction of immunologic tolerance (e.g. where the
expression of the antigen in the host may lead to
specific non-responsiveness to that antigen)

23. 23
DNA vaccine – to further improve
• CpG motifs inclusion
• Cytokines inclusion
• Co-stimulatory molecules inclusion
• Conventional adjuvants
• Prime boost approach –Priming with DNA vaccine but
boost with protein or some other recombinant virus
vector expressing the protein

24. 24

25. Vaccines against Johne’s disease
Vaccine Vector SpeciesVac
cination
ages
Dosage Immunity
DNA
vaccine
(Kathaper
umal et
al. 2008)
Four rAgs (85A,
85B, 85C
and superoxide
dismutase)
with two adjuvants
(monophosphoryl
lipid A
and bovine IL-12)
Cattle5–10
days
100 μg of
each
antigen
and 100
μg
of IL-12 im
Antibodies within 3 weeks;
significant IFN-g production
within 11 weeks Significant
increases in CD4+ and CD8+ T
cells against all four rAgs;
rAg-specific expression of IL-2,
IL-12 and TNF-α. 4/8 animals
did not show bacteria in tissue
DNA
vaccine
(Sechi et al.
2005)
Three rAgs
(Mycobacterium
avium 85A, BCG
85A
and 65K)
Sheep5
months
Three
doses
of 1 mg of
each
antigen
im. 20
days
apart
Increased IFN-g and IL-10
expression, increased CD4+ T
cells,Absence of lesions and
bacteria in tissues

27. New generation vaccine-IIIrd generation
• Sequencing of whole bacterial genomes has led to new
approaches to vaccine design (Scarselli et al., 2005), and a
‘‘third generation’’ of vaccines
• New methods of antigen discovery and design including
reverse vaccinology, structural biology, and systems biology
(Rinaudo et al. 2009)
• First example of the use of a genome sequence to produce
vaccine antigens was Neisseria meningitidis (Pizza et al.
2000)

28. Reverse vaccinology

29. Application of Reverse vaccinology in vaccine
design
Genome-based approaches: strategies in
selecting protective antigens
– In silico analysis
– for detection of virulence factors
– for detection of secreted or surface-associated
proteins
– for prediction of T cell and B cell epitopes
– Functional genomics in vaccine design
– Proteomics
– DNA microarray analysis
– Other technologies
– Pan-genomic approach in vaccine design

31. In silico analysis for detection of virulence
factors
• Comparison of the predicted coding sequence with the
known genes in a database using BlastP or BlastN homology
search tools is a convenient way to identify a putative
virulence gene
• Blast sequence comparison cannot be used for prediction of
new families of virulence factors (Grandi, 2001)

32. Programme Location and/or function Reference
PSORTb
(http://www.psort.org
/psortb/)
For predicting the location of proteins in
Gram-negative bacteria (cytoplasm,
cytoplasmic membrane,periplasm, and
outer membrane or extracellular space)
Nakai (2000)
SignalP
(http://www.cbs.dtu.d
k/services/SignalP/)
For predicting the presence and
location of signal peptidase I (SPaseI)
cleavagesites within the N-terminal 70
amino acids of secreted proteins
Bendtsen et
al. (2004)
TMpred
(http://www.ch.embne
t.org/
software/TMPRED_for
m.html)
For detecting N-terminal
trans-membrane helices
Hoffman and
Stoffel (1993)
In silico analysis for detection of secreted or
surface-associated proteins

34. Functional genomics in vaccine design
• Techniques-
 Proteomics
 DNA microarray analysis
 Other technologies
• Aim of functional genomics is to reveal the links between
a specific genotype and its corresponding phenotype
• Phenotype results from the expression of genes through
conversion into systemic, catalytic and regulatory
products, and is a complex function of genotype and
environment (Dharmadi and Gonzalez, 2004)

35. Proteomics
Proteomics can be divided into three main areas:
• ‘‘Protein micro-characterization’’, which deals with large-scale
identification of proteins and their post-translational modifications
• ‘‘Differential display’’ proteomics, a way to compare protein
quantities, and which can be used to investigate the microbial
pathogenicity in regards to protein expression levels
• ‘‘Protein–protein interactions’’ using techniques such as mass
spectrometry (Pandey and Mann, 2000)
These three applications in conjunction with the characterization of
membrane and surface-associated proteins are important for vaccine
development (Serruto et al., 2004)

36. Proteomics approach to bacterial vaccine
development
• Either all the bacterial proteins, or preferably only the
surface proteins (the ‘‘surfaceome’’Cullen et al., 2005), are
first resolved into their individual components using 2DE
• Each separated protein is digested into its discrete peptide
fragments using a suitable enzyme, and the molecular
mass of each proteolytic digested fragment is then
accurately measured using matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS)

