Transcript Adenovirus vectors - Baylor College of Medicine

Adenovirus vectors
Gene Therapy-2008
Virology 1

Infectivity
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Viral particles
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How do you count them?
Quality control
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How do you measure it?
Why is it important?
Host range
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What determines it?
Infectivity
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Measure by infecting dilutions of the virus stock
on permissive (e.g. 293) cells.
Plaque assay (1-2 weeks)
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Rapid titer (2 days)
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Measure by immunostaining viral antigens
Endpoint dilution
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Foci of infection
TCID50
FACS after immunostaining viral antigens
Infectivity (continued)

Infectivity is variable and dependent upon:
Passage number and health of cell substrate
 Time of virus adsorption
 Volume of virus adsorption
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Poisson distribution
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The equation for the Poisson distribution is:
Px=(mx.e-m) / x!
Where Px = the probability of a cell being "hit"
by x viruses
m = mean hits per cell = multiplicity of
infection (moi).
e = natural logarithm.
Poisson distribution (continued)
Therefore at an MOI of 1, P(0)=0.37
This means that at 1 PFU/cell approx. 63% of the
cells are infected.
Therefore one typically infects at an MOI of 10 to
infect all the cells.
Virus purification

Ultracentrifugation

Rate zonal
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Dependent upon sedimentation velocity (size and shape)
of virus particle. Underutilized.
Isopycnic
Dependent on equilibrium density of virus particle.
 CsCl typical
 Buoyant density of adenovirus is 1.35 g/ml
 Determine by refractometry
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Particle number
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Measured by A260
Quantitative PCR
Is invariant and therefore a good number to use
when infecting cells
Particle/PFU ratio typically 10-100
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Doesn’t necessarily infect amount of “dead” virus in
preparation
Quality Control
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Replication competent adenovirus
Sterility
Titer
Seed lot system
Virus
 Cells
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Adenovirus structure
Adenovirus ds
DNA approx. 36
kd with a
terminal protein
attached
Adenovirus transciption
Function of transcripts
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E1: Transactivation of early genes
E2: Adenovirus DNA replication
E3: Prevention/modulation of host immune
response
E4: Prevention/modulation of CTL response
Adenovirus receptors

Fiber
Spike of the virus
 Binds coxsachie-adenovirus receptor (CAR) in subgroup C
 Binds CD46 in subgroup B
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
Penton base
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Co-receptor-binds RGD (integrin) for internalization
of virion
Adenovirus Receptors (continued)

