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MICR 306 Advanced
Applications of Viruses
(Part 4.1)
Prof. J. Lin
University of KwaZulu-Natal
Westville campus
Microbiology Discipline
2014
School of Life Sciences
New findings
• Gene x Gene x Environment
The transmission of chikungunya, a mosquitoborne disease that is spreading quickly on a
global scale, depends strongly on local mosquito
populations/genomes and mean environmental
temperature.
• Researchers detect traces of the Middle East
respiratory syndrome coronavirus (MERS) in
the air of a barn housing an infected camel.
 Air-borne infection?
• Holistic approaches - Ecology !!
Treatment of Viral Infections?
•
Initiation phase:
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Replication phase:
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attachment
penetration
uncoating
DNA synthesis
RNA synthesis
protein synthesis
Release phase:
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assembly
maturation
exit from cell
Zaire ebolavirus and the
family of filoviruses to which it
belongs owe their virulence to
mechanisms that first disarm
the immune response
(defective dendritic cells)
and then dismantle the
vascular system. The virus
progresses so quickly that
researchers have struggled to
tease out the precise
sequence of events,
particularly in the midst of an
outbreak. Much is still
unknown.
VLP Applications
1) Their natural immunogenic properties
make them attractive candidates for vaccine
strategies still ranking first among molecular
VLP-based applications.
2) VLPs have also established themselves in
other branches of biotechnology taking
advantage of their structural stability and
tolerance towards manipulation to carry and
display heterologous molecules or serve
as building blocks for novel nanomaterials.
Focus points
Host Specificity: species (strain), organ, tissue
Unique morphology: nm ranges, self assembly
Unique replication cycle and rate
 Biological controls, Drug delivery, Vaccine
 Nanotechnology,
 Protein engineering, Industrial productions
Mutation: Efficiency???
Host immunity: Efficiency???
Viral genetic elements used to construct
Eukaryotic expression plasmid vectors
 Viruses are highly efficient replicators & viral
gene expression is adapted to eukaryotic
systems
– very strong promoters (CMV immediate / early promoter)
– small introns (CMV intron)
– regulatory elements often constitutive - require only host
factor binding (porcine circovirus (PCV) capsid promoter /
enhancer)
 Minimal regulatory elements from viruses
– Promoters, enhancers, polyadenylation signals,
introns, replication origins, IRES elements.
APPLICATIONS
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Molecular cloning (RDNA 220)
Vaccine Development
Gene Therapy
Cancer therapy
Phage Therapy (Drug Resistance Problem)
Nanoscience – where physics, chemistry and biology
collide
• Synthetic Biology
• Biological Control Agents
• Protein Engineering
Plasmids
T7 & M13 Promoters
Vaccine Development
Vaccine Development
• Inactivated viruses
• Attenuation viruses
Sub-viral particle concepts
Single peptide
• Virosomes (Virus like particles)
3) Anti-idiotype vaccines
An antigen binding site in an antibody is a reflection of the threedimensional structure of part of the antigen, that is of a particular
epitope. This unique amino acid structure in the antibody is
known as the idiotype which can be thought of as a mirror of the
epitope in the antigen. Antibodies (anti-ids) can be raised against
the idiotype by injecting the antibody into another animal. This
gives us an anti-idiotype antibody and this, therefore, mimics part
of the three dimensional structure of the antigen, that is, the
epitope. This can be used as a vaccine. When the anti-idiotype
antibody is injected into a vaccinee, antibodies (anti-anti-idiotype
antiobodies) are formed that recognize a structure similar to part
of the virus and might potentially neutralize the virus. This
happens: Anti-ids raised against antibodies to hepatitis B S
antigen elicit anti-viral antibodies.
4) Recombinant DNA techniques
a) Attenuation of virus
Deletion mutations can be made that are large enough that they are
unlikely to revert. the Nef deletion mutants as potential anti-HIV
vaccines. Flu viruses  six different crucial mutations.
Another problem: that the virus could still retain other unwanted
characteristics such as oncogenicity (e.g. with adenovirus, herpes
virus, HIV).
b) Single gene approach (usually a surface glycoprotein)
A single gene (for a protective antigen) can be expressed
in a foreign host. Yeast are better for making large
amounts of antigen for vaccines since they process
glycoproteins in their Golgi bodies in a manner more
similar to mammals. It have the same disadvantages of a
killed vaccine. current hepatitis B vaccine. Several
potential HIV vaccines but they provoke little cell-
c) Cloning of a gene into another virus
By cloning the gene for a protective antigen into another
harmless virus. Cells become infected, leading to cell-mediated
immunity. Vaccinia (the smallpox vaccine virus) is a good
candidate since it has been widely used in the human
population with no ill effects. We can make a multivalent
vaccine virus strain as Vaccinia will accept several foreign
genes A candidate HIV vaccine has undergone clinical trials.
However, the use of vaccinia against smallpox has shown rare
but serious complications in immuno-compromised patients and
alternatives have been sought  canary pox virus that does
not replicate in humans but does infect cells. Recombinant
canary pox vector expressing the HIV envelope gene (gag,
protease, nef and parts of pol genes) has induced an HIV-1
envelope specific CTL response.
VLP-based strategies
for vaccine design
Principle:
Cell transcribes DNA.
Vaccine protein is
expressed on cell surface
Mammalian expression
control elements
l DNA
l DNA
Antigen gene
Phage broken down.
Vaccine expression cassette
cloned into bacteriophage l DNA
Vaccine-encoding DNA
released
Immune response
Grow l phage in E. coli & purify
Macrophage
Dendritic
cell
Inoculate - injection / oral
Antigen –
presenting cells
engulf l particles
DNA Vaccines
The Third Vaccine Revolution: deliberate introduction of a
DNA plasmid into the vaccinee. The plasmid carries a proteincoding gene that transfect cells in vivo at very low efficiency
and expresses an antigen that causes an immune response.
DNA-mediated or DNA-based immunization. Usually,
muscle cells do this since the plasmid is given
intramuscularly. The plasmid DNA is taken up by muscle cells
after injection, by bombarding the skin with DNA-coated gold
particles or by introducing DNA into nasal tissue in nose
drops. In the case of the gold bombardment method, one
nanogram of DNA coated on gold produced an immune
response. One microgram of DNA could potentially introduce
a thousand different genes into the vaccine.
Advantages of DNA vaccines
 Plasmids are easily manufactured in large amounts  DNA is very stable
 DNA resists temperature extremes and so storage and transport are
straight forward  A DNA sequence can be changed easily in the
laboratory  can respond to changes in the infectious agent
 By using the plasmid in the vaccine to code for antigen synthesis, the
antigenic protein(s) that are produced are processed (post-translationally
modified) in the same way as the proteins of the virus against which
protection is to be produced a far better antigen than using a
recombinant plasmid to produce an antigen in yeast (e.g. the HBV
vaccine), purifying that protein and using it as an immunogen.
 Mixtures of plasmids could be used that encode many protein fragments
from a virus or viruses a broad spectrum vaccine could be produced
 The plasmid does not replicate and encodes only the proteins of interest
There is no protein component and so there will be no immune response
against the vector itself
DNA vaccines against viruses
DNA-based plasmid immunization actually resembles virus
infection the immune responses are broad-based and
mimic the situation seen in a normal infection by the
homologous virus.long lasting. Cytotoxic T lymphocyte
(CTL) responses.
e.g. a DNA vaccine has been the induction of cytotoxic
cellular immunity to a conserved internal protein of
influenza A to overcome the annual variation (antigenic
drift and shift) of the virus.
The current influenza vaccine is an inactivated preparation
containing antigens from the flu strains that are predicted to
infect during the next flu season.  IgG response
neutralization change as a result of reassortment
Possible Problems
 Potential integration of plasmid into host genome
leading to insertional mutagenesis
 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)
Vaccines Backfire
Two different vaccine
viruses, used
simultaneously to
control the same
condition in chickens,
have combined to
produce new
infectious viruses.
Anti-HIV-1
Vaccines
Plasmid DNA
makes encoded HIV
protein in cells of the body
Virus-like
particle with
outer surface
display of
epitopes
Epitope
Display
Vectors
Live
Attenuated
Viral
Vectors
Adenovirus
Modified Vaccinia (MVA)
Replicon Vaccines:
DNA from HIV is Cloned
into Various Vectors
Virally encapsidated
plasmid vaccine
Viruses for Peptide display: M13 Phage or
plant virus (TMV) Coat Protein Fusions
Need :
non-enveloped virus
many repeat capsid subunits
ordered capsid array - amplified display
external loops or termini available for
peptide addition via gene fusion
Mass peptide display
on outer surface of
TMV particle
N
C
60S
loop
Tobacco mosaic virus
TMV
VIRION
Assembly of mixed TMV capsids
carrying epitope variants = useful
vaccine vs highly variable pathogen
Live Attenuated Viral Vectors at UCT
Modified Vaccinia Ankara (MVA)
Recombinant MVA (rMVA) expressing HIV-1C gag and env genes
 Used in a Prime-Boost immunisation regimen
 prime immune response with plasmid vaccine expressing gag and env
 boost to broaden / increase response with rMVA expressing gag and env
DNA prime
rMVA boost
Gene Therapy
Gene therapy potentially represents one of the most
important developments to occur in medicine.
In order to modify a specific cell type or tissue, the
therapeutic gene must be efficiently delivered to
the cell. Two broad approaches have been used to
deliver DNA to cells, namely viral vectors & nonviral vectors
Viral Vectors
Viruses are obligate intra-cellular parasites, designed
through the course of evolution to infect cells, often with
great specificity to a particular cell type. They tend to be
very efficient at transfecting their own DNA into the host
cell, which is expressed to produce new viral particles. By
replacing genes that are needed for the replication
phase of their life cycle (the non-essential genes) with
foreign genes of interest, the recombinant viral vectors
can transduce the cell type it would normally infect.
Though a number of viruses have been developed, interest
has centred on four types; retroviruses (including
lentiviruses), adenoviruses, adeno-associated viruses &
herpes simplex virus type 1.
Gene targeting with rAAV vectors
The ideal vector
1) It should be relatively simple (e.g. not involving multiple
steps such as attachment of a ligand targeting a particular
cell type) & result in high vector concentrations (>108
particles/ml).
2) To allow subsequent readministration & avoid
undesired host reactions there would be no significant
immune response to any component of the vector. The
lack of an immune response may allow transgene
expression to be sufficiently prolonged from episomal
systems, such that readministration is not necessary.
Alternatively, integration into the host genome, preferably
in a site specific location, would ensure that the
transgene is not lost during the lifetime of the cell.
Adenoviruses
Adenoviruses: non-enveloped with a linear double stranded DNA genome
35 kb  most cause respiratory tract infections in humans.
subgroup C serotypes 2 or 5 are predominantly used as vectors. They replicate as
episomal elements in the nucleus of the host cell & consequently there is no
risk of insertional mutagenesis.
Up to 30 kb can be replaced with foreign DNA.  very efficient at
transducing target cells in vitro & vivo & can be produced at high titres
(>1011/ml).  successful in prolonging transgene expression &
achieving secondary gene transfer. fewer genes has resulted in
prolonged in vivo transgene expression in liver tissue.
 the majority of adenoviral proteins will be degraded & presented to
the immune system  cause problems for clinical trials. Moreover the
human population is heterogeneous with respect to MHC haplotype & a
proportion of the population will have been already exposed to the
adenoviral strain.
Methods of increasing viral uptake include stimulating the target cells to express an
appropriate integrin & conjugating an antibody with specificity for the target cell type to
the adenovirus.
Cytomegalovirus  E1 expressing cells such as HEK293 cells
Adeno-associated viruses (AAV)
Adeno-associated viruses (AAV) are non-pathogenic human
parvoviruses, dependant on a helper virus, usually
adenovirus, to proliferate. They are capable of infecting
both dividing & non dividing cells, & in the absence of a
helper virus integrate into a specific point of the host
genome (19q 13-qter) at a high frequency. When used as a
vector, the rep & cap genes are replaced by the
transgene & its associated regulatory sequences.
Interest in AAV vectors has been due to their integration
into the host genome allowing prolonged transgene
expression. Gene transfer into vascular epithelial cells,
striated muscle & hepatic cells has been reported, with
prolonged expression when the transgene is not derived
from a different species.
• The main factor limiting the
utility of AAVs as gene vectors
is their small size—no larger
than a nanoparticle. This
means they can only carry
about 4.7 kilobases of DNA,
and that must include any
promoters needed to regulate
the expression of the
therapeutic DNA.
Retroviruses
Retroviruses: enveloped viruses containing a single stranded RNA
molecule as the genome. Following infection, the viral genome is
reverse transcribed into double stranded DNA, which integrates
into the host genome & is expressed as proteins. To prevent
recombination resulting in replication competent retroviruses, all
regions of homology with the vector backbone should be
removed & the non-essential genes should be expressed by at
least two transcriptional units. The retroviral envelope interacts
with a specific cellular protein to determine the target cell range.
Altering the env gene or its product has proved a successful
means of manipulating the cell range.
Lentiviruses are a subclass of retroviruses which are able to infect both
proliferating & non-proliferating cells. They are considerably more
complicated than simple retroviruses. Mutants of vpr & vif are able
to infect neurones with reduced efficiency, but not muscle or
liver cells.
• 9 kilobases of genetic material.
Researchers only have to delete
a few genes to get lentivirus to
carry twice as much as AAVs
• transform cells by integrating near
to a cellular protooncogene &
driving inappropriate expression
from the LTR, or by disrupting a
tumour suppresser gene.
Herpes simplex virus type 1 (HSV-1)
HSV-1: a human neurotropic virus  a vector for gene
transfer to the nervous system. Latently infected neurones
function normally & are not rejected by the immune system.
There are up to 81 genes (40-50 kb of foreign DNA), of
which about half are not essential for growth in cell
culture.  some success in Parkinsons disease by
expressing tyrosine hydroxylase in striatal cells.  strong
inflammatory responses to HSV-1 amplicon vectors, both at
the primary site of the injection & at secondary sites were
observed.
Characteristics of viral vector systems
Viral vectors in use for clinical trials of
gene therapy (2011)
Strategies:
Gene therapy can either be applied ex vivo or in vivo. Ex vivo methods
are technically simpler with regard to vector transfer & gene
expression, but surgery is required to obtain & replace the cells. To
enhance in vivo delivery the target organ may be stimulated, for
example a partial hepatectomy will improve retroviral transduction to
the liver.
For some diseases the pathology affects the function of a
particular organ which must be directly treated. The common
sites for therapy are the liver, gut & muscle. These sites are chosen
for their ease of access, bulk & metabolic activity. Monogenic
recessive diseases only require the functional gene to be
expressed, often therapeutically useful levels being much lower than
that those found in normal individuals. Monogenic dominant
diseases require that the aberrant gene is silenced, usually by
means of an anti-sense DNA which is complementary to the
aberrant gene.
Gene Therapy for Cancer:
Cancer.  mutation to a protooncogene (yielding an oncogene) & to a
tumour suppressor gene, allowing the cancer to proliferate.  a variety
of strategies for gene therapy namely; immunopotentiation, oncogene
inactivation, tumor suppressor gene replacement, molecular
chemotherapy & drug resistance genes.
The aim of immunopotentiation is to enhance the response of the immune
system to cancers. Passive immunotherapy to increase the preexisting immune response to the cancer; active immunotherapy initiