Download Innate Immune responses to Adenovirus based vectors: Ad

Concepts Regarding Adenovirus based vaccine
systems.
By:
Andrea Amalfitano D.O., Ph.D.
Osteopathic Heritage Foundation Professor
Dept of Microbiology and Molecular Genetics and Pediatrics
Michigan State University
Objectives:

Adenovirus Biology: Basics
 Adenovirus Vectors
– Construction
– Propagation
– Purification

Utilization of Adenovirus Vectors as a
Vaccine platform
 Limitations of Adenovirus Vectors
Adenovirus:
-7 Human subgroups , ~42 serotypes
- primate, porcine, ovine, avian,
murine
-70-90 nanometers
-36 kb dsDNA linear genome
Adenovirus –Pathology/Facts

5% of all Acute respiratory illnesses(~30,000,000 cases/yr)
 Pharyngitis-infants
 Gastroenteritis-infants
 Conjunctivitis-All
 Pneumonia- Infants, Military Recruits
– (Ad 4, 7,21)
– Literally millions vaccinated with large oral
doses of these wild-type viruses
Adenovirus Genomic
Organization reflects life cycle
Adenovirus life cycle
ssDBP
E1
pol
pTP
E2
E1
E3
E4
E1
E2
E3
E4
ssDBP
pTP
pol
ΨΨ
ΨΨ
ΨΨ
Ψ
Late gene expression
(structural proteins)
100K
Preformed
empty capsids
Adenovirus as a “work-horse” gene
transfer vector:
Your favorite antigen
Construction of [E1-]Ad vectors


Gene X
Homologous recombination
ΔE1

Gene X
Pac I Enhancer/Promoter
Pac I
ori
Kan
GENE X

Poly A
Pme I
Ampr
Pac I
Pme I
Newer methods for
creating modified Ad
vectorsbacterial recombination
pAd[E1-]

Pac I
Pac I
Kan

ori
Gene X
E1
GENE X
PacI
E3
{
}
E1+,E2b+
cells
pAd[E1-]
GENE X
Ad[E1-]ANTIGEN X
Viral Particles
[E1-] Ad Vector Production
ssDBP
E2
Gene
X
pTP
pol
E3
E4
293 cells
E1
100K
Ease of scale-up for production of
modified Ad vectors
Gene
X
X
X
X
X
X
X
X
X
X
X
x
x x x
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x
Large-scale purification:
independent of CsCl2
centrifugation
CsCl2 purified Adenovirus vector:
Adenovirus vector production,
purification, and storage:


Serum Free Cell Culture
Large Scale Cultivation Systems:
– cell suspension based systems
– Micro-carrier bead based
– Hollow fiber
 Isayeva et.al.: BioProcessing Journal p.64-70: 2003

Anion Exchange Based Column Chromatography
Purification



Huyghe et.al.: Human Gene Therapy 6:11403-1416: 1996
Green et.al.: Human Gene Therapy 13:1921-1934: 2002
Stability and long term storage
– Long term stability and lyophilization capability
 Croyle et.al.: Gene Therapy 8(17):1281-90: 2001
Efficacy of Adenovirus based Vaccine:
Humoral and Cellular Immune
response induction

“Phase 1 safety and immunogenicity evaluation of a
multiclade HIV-1 candidate vaccine delivered by a
replication-defective recombinant adenovirus vector”
–

“Protection of mice and poultry from lethal H5N1 avian
influenza virus through adenovirus-based immunization”
–

Catamzaro et.al.: J. Infectious Diseases: 194(12):1638-1649: 2006
Gao et.al.: J. Virology 80(4): 1959-1964:2006
“Safety and immunogenicity of Adenovirus vectored nasal
and epicutaneous influenza vaccines in humans.”
–
Van Kampen et. al.: Vaccine 23: 1029-1036: 2005
Why are Adenoviruses so
potent?

The Ad capsid is intrinsically capable of
inducing potent innate immune responses.
– In part, these responses are TLR mediated.
 Harman et.al. J. Virology : in press
Ad induced innate immune responses are
in part, MyD88 dependent
Ad dyregulated Liver Transcriptome is significantly
impacted upon by lack of MyD88 functionality:
Ad infected
Wt mice
6 hpi

Ad treated Wild-Type mice exhibit
significantly higher activation of immune
response, nucleotide binding, RNA
processing, and Extracellular and Adhesion
Genes relative to Ad treated, MyD88KO
mice.

Ad treated Wild-Type mice exhibit
significantly greater repression of
mitochondria-related genes as well as cell
cycle and growth gene groups relative to
Ad treated, MyD88KO mice.
– Hartman et.al.: J. Virology: in press.

Statistically different genes between Ad and
mock infected samples assessed using a 1-way
ANOVA, p = .05 with Benjamini and Hochberg
Multiple Testing Correction.
Adenovirus Limitations:
“Toxicity” due to continued expression of multiple Ad
encoded genes present in [E1-]Ad based vectors:
ssDBP
pTP
polGene X E2
Protein
X
E3
“E1
like
”
E4
100K
Pre-existing Immunity hampers
efficacy and might increase
toxicity

Upwards of 50-85% of adults have some
pre-existing immunity to Adenoviruses



Piedra et.al.: Pediatrics 101:1013-1019: 1998
Chirmule et.al.: Gene Therapy 6: 1574-1583: 1999
Varnavski et.al.: J. Virology 76(11): 5711-5719:2002
Wild-type Ad (RCA) reversion
during production
293 cells:
0
4344
Ψ
E1a and E1b
RCA due to recombination with
Ad sequences present in 293
cells
0
480
Ψ
4344
Your Favorite Antigen
Wild-type Ad=RCA
0 E1a and E1b
Ψ
0
4344
Ψ
E1a and E1b
4344
Conclusions:

Great potential
 Several Obstacles /Limitations must be
considered