Receptors are not fully understood
In vivo distribution doesn’t match receptor
distribution
 Other serotypes (e.g.type 37, ocular) may have other
receptors (e.g. sialic acid)
 Certain serotypes bind clotting factors
 Small molecules may be involved in adenovirus host
range (adamantine).
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Adenovirus Host Range
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Adenovirus infects humans
No other good animal model
Chimpanzee
 Monkeys are non-permissive
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Mouse model is poor because most murine cells
lack viral receptors
Host range can be enhanced by using lipids
containing polyamines (see Davis, Human Gene
Therapy, 2005)
Subgroups, serotypes, and disease
Adenovirus serotypes and clinical disease
Adenovirus in gene therapy
Initial safety and immunogenicity
Vaccine (1992)
studies of an oral recombinant
adenohepatitis B vaccine
Carol 0. Tacket*i, Genevieve Losonsky*, Michael D. Lubeck?, Alan R. Davis?,
Satoshi Mizutani+, Gary Horwith+, Paul Hung+, Robert Edelman* and
Myron M. Levine*
Orally administered adenovirus may be a useful vaccine carrier of cloned antigens of
other
pathogens. A recombinant adenohepatitis vaccine Wy-Ad7HZ6-I, which expressed
hepatitis B surface antigen and contained a large deletion in early region 3 (E3), was
constructed and studied in humans. Volunteers received Wy-Ad7HZ6-I (n = 3),
adenovirus type 7 vaccine (n = 3) or placebo (n = 3). Recipients of Wy-Ad7HZ6-1 shed
less vaccine virus in the stool for a shorter period and had a lower titre of anti-adenovirus
type 7 antibodies than recipients of the adenovirus 7 vaccine. None of the three
Wy-Ad7HZ6-I vaccinees developed antibody to hepatitis B surface antigen after this one
dose primary immunization regimen. The E3 region may be required for optimal enteric
growth of adenovirus-vectored vaccines.
*Center for Vaccine Development, Department of Medicine,
University of Maryland School of Medicine, Baltimore, MD
21201, USA. +Wyeth-Ayerst Research, Philadelphia, PA
19101, USA. *To whom correspondence should be
addressed. (Received 10 December 1991; revised 7 February
1992; accepted 10 February 1992)
Adenovirus-HIV vaccine
Title:
Recombinant adenovirus vaccines
Document Type and Number:
United States Patent 20040170647
Kind Code:
A1
AR Davis, MD Lubeck, RJ Natuk, PK Chanda,
Abstract:
This invention provides a method of protecting a primate from an infectious organism by
stimulating the production of antibodies or cell mediated immunity to the infectious
organism which comprises administering to said primate intranasally, intramuscularly, or
subcutaneously, live recombinant adenoviruses in which the virion structural protein is
unchanged from that in the native adenovirus from which the recombinant adenovirus is
produced, and which contain the gene coding for the antigen corresponding to said
antibodies or inducing said cell mediated immunity. Preferably, the infectious organism is
HIV and the primate is a human.
June 1997 Volume 3 Number 6 p651
Nature Medicine
Long-term protection of chimpanzees against highdose HIV-1 challenge induced by immunization
Michael D. Lubeck1, Robert Natuk1, Maria Myagkikh2, Narender Kalyan1, Kristine Aldrich2,
Faruk Sinangil3, Shabnam Alipanah2, Shri C.S. Murthy1, Pranab K. Chanda1, Stephen M.
Nigida, Jr.4, Phillip D. Markham5, Susan Zolla-Pazner6, Kathy Steimer3, Mark Wade1, Marvin
S. Reitz, Jr.2, Larry O. Arthur4, Satoshi Mizutani1, Alan Davis1, Paul P. Hung1, Robert C.
Gallo2, Jorg Eichberg1, Marjorie Robert-Guroff2
A combination AIDS vaccine approach consisting of priming with adenovirus-HIV-1MN gp160
recombinants followed by boosting with HIV-1SF2 gp120 was evaluated in chimpanzees.
Long-lasting protection, requiring only three immunizations, was achieved against a lowdose challenge with the SF2 strain of HIV-1 and a subsequent high-dose SF2 challenge
