My Minor Credit Seminar During MVSc on Expression Library Immunization Against Bacterial Diseases

1. Presented By:
Sangram P. Ramane
MVSc
Div. of VBM
4673

2. CONTENTS
Introduction
Historical events
What is and why for ELI?
Main approaches for ELI
Vector design
Library construction and library deconvolution
Improving immune response against ELI antigens
Limitations
Conclusion

3. INTRODUCTORY TERMS
 Expression library is a library of
DNA fragments which was created
with an expression vector so that
any genes present in the library are
expressed.
 Expression vectors are a specialized
type of cloning vector in which the
transcriptional and translational
signals needed for the regulation of
the gene of interest are included in
the cloning vector.
 Plasmid vaccine:
Mammalian expression
vector with vaccine gene

4. HISTORICAL EVENTS
 1990: Wolf et. al. found that in vivo injection of DNA plasmids led to
their entry into mammalian cells with subsequent synthesis of proteins
encoded in them
 1995: Barry et al. gives first time basic concept of ELI.
 1995: First ELI libraries were built for Mycoplasma pulmonis by Barry
et. al.
 1998: Brayton et. al. constructed the ELI libraries for Cowdria
ruminantium.
 2003: For Brucella abortus, Leclereq et. al.
 2006: Immunization of mice with a Mycobacterium tuberculosis genomic
expression library by Hernández et. al.

5. System of discovering vaccines
against pathogen
Whole genome of pathogen is
converted into fragments
Cloned into expression vectors to
create expression library
Entire library is delivered into
host
Screened to identify individual
protective clones
Sequenced and then used as
vaccine candidate
WHAT IS ELI?

6. WHY FOR ELI?
 Simple design and allows us the rapid discovery of vaccines.
 Can completely define the vaccinome of any pathogen.
 Possible to create a vaccine against sequenced as well as unsequenced
pathogen.
 Can convert immunoevasive pathogen protein to immunostimulatory
vaccine antigen.
 Non expressed genes from bacterial genome as vaccine candidate.
 Pathogen challenge model system in which humanized mice can be
used to screen human leucocyte antigen (HLA) restricted responses.

7. Takes advantage of a well-
established technology of
cDNA cloning into
mammalian expression
vector
All ORFs in a genome
are amplified by PCR
and cloned to a
mammalian expression
vector
Randomly shear genomic
DNA and to clone the
fragments into an
appropriate mammalian
expression vector
MAIN APPROACHES FOR ELI
Random ELI Directed ELI cDNA directed ELI
rELI cDELIdELI
Mycoplasma pulmonis,
Brucella abortus,
Toxoplasma gondii,
Trypanosoma cruzi, SIV
HIV-1, HIV-2
For fungal and
protozoal pathogens
incluide C. immitis, I.
scapularis, L. donovani

8. AN OUTLINE OF ELI PROTOCOL
LIBRARY
DECONVOLUTION
VECTOR
DESIGN
LIBRARY
CONSTUCTION

9. TYPICAL VECTOR DESIGN
The antigen expression
(vaccine synthesis) unit
composed of
promoter/enhancer sequences,
followed by antigen-encoding
and polyadenylation sequences
The production (plasmid
propagation) unit composed of
bacterial sequences necessary
for plasmid amplification and
selection.

10. Viral - hCMV, RSV, SV40, MMTV, HIV-LTR
Mammalian – MHC1, β Actin, CK, Desmin,
Heavy chain of myosin gene promoters.
Intron A from hCMV.
Can be attributed to an enhanced rate of
RNA polyadenylation and/or nuclear
transport linked to RNA splicing.
Usually derived from bovine growth hormone
(BGH), Rabbit β globin, late SV40-derived
sequence terminators.
Enhance RNA stability and translation.
Facilitates efficient processing of cloned genes.
Protect mRNA from degradation.
Can induce cytokine secretion and
lymphocyte activation
Certain CpG motifs in bacterial DNA
(In MCS)
ColE1
Drug resistant
Auxotrophic
signals
…..contd

11. LIBRARY CONSTRUCTION
 Depending on the approach for ELI
 Mammalian expression vectors are
used
e.g. pVAX, pCMV (pCMV-Ubi,
pCMV-hGH), pCI30, pVAC1-mcs
 Library size depending on the size of
genome
M. pulmonis – 1Mb – 3000 clones
(Barry. et. al.)
B. abortus – 3Mb – 20,000 clones
(Leclereq, et. al.)

