Viruses in Biotechnology
Dr. James Maruniak
Animal Viruses - Vaccines
• Mutated viruses- generated by recombinant
DNA techniques
• Vaccine vectors- Yellow fever chimeric
vaccines
Other ENV gene
YFV
• Vaccinia used for
– Cytokine expression- to boost immunity
– Subunit vaccines- envelope proteins from other
viruses
– Problems with low antigenicity- add enhancers or
try other promoters
– Multiple vaccines in it- influenza & Hepatitis B env
– Disseminated vaccinia- is a problem
Foreign gene
Positive control
+ control
For treatment of existing HPV infection, techniques to improve cellular immunity by
enhancing viral antigen recognition are being studied. For this purpose, the oncogenic
proteins E6 and E7 of HPV-16 and -18 are the focus of current clinical trials for cervical
cancer patients.
Clinical Pharmacology & Therapeutics (2007) 81, 259–264.
GARDASIL®: Prophylactic Human Papillomavirus Vaccine Development –
From Bench Top to Bed-side
L. Shi, H. L. Sings, J. T. Bryan, B. Wang, Y. Wang, H. Mach, M. Kosinski, M.
W. Washabaugh, R. Sitrin and E. Barr
Merck Research Laboratories, West Point, Pennsylvania, USA
Abstract
GARDASIL® (Merck, Whitehouse Station, NJ) is a non-infectious
recombinant, quadrivalent vaccine prepared from the highly purified
virus-like particles (VLPs) of the major capsid proteins of human
papillomavirus (HPV) types 6, 11, 16, and 18.
GARDASIL® is the first vaccine approved for use in women aged 9–26
years for the prevention of cervical cancer and genital warts, as well as
vulvar and vaginal precancerous lesions. This report describes some of
the key preclinical efforts, achievements in pharmaceutical development,
in vivo animal evaluation, and clinical trial data.
Title: Papillomavirus vaccine
Document Type and Number: United States Patent 7169585
Abstract:
A method of providing papillomavirus like particles which may be used for
diagnostic purposes or for incorporation in a vaccine for use in relation to
infections caused by papillomavirus.
The method includes an initial step of constructing one or more recombinant
DNA molecules which each encode papillomavirus L1 protein or a
combination of papillomavirus L1 protein and papillomavirus L2 protein
followed by a further step of transfecting a suitable host cell with one or
more of the recombinant DNA molecules so that virus like particles (VLPs)
are produced within the cell after expression of the L1 or combination of L1
and L2 proteins.
The VLPs are also claimed per se as well as vaccines incorporating the VLPs.
Virus-like particles
(VLPs) assembled
from the L1 protein
of Human
Papillomavirus 16
HPV-assembly.
•
Five L1 main structure
proteins (monomers)
are assembling to one
pentamer (capsomere).
Finally, 72 capsomeres
are building one viruslike particle (VLP)
(according to Lutz
Gissmann, modified).
The HPV major capsid protein L1 can
fold correctly and self-assemble into
VLPs when expressed in eukaryotic
cells. VLPs aim to protect against the
development of cervical cancer;
protection would be mediated by the
induction of high titres of neutralizing
antibodies against the HPV
genotypes in the vaccine that prevent
the virus infecting the transformation
zone, a metaplastic area between the
squamous and columnar epithelia in
the cervix, where most cancers arise.
Delivering on the promise: HPV vaccines
and cervical cancer
John T. Schiller & Philip Davies
Nature Reviews Microbiology 2, 343-347
(April 2004)
HPV L1-VLP specific IgM, IgA, IgG1 and IgG4 antibody titers. Sera from rhesus macaques immunized at week 0, 8 and 24
with 2 μg each of HPV 6, 11, 16 and 18 L1-VLPs formulated with Merck Aluminum Adjuvant (+MAA) (-◆-) or 2 μg each of HPV 6,
11, 16 and 18L1-VLPs alone (-MAA) (-◊ -) were collected at the indicated time points and tested for HPV L1 VLP specific (A) IgM,
(B) IgA, (C) IgG1 and (D) IgG4 titers in a multiplexed detection assay. Responses are reported as GMTs (n = 5 monkeys per
group) for HPV 16. Arrows indicate vaccination boosts at weeks 8 and 24. Graphs shown are for HPV 16 and are representative
of HPV 6, 11, and 18.
•
The Journal of Infectious Diseases
2001;183:1485-1493
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A Phase 1 Study of a Recombinant Virus-like Particle Vaccine against Human
Papillomavirus Type 11 in Healthy Adult Volunteers
Thomas G. Evans,1,a William Bonnez,1 Robert C. Rose,1,2 Scott Koenig,3
Lisa Demeter,1,2 JoAnn A. Suzich,3 Diane O'Brien,1 Meredith Campbell,1
Wendy I. White,3 James Balsley,3 and Richard C. Reichman1,2
1Infectious Diseases Unit, Department of Medicine, and 2Department of Microbiology and
Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York;
3MedImmune, Gaithersburg, Maryland
Received 17 March 2000; revised 31 January 2001; electronically published 24 April 2001.
Virus-like particles (VLPs) produced from the L1 protein of several papillomaviruses
have induced protection from infection after live challenge in animal models. In the
present study, the safety and immunogenicity of a human papillomavirus (HPV)11 L1
VLP candidate vaccine were measured in a phase 1, dose-finding trial in humans. The
vaccine was well tolerated and induced high levels of both binding and neutralizing
antibodies. Marked increases in lymphoproliferation to HPV-11 L1 antigens were noted
after the second vaccination.
In addition, lymphoproliferation was induced after vaccination in peripheral blood
mononuclear cells (PBMC) stimulated with heterologous L1 VLP antigens of HPV types 6
and 16. Statistically significant increases in HPV antigen specific interferon and
interleukin-5 production were measured from PBMC culture supernatants.
This candidate HPV VLP vaccine induced robust B and T cell responses, and T cell
helper epitopes appear to be conserved across HPV types.
L1 VLPs corresponding to HPV-6, -11, -16, and -18 and to parvovirus B19 VP2 were
produced at MedImmune, using the baculovirus expression system. Purification was
accomplished by cesium chloride and sucrose gradient centrifugation and by phenyl
hydrophobic interaction chromatography. The final bulk product was dialyzed and sterilized by
filtration.
A Controlled Trial of a Human Papillomavirus Type 16 Vaccine
Laura A. Koutsky, Ph.D., Kevin A. Ault, M.D., Cosette M. Wheeler, Ph.D., Darron R. Brown, M.D., Eliav Barr, M.D.,
Frances B. Alvarez, R.N., Lisa M. Chiacchierini, Ph.D., Kathrin U. Jansen, Ph.D., for the Proof of Principle Study
Investigators
Background Approximately 20 percent of adults become infected with human papillomavirus type 16 (HPV16). Although most infections are benign, some progress to anogenital cancer. A vaccine that reduces the
incidence of HPV-16 infection may provide important public health benefits.
Methods In this double-blind study, we randomly assigned 2392 young women (defined as females 16 to 23
years of age) to receive three doses of placebo or HPV-16 virus-like–particle vaccine
(40 µg per dose), given at day 0, month 2, and month 6. Genital samples to test for HPV-16 DNA were
obtained at enrollment, one month after the third vaccination, and every six months thereafter. Women were
referred for colposcopy according to a protocol. Biopsy tissue was evaluated for cervical intraepithelial
neoplasia and analyzed for HPV-16 DNA with use of the polymerase chain reaction. The primary end point
was persistent HPV-16 infection, defined as the detection of HPV-16 DNA in samples obtained at two or
more visits. The primary analysis was limited to women who were negative for HPV-16 DNA and HPV-16
antibodies at enrollment and HPV-16 DNA at month 7.
Results The women were followed for a median of 17.4 months after completing the vaccination regimen.
The incidence of persistent HPV-16 infection was 3.8 per 100 woman-years at risk in the placebo group and
0 per 100 woman-years at risk in the vaccine group (100 percent efficacy; 95 percent confidence interval,
90 to 100; P<0.001). All nine cases of HPV-16–related cervical intraepithelial neoplasia occurred among the
placebo recipients.