administered 1 year later without an intervening boost. Notably, neutralizing antibody
responses against both clinical and laboratory isolates developed in three chimpanzees
and persisted until the time of high-dose challenge. The possibility that cytotoxic Tlymphocytes contribute to low-dose protection of a chimpanzee lacking neutralizing
antibodies is suggested. Our results validate the live vector priming/subunit booster
approach and should stimulate interest in assessing this combination vaccine approach in
humans.
1Wyeth-Ayerst
Research, 145 King of Prussia Road, Radnor, PA 19087, USA, 2Laboratory of Tumor Cell Biology,
National Cancer Institute, Building 37, Room 6B03, National Institutes of Health, Bethesda, MD 20892-4255, USA,
3Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608-2916, USA, 4Program Resources, Inc., Frederick
Cancer Research and Development Center, National Cancer Institute, P.O. Box B, Frederick, MD 21702, USA,
5Advanced BioScience Laboratories, 5510 Nicholson Lane, Kensington, MD 20895-1078, USA, 6Veterans Affairs
Medical Centers, New York, NY 10010, USA, M.D.L. present address: Wyeth-Lederle Vaccines & Pediatrics, P.O. Box
304, Marietta, PA 17547, USA, R.N., N.K., S.M. present address: Wyeth-Lederle Vaccines, 401 North Middleton Road,
Pearl River, NY 10965 USA, A.D. present address: Institute for Gene Therapy, University of Pennsylvania, 601
Maloney Building, 36th & Spruce Streets, Philadelphia, PA 19104, USA, P.P.H. present address: 506 Ramblewood,
Bryn Mawr, PA 19010, USA, M.S.R., R.C.G. present address: Institute of Human Virology, Medical Biotechnology
Center, University of Maryland at Baltimore, 725 West Lombard Street, Baltimore, MD 21201, USA, J.E. present
address: Dutch Primate Centre, Biomedical Primate Research Centre, Lange Kleiweg 151, 2280HV Rijswijk, the
Netherlands
Clinical Testing
Pre-clinical
testing
Phase I
Phase II
Phase III
Test
Population
Laboratory
and animal
studies
20 to 80
healthy
volunteers
100 to 300
patient
volunteers
1000 to
3000
patient
volunteers
Purpose
Assess safety
and biological
activity
Determine
safety and
dosage
Evaluate
Verify
effectivene effectivene
ss, look
ss, monitor
for side
adverse
effects
reactions
from longterm use
5,000
compounds
evaluated
5 enter
trials
FDA
Phase IV
Additional
Post
marketing
testing
required by
FDA
Review
process/ap
proval
1
approved
Current Gene Therapy Clinical Studies Using Adenovirus Vectors
1. RecruitingAdenovirus and Fungal Load in Pediatric Stem Cell Transplant Patients
Conditions: Adenovirus; Other Mycoses
2.RecruitingVirus-Specific Cytotoxic T-Lymphocytes (CTLs) for Adenovirus Infection Following an Allogeneic Stem Cell Transplant
Condition: Adenoviridae Infections
3.RecruitingSafety and Immunogenicity of Recombinant DNA and Adenovirus Expressing L523S Protein in Early Stage Non-Small Cell Lung Cancer
Condition: Non-Small Cell Lung Cancer
4.RecruitingSafety of and Immune Response to a DNA HIV Vaccine Followed by an Adenoviral Vaccine Boost Given 3 Different Ways to HIV
Uninfected Adults
Condition: HIV Infections
5.RecruitingAdenovirus Vaccine for Malaria
Condition: Malaria
6.RecruitingSafety and Efficacy of a Three-Dose Regimen of an Adenoviral HIV Vaccine (MRKAd5 HIV-1 Gag/Pol/Nef) in HIV Uninfected South
African Adults
Condition: HIV Infections
7.RecruitingAdministration of Autologous DCs Infected with an Adenovirus Expressing Her-2
Conditions: Inclusion criteria: Patients with metastatic breast cancer who are HER2/neu positive (3+ by immunohistochemistry or FISH
positive); and either,; ...
8.RecruitingAdenovirus Encoding Rat HER-2 in Patients With Metastatic Breast Cancer (AdHER2.1)
Conditions: Metastatic Breast Cancer; Recurrent Breast Cancer
9. .RecruitingWild Type p53 Adenovirus for Oral Premalignancies
Conditions: Mouth Cancer; Dysplasia/Carcinoma in Situ (CIS) of the Oral Cavity; Dysplasia/Carcinoma in Situ (CIS) of the Oral Pharynx
10.RecruitingStudy to Evaluate the Safety and Efficacy of Adeno-IFN Gamma in Cutaneous B-Cell Lymphoma