37. Proteomics approach to bacterial vaccine
development
• SERological Proteome Analysis (SERPA)- Combining proteomics
with serological analysis is another useful refinement for
identifying potential vaccine candidates, used by Vytvytska et al.
(2002) for identification of vaccine candidates against S. aureus
• In SERPA the bacterial surface proteins were first resolved by 2-DE
and then electro-transferred onto a membrane that was blotted
with different serum pools of S. aureus infections
• Identified a set of highly immunoreactive staphylococcal proteins

38. Proteomics approach to bacterial vaccine
development
• Proteome microarrays
– Used with serum from immunized or infected animals to help
identify immunodominant antigens of bacteria
– Method has been applied to identify antigens of Francisella
tularensis that are of relevance for vaccine development
(Eyles et al., 2007)
Disadvantages-limitation of the proteomic approach is the large
number of proteins expressed by the pathogen under in vitro
growth conditions (Etz et al., 2002a)

39. DNA microarray analysis
• Preparation of DNA microarrays from whole genome sequence
data that represent the whole bacterial genome
• Microarrays can be hybridized with cDNA that is prepared from
extracted RNA of a microorganism grown under different growth
conditions (e.g. in vivo versus in vitro growth)
• In this way analysis of the transcriptome allows identification of
genes that are up regulated in vivo (Rappuoli, 2000a)
– N. meningitidis - (Grifantini et al., 2002)
– P. multocida - to identify a core set of 13 upregulated and 16 down-
regulated genes in isolated from the liver and bloodstream of infected
chickens (Boyce et al.,2004)

40. Disadvntages of DNA microarray
(1) Use of DNA microarrays is a semiquantitative method
due to the lack of linearity and proportionality of
mRNA/ cDNA concentration to signal intensity at high
concentration
(2) An inherent limitation of DNA microarrays is that the
resulting transcriptome does not account for post-
transcriptional events
(3) Processing and analysing transcriptome is difficult

41. Reverse vaccinology approach used with bacterial pathogens include
– Porphyromonas gingivalis (Ross et al., 2002)
– Streptococcus pneumoniae (Wizemann et al., 2001)
– B. anthracis (Ariel et al., 2002)
– Chlamydia pneumoniae (Montigiani et al., 2002)
– Mycobacterium tuberculosis (Betts, 2002)
– Staphylococcus aureus (Vytvytska et al., 2002)
– Edwardsiella tarda (Srinivasa Rao et al., 2003)
– Leptospira interrogans (Gamberini et al., 2005)

42. Other technologies for functional genomics
• ‘‘In vivo induce antigen technology’’ (IVAT)-convalescent sera from several
naturally infected individuals are pooled and pre-adsorbed with surface
expressed antigens of the target pathogen grown in vitro, to remove antibodies
recognizing antigens expressed in vitro
• ‘‘Expression library immunization’’ (ELI)-
– a genomic DNA expression library from the pathogen of interest is prepared
and divided into pools
– Plasmid DNA from each pool of clones is used to immunize a model
subsequently used as protein subunits or as components of DNA vaccines
(Barry et al., 2004)
 Protective antigens in Mycobacterium avium subspecies paratuberculosis, the
agent of Johne’s disease (Huntley et al., 2005)

43. Pan-genomic approach in vaccine design
• Genomic sequence of a single strain provides a huge potential
resource to determine relationships between genotype and
phenotypes within the species, but it fails to differentiate genetic
variation between strains (e.g. avirulent and virulent strains)
• All available genome sequences of different strains of pathogen
were divide into two subgenomes:
– A core genome containing genes present in all strains -
responsible for the basic bacterial metabolisms
– A dispensable genome composed of genes that are unique to
each strain –responsible for genetic diversity, pathogenicity,
colonization,and antibiotic resistance
• Vaccine candidates against Streptococcus agalactiae (Medini et
al., 2005)
• Recombinant subunit vaccine against rickettsial septicaemia in
fish caused by Piscirickettsia salmonis (Wilhelma et al., 2006)

47. Adjuvants-Immunomodulators

49. Conclusion
• Vaccines are valuable and specialized products, of great diversity
have already achieved great success in controlling many diseases of
economics importance in farm and companion animals
• While present they do not cover all infections, access to modern
techniques are used for designing to new vaccine ,not only
prolongation of immunity, but also to better practical aspects, such
as product stability and less dependence on cold-storage
• With improvements in vaccines and reduction in “cold-chain”
requirements will contribute to better standards of animal health
and farming prosperity, which in turn benefit human health and the
cost of producing safe food