Administration of this HPV-16 vaccine reduced the incidence of both HPV-16 infection and HPV-16–
related cervical intraepithelial neoplasia. Immunizing HPV-16–negative women may eventually
reduce the incidence of cervical cancer.
Vaccine Promising Against Disease-Causing HPV
NEW YORK (Reuters Health) - April 7, 2005
Certain strains of the virus responsible for causing genital warts -- human papillomavirus or HPV -are also likely to trigger cervical cancer. Now comes news that a vaccine designed to combat four
of these strains can dramatically reduce persistent infection and the risk of developing warts and
cancer.
The findings are similar to results seen when a two-hit vaccine was tested in North America and
Brazil last year. However, in that study, the vaccine targeted only HPV-16 and HPV-18, the main
cancer-causing types, whereas the new four-strain 'quadrivalent' vaccine targets these types as
well as HPV-6 and HPV-11, the ones most often linked to genital warts.
The study involved 552 young women who were not pregnant, had no history of abnormal Pap
smears, and had had no more than four sexual partners. Women with previous HPV infection were
not excluded from the study.
The women were given three shots of the quadrivalent vaccine or placebo injections over a period
of six months. They were then followed for 36 months with regular gynecologic exams, cervical
testing for HPV, and Pap smear tests.
Compared with placebo injections, the vaccine led to 90 percent fewer cases of persistent infection
or disease due to HPV types 6, 11, 16, or 18, lead study author Dr. Luisa L. Villa, from the Ludwig
Institute for Cancer Research in Sao Paulo, Brazil, and colleagues report in the medical journal
Lancet Oncology.
The vaccine was 89 percent effective in preventing infection with the four HPV types, and 100
percent effective in preventing the diseases associated with these types.
The study was funded by Merck Research Laboratories, which is developing the vaccine.
SOURCE: Lancet Oncology, online April 7, 2005.
Expert Opin Emerg Drugs. 2005 Feb;10(1):5-19.
Emerging human papillomavirus vaccines.
Christensen ND.
The Pennsylvania State University College of Medicine, The Department of Microbiology and
Immunology, Hershey, PA 17033, USA.
Human papillomavirus (HPV) infections are a leading cause of virus-associated cancers of the
anogenital, oropharyneal and cutaneous epithelium. The most prevalent of these is cervical cancer,
which is responsible for approximately 500,000 deaths annually worldwide. A group of about 15
serologically unrelated 'high-risk' HPV types are responsible for almost all HPV-associated cancers.
Prevention of papillomavirus infection can be achieved by induction of capsid-specific
neutralising antibodies in preclinical animal papillomavirus models and in recent human clinical trials.
High titres of conformationally-dependent, type-specific HPV-neutralising antibodies are triggered by
HPV virus-like particle (VLP) vaccines. Overcoming the problems of type-specificity of the responses to
these VLP vaccines is a potentially important area of current HPV vaccine research, with an emphasis
on induction of more broadly cross-protective neutralising responses. Viral oncogenes E6 and E7 are
continuously present in HPV-associated cancers and are prime targets for HPV therapeutic vaccines. A
variety of approaches are being tested in therapeutic vaccine clinical trials and in various preclinical
animal papillomavirus models for efficacy.
Approaches include genetic vaccines, recombinant virus vaccines, dendritic cell-based strategies,
immunomodulatory strategies and various combination strategies to maximise cell-mediated immunity
to papillomavirus proteins present in HPV infections and cancers. The success of preventive HPV VLP
vaccines in clinical trials is clear. However, current therapeutic vaccine trials are less
effective with respect to disease clearance.
Animal Viruses - Vaccines
• Avipoxvirus
– Results in abortive virus infection in humans
– Not highly immunogenic, must use multiple
times
– Canarypox - attenuated and used for rabies
– Canarypox expresses measles glycoproteins
• Entomopoxvirus
– Abortive infection in mammalian cells can be
engineered to transiently express proteins
Animal Viruses - Vaccines
• Poliovirus
– Sabin vaccine attenuated live virus has been
engineered to express foreign proteins
– Insertion of large foreign DNA into VP1 to
make antigen on virus surface, but requires
helper poliovirus
– Insert small DNA (60 bp) in loop region of
VP1 to express epitope in highly
immunogenic region on virion
– Foreign antigens can be part of the poliovirus
polyprotein that is cleaved
Animal Viruses - Vaccines
• DNA vaccines
– Stimulate humoral and cell-mediated
immunity
– May not prime deleterious immune
response due to incoming virus proteins
– Bypasses toxic effect of virion proteins
such as: ???
*
Pcmv=cytomegalovirus promoter, pA=polyadenylation signal, f1 ori &
ColE1=bacterial origins of replication, SV40=promoter, amp=ampicillin resistance,
RSV LTR=Rous sarcoma virus promoter, plus the indicated restriction sites
P CMV=cytomegalovirus promoter, pA=polyadenylation signal, oriP & ColE1=bacterial origins of replication,
SV40=promoter, amp=ampicillin resistance, pRSV=Rous sarcoma virus promoter, EBNA=Epstein Barr Virus
Nuclear Antigen, PTK= Thymidine Kinase promoter, plus the indicated restriction sites
The Making of a DNA Vaccine
Against West Nile Virus
http://www3.niaid.nih.gov/news/newsreleases/2005/wnvgraphic.htm
Comparison of Viral-Based Vectors for Vaccine Applications
PARENT VIRUS
Retrovirus Adenovirus
Nucleic Acid
AAV
VEE
RNA->
DNA
DNA
DNA
RNA
YES
YES
YES
NO
MEDIUM
MEDIUM
MEDIUM
HIGH
Lymph Node Targeting
NO
NO
NO
YES
Pre-existing Vector-specific Immune Response
NO
YES
YES
NO
Ab, CTL
Ab, CTL
Ab, CTL
YES
NO
Chromosomal Integration/ Persistence
Expression Level
Induction of Ab, CTL, and Mucosal Immune
Responses
Tested in Human Clinical Trials
Ab, CTL, and Mucosal
* Live attenuated VEE vaccine strain TC83 used extensively in humans.
Ab, CTL, and Mucosal
YES*
Animal Viruses
• Diagnostic Virology make reagents
– To test for recent infections for antibodies
– Genomes in polymerase chain reaction for
diagnostics
• Expression vectors for protein production
– Cytomegalovirus constitutive promoters
– SV40 promoters and signal sequences used
– Adenovirus genes such as E1A
Make viral proteins for diagnostic assays
Protein or
Pharmaceutical development
• Develop heart disease drugs against
C-reactive protein induced by Herpesvirus
cultured in the laboratory for this assay
• Kaposi's sarcoma linked with a herpesvirus.
Use virus to test drugs.
• Brain tumors targeted with engineered
adenovirus
• Genital herpes need new antiviral drugs
tested
Evaluation of a test based on baculovirus expressed glycoprotein G for
detection of herpes simplex virus type specific antibodies.
Division of Viral and Rickettsial Diseases, Centers for Disease Control,
Atlanta, Georgia 30333.
J Infect Dis. 1991 Dec;164:1196-9.
An immunoblot assay for discrimination of antibodies to herpes simplex
virus (HSV) types 1 and 2 was devised using extracts of recombinantbaculovirus-infected insect cells expressing HSV-1 or -2 glycoprotein G (gG1
or gG2).
The assay was evaluated by comparing its results with those obtained by
using an immunodot assay based on gG immunopurified from HSV-1- and
HSV-2-infected cells.
The assay is accurate and reproducible. The ease of antigen production
should allow the test to become widely available
Viruses used in Biological
Control
BACULOVIRUS
EPA Registered wt Baculoviruses in USA
• Autographa californica MNPV – vegetable insects
• Spodoptera exigua MNPV - cotton and vegetable
caterpillars
• Neodiprion sertifer SNPV – forest caterpillars
• Lymantria dispar MNPV – forest caterpillars
• Cydia pomonella GV – fruit caterpillars
• Helicoverpa zea SNPV - cotton and corn insects
• Orgyia pseudotsugata MNPV - forest caterpillars
After recombination
with the baculovirus
genome and insect
cell infection
P ph= polyhedrin promoter, P p10= promoter,
ph pA=polyadenylation signal, Recombination
sequences from part of the baculovirus
genome, ColE1=plasmid origin of replication in
bacteria, Amp=ampicillin resistance
for protein expression after
recombination with the
baculovirus genome and insect
cell infection
P ph= polyhedrin promoter, PETL= promoter,
Recombination sequences from part of the
baculovirus genome, ColE1=plasmid origin of
replication in bacteria, 5’ lacZ Fragment = β
galactosidase gene for recombination in Baculovirus
genome, (His)6= 6 histidine residues for nickel
column purification of fused protein
pGenBac2.1 is a transfer vector for high level eukaryotic expression of recombinant proteins in
insect cells. This vector is compatible with Invitrogen Bac-to-Bac Baculovirus Expression System.
The gene of interest can be easily cloned downstream of polyhedron promoter. This transfer
vector recombines with a parent bacmid in E. coli cells to form an expression bacmid. High level
gene expression is achieved by infecting insect cell lines with the recombinant bacmid.
A simplified schematic of the capabilities for glycosylation in most lepidopteran cell lines. Note
that there is evidence that glycosidases do some trimming of these structures, particularly the
GlcNAc residues from the intermediate structure.
Baculovirus
wild type
and
recombinant
plaques
Protein Sciences collaborates with some of the largest pharmaceutical and
biopharmaceutical firms in the world, as well as with (virtual) biotech companies,
and academic and government institutions.
A selection of our customers:
Rabbit Calicivirus
Behind CMV
promoter
CaMV 35S is Cauliflower Mosaic Virus promoter used
in RNA silencing Tobacco Mosaic Virus in plants
Tobacco Mosaic Virus BCI
CaMV 35S
Enhancement of freezing tolerance of plants
CaMV 35S=
Cauliflower
Mosaic Virus
35S promoter
Genetic Transformation for
Virus Resistance
• Insert DNA sequences isolated from WMV
(watermelon mosaic virus) and other viruses
into plant cell cultures
• Select transformed plants from cell cultures
• Test plants and progeny for resistance in
greenhouse and field
• Integrate progeny into patented process to
develop new seedless varieties
Foreign Genes Commonly
Used in Transgenic Organisms
• Plants
– Bt (Bacillus thuringiensis) delta endotoxins
– against lepidoptera, coleoptera
– herbicide tolerance
– plant virus resistance
Plant Made Antibody Targets Hepatitis B Virus
NEW YORK (Reuters Health) - June 23, 2004
Japanese scientists have successfully used genetically engineered cells from
the tobacco plant to produce a human antibody that homes in on a molecule
on the surface of the hepatitis B virus (HBV).
Currently, treatment of HBV may include infusion of serum containing
antibodies, called immunoglobulin, collected from blood donors.
The new results demonstrate the feasibility of producing anti-hepatitis
antibodies in plants "as an alternative to anti-HBV human
immunoglobulins," Dr. Akira Yano from the National Institute of Public Health
in Tokyo and colleagues write in the Journal of Medical Virology.
"Our plant-derived (antibody) has the potential to be a cheap and effective
pharmaceutical" for the prevention and treatment of HBV infection, Yano told
Reuters Health.
Transgenic Plants in Agriculture
• Example of companies involved in
producing transgenic crops
– Novartis, Monsanto, Pioneer, Cargill,
DeKalb, AgrEvo, Calgene, DuPont,
Mycogen, Garst
• Commodities
– corn, soybean, potato, sunflower, cotton,
rice, peanut, tomato
• Insects controlled
– Lepidoptera, Coleoptera
Which of the following steps is not used in
construction of a vaccinia vaccine vector?
1. cloning of foreign gene
2. transfection of vaccinia genome
3. transfection of recombinant plasmid
4. infection of tissue culture cells
5. homologous recombination