Condition: Lymphoma, B-Cell
11.RecruitingNMRC-M3V-Ad-PfCA Vaccine - Clinical Trial 1
Condition: Plasmodium Falciparum
12.RecruitingChemotherapy Followed By Vaccine Therapy in Treating Patients With Extensive-Stage Small Cell Lung Cancer
Condition: Lung Cancer
13.RecruitingVaccine Therapy With Either Neoadjuvant or Adjuvant Chemotherapy and Adjuvant Radiation Therapy in Treating Women With p53Overexpressing Stage II or Stage III Breast Cancer
Condition: Breast Cancer
14.Not yet recruitingSafety of and Immune Response to an HIV DNA Plasmid Vaccine Followed by HIV Adenoviral Vector Vaccine in Healthy
African Adults
Condition: HIV Infections
15.RecruitingSafety of and Immune Response to a DNA HIV Vaccine Followed By an Adenoviral Vector HIV Vaccine in
Healthy Adults
Condition: HIV Infections
16.RecruitingInterleukin-12 Gene in Treating Patients With Liver Metastases Secondary to Colorectal Cancer
Conditions: Colorectal Cancer; Metastatic Cancer
17.RecruitingBiological Therapy in Treating Women With Breast Cancer That Has Spread to the Liver
Conditions: Breast Cancer; Metastatic Cancer
18.RecruitingPhase I Study of Vaccination Schedule of Experimental HIV Vaccines
Condition: HIV Infections
19.RecruitingPhase I Study Combining Suicide Gene Therapy With Neoadjuvant Chemoradiotherapy in the Treatment of
Potentially Resectable Pancreatic Adenocarcinoma
Condition: Pancreatic Cancer
20.RecruitingGene Therapy for Prostate Cancer That Returns After Radiation Therapy
Conditions: Prostatic Neoplasms; Neoplasm Recurrence, Local
21.Not yet recruitingSafety Study on the Transfer of the CD40 Ligand Gene (AdcuCD40L) to Patients With Esophageal
Carcinoma
Condition: Esophageal Neoplasms
22.RecruitingNeoadjuvant Capecitabine and Radiation Therapy With or Without TNFerade Followed By Surgical
Resection in Treating Patients With Stage II, Stage III, or Locally Recurrent Rectal Cancer
Condition: Colorectal Cancer
23.RecruitingEffect of AdhAQP1 on Salivary Flow in Patients Treated With Radiation for Head and Neck Cancer
Condition: Parotid Salivary Dysfunction
24.RecruitingGene Therapy for Pleural Malignancies
Conditions: Pleural Mesothelioma; Metastatic Pleural Effusions
25.RecruitingSafety and Immune Response to a Prime-Boost Vaccination Schedule in HIV-Infected Patients
Condition: HIV
26.RecruitingA Clinical Trial to Evaluate the Safety, Efficacy, and Immunogenicity of DR-5001
Condition: Respiratory Tract Diseases
27.RecruitingVector Delivery of the IL-12 Gene in Men With Prostate Cancer
Conditions: Prostatic Neoplasms; Prostate Cancer
28.RecruitingVaccine Trial for Clear Cell Sarcoma, Pediatric Renal Cell Carcinoma, Alveolar Soft Part Sarcoma and
Children With Stage IV Melanoma
Conditions: Sarcoma, Clear Cell; Sarcoma, Alveolar Soft Part; Renal Cell Carcinoma; Secrete Granulocyte-Macrophage
Colony-Stimulating Factor (GM-CSF Melanoma
29.RecruitingVaccination With Autologous Breast Cancer Cells Engineered to ) in Metastatic Breast Cancer Patients
Condition: Breast
Cancer
30.RecruitingHer-2/Neu in Patients With Metastatic Breast Cancer (AdHERe)
Conditions: Metastatic Breast Cancer; Locally Recurrent Breast Cancer
31.RecruitingStudy of Treatment and Metabolism in Patients With Urea Cycle Disorders
Condition: Amino Acid Metabolism, Inborn Errors
32.RecruitingSafety of and Immune Response to an HIV-1 Vaccine (VRC-HIVDNA016-00-VP) and a Vaccine Booster
(VRC-HIVADV014-00-VP) in HIV Uninfected East African Adults
Condition: HIV Infections
33.RecruitingDose-Escalation Study of CG0070 for Bladder Cancer After BCG (Bacillus Calmette-Guerin) Failure
Conditions: Carcinoma, Transitional Cell; Bladder Neoplasms
34.RecruitingSafety and Efficacy Study of INGN 241 Gene Therapy in Patients With In Transit Melanoma
Conditions: Malignant Melanoma; Neoplasm Metastasis
35.RecruitingExperimental Vaccine for Prevention of Ebola Virus Infection
Conditions: Ebola Hemorrhagic Fever; Ebola Virus Disease; Ebola Virus Vaccines; Envelope Glycoprotein, Ebola Virus;
Filovirus
36.RecruitingGene Therapy in Preventing Cancer in Patients With Premalignant Carcinoma of the Oral Cavity or
Pharynx
Condition: Head and Neck Cancer
37.RecruitingStudy to Compare the Overall Survival of Patients Receiving INGN 201 (Study Drug) With Patients
Receiving Methotrexate
Condition: Carcinoma, Squamous Cell
38.RecruitingEffectiveness and Safety of INGN 201 in Combination With Chemotherapy Versus Chemotherapy Alone
Condition: Carcinoma, Squamous Cell
39.RecruitingPhase I - Pre-Radical Prostatectomy RTVP-1 Gene Therapy for Prostate Cancer
Condition: Prostatic Neoplasms
40.RecruitingA Study of TNFerade™ Biologic With 5-FU and Radiation Therapy for First-Line Treatment of
Unresectable Locally Advanced Pancreatic Cancer
Condition: Pancreatic Cancer
Safety and Efficacy of a Three-Dose Regimen of an
Adenoviral HIV Vaccine (MRKAd5 HIV-1 Gag/Pol/Nef)
in HIV Uninfected South African Adults
This study is currently recruiting patients.
Verified by National Institute of Allergy and Infectious Diseases (NIAID) December 2006
Sponsors and
Collaborators: National Institute of Allergy and
Infectious Diseases (NIAID)
HIV Vaccine Trials Network
Merck
Information provided
National Institute of Allergy and
by:
Infectious Diseases (NIAID)
ClinicalTrials.gov
NCT00413725
Identifier:
Safety and Efficacy of a Three-Dose Regimen of
an Adenoviral HIV Vaccine
(MRKAd5 HIV-1 Gag/Pol/Nef) in HIV Uninfected South African Adults
This study has been suspended.
( Based on an interim data review, the DSMB concluded that the vaccine cannot
be shown in this trial
to prevent HIV infection or affect the course of the disease. )
Bone formation in a wild type mouse
2500 vp/cell
Ad5BMP2
C57BL\6
Genejammer®
+ Genejammer
C57Bl\6 derived
cell line.
• Bone formation only occurs if the
cells are effectively transduced with the
virus. This step is critical.
- Genejammer
Fouletier-Dilling et al Human
Gene Therapy 16(11): 1287-1298.
Efficient and Rapid Osteoinduction
in an Immune Competent Host
1.
No differences in host cell survival
2.
No differences in the volume of bone formed
3.
No differences in stages of bone formation
4.
No differences in the timing of bone formation
Fouletier-Dilling et al Human Gene
Therapy 18: .
Stages of Rapid Endochondral Bone Formation
VWF
Day 1: Appearance of
brown fat
Day 3-4: Angiogenesis and
stem cell extravasation
Collagen type II
Day 5-6: Appearance of
cartilage
Day 7: Mineralized bone
Hydrogel encapsulation of Ad5BMP2
transduced cells
Osteoclast
Osteoclast specific degradation sites
PEG-DA
polymer chain
cells
+
PEG-DA +
cell
photoinitiator suspension
solution
Encapsulated BMP2
producing cell
photopolymerized
PEG-DA
chains
aliquots
added to
cell culture
dish
exposure to
white light, ~11.5 min
cells
encapsulated
within PEG-DA
Development of target delivery of the
AdBMP2 transduced cells
Ad5F35BMP2
Encapsulation
Transduce
30µL hydrogel disks
150µL hydrogel disk in a sheet structure
8000
7000
BMP-2 (pg/mg Total Protein)
PEG-DA (10k) - 100uL disks
6000
Plated tMRC-5 - 10m
5000
4000
3000
2000
1000
0
1
3
5
7
9
15
Time (days)
Comparison of BMP2 secretion from the
hydrogel encapsulated and unencapsulated
cells
Implant the disks near the critical
size defect
Hydrogel encapsulation of Ad5BMP2
transduced cells
Osteoclast
Osteoclast specific degradation sites
PEG-DA
polymer chain
cells
+
PEG-DA +
cell
photoinitiator suspension
solution
Encapsulated BMP2
producing cell
photopolymerized
PEG-DA
chains
aliquots
added to
cell culture
dish
exposure to
white light, ~11.5 min
cells
encapsulated
within PEG-DA
Other
Slides of this lecture are available on our website
http://vector.bcm.tmc.edu/
 References (will be fair game on exam)


Biology of adenovirus and its use as a vector for gene
therapy. Hum Gene Ther. 2004 Nov;15(11):1022-33.
McConnell MJ, Imperiale MJ

Adenovirus: from foe to friend. Rev Med Virol. 2006
May-Jun;16(3):167-86 Goncalves MA, de Vries AA.