12. …..contd

13.  “Screening of successively smaller pools of clone in animal
challenge model to identify clone that induce protective
immunity.”
 Laboratory animal model (e.g. BALB/c mice).
 Technologies available to design humanized or transgenic
mice.
 Mouse model is used as a first screen for the library pools
before fewer numbers of plasmids are selected for testing in a
more expensive or sophisticated animal model.
LIBRARY DECONVOLUTION

15. IMPROVING ELI ANTIGEN PRESENTATION
 Pathogen proteins traffic poorly inside mammalian cells
so actively evades immune presentation are difficult tools
to develop into potent vaccines for ELI.
 Improvement in immune response against ELI antigen
by protein targeting done by:
1. Targeting for CD8 responses
2. Targeting for CD4 responses

16. TARGETING FOR CD8 RESPONSES
 CD8 response recognize vaccine epitope by display on
MHC class I molecules.
 These epitopes are generally supplied by proteasome
degradation of intracellular proteins.
 The carboxy terminus of antigen ubiquitin is added to
translate antigens already ubiquitinated and targeted to the
proteasome.
 Also, Cytotoxic T lymphocyte antigen 4 (CTLA4), Dendritic
cell targeting moieties
…..contd

17. TARGETING FOR CD4 RESPONSES
 CD4 stimulation to provide helper responses to CD8 cells
and for antibody production.
 Antigen fused to the N terminus of lysosome associated
membrane protein type 1 (LAMP1), antigen can be fused
with the invariant chain.
 Fused with Tissue plasminogen activator (TPA), Fc domain
of immunoglobulin.
…..contd

18. …..contd

19. No. Bacteria Animal ELI Genome Library size Outcome
1. Mycoplasma
pulmonis
Mouse rELI 1 Mb 3,000 clones Complete
protection
2. Neisseria
menigitidis
Rat rELI 2.2Mb 10 pools of 600
clones each
6 pools induce
humoral
immune
response
3. Chlamydophila
abortus
Mice rELI 1.1 Mb 27 pools of 300
clones each
5 gene fragments
induce
protective
immunity
4. M. avium sub.
paratuberculosis
Mice rELI 4.8 Mb 11 pools of
1500 clones
each
Protection
(Reduced
colonization)
LIST OF BACTERIA TO WHICH ELI APPLIED

20. No. Bacteria Animal ELI Genome Library size Outcome
5. Chlamydia
muridarum
Mice rELI 0.107 Mb Nearly 21,000
clones
Partial protection
6. Brucella
abortus
Mouse rELI 3 Mb 1 library with
20,000 clones
Partial protection
7. Cowdria
ruminantium
Mouse rELI 1.57 Mb 22 pools with
3,000 clones
each
Partial protection
8. Mycoplasma
hyopneumoniae
Pig rELI 0.9Mb About 20,000
clones
Used a new in-
frame cloning
vector
9. Piscirickettsia
salmonis
Coho
salm
on
rELI Unkonwn 22,000-28,000
clones
Partial protection
LIST OF BACTERIA TO WHICH ELI APPLIED

21. LIMITATIONS
 Laborious and expensive.
 Only one in six clones are in correct orientation and reading frame for
expression.
 Dilution of protective antigen.
 During screening there is no clear demarcation between the genes that
mediate total protection and that which does not mediate protection.
 Codon bias
e.g. UGA = STOP (Mammals) but Tryptophan (Mycoplasma).
 During random shearing of pathogen genomic DNA, likelihood capturing
of start methionine codon in correct orientation is small.

22. CONCLUSION
 So to make safer and more-effective vaccines, we need to find an
array of protective antigens for a given pathogen.
 In vaccinology, ELI protocols have a good chance to contribute
tremendously to the discovery of novel antigens that can serve as
vaccines.
 Till date ELI have been applied to more than 40 pathogens.
 The antigens expressed by an ELI vaccine can be engineered to
drive potent immune response.
 It is a ‘Road-map’ for discovery of Vaccines against Infectious
diseases.

23.  Barry et. al. 2004. Expression library immunization to discover and improve
vaccine antigens. Immunol. Rev. 199:68-83.
 Barry et.al. 1995. Protection against mycoplasma infection using expression-
library immunization. Nature 377:632-635.
 Tallat and Stemke-Hale. 2005. Exprssion Library Immunization: A Road for
Discovery of Vaccines against Infectious Diseases. Inf. & Immu. 7089-7098
 Brayton, K. A., S. W. Vogel, and B. A. Allsopp. 1998. Expression library
immunization to identify protective antigens from Cowdria ruminantium. Ann.
N. Y. Acad. Sci. 849:369-371.
 Moore, R. J., C. Lenghaus, S. A. Sheedy, and T. J. Doran. 2001. Improved
vectors for ELI application to Mycoplasma hyopneumoniae infection in pigs.
Vaccine 20:115-120.
 Stemke-Hale et. al. 2005. Screening the whole genome of a pathogen in vivo for
individual protective antigens. Vaccine 23:3016-3025.
REFERENCES

24.  V. Azevedo, G. Levitus. 1999. Main features of DNA-based immunization
vectors. Brazilian Jr. of Med. & Biological Res. 32, 147-153
 Garmory, Brown and Titball. 2003. DNA vaccines: improving expression of
antigens. Gen. Vaccines & Therapy. 1-5
 McNeilly et. al., 2007. ELI confers partial protection against Chlamydia
muridarum genital infection. Vaccine, 25:2643-2655
 Huntley et. al., 2005. ELI confers protection against Mycobacterium avium sub
paratuberculosis Infection, Inf. & Immu., 6877-6884
 Yero et. al., 2005. Immunization of Mice with Neiseria menigitiis serogroup B
genomic expression libraries elicit functional antibodies and reduces the level
of Bacteremia in an infant rat infection model, Vaccine, 23:932-939
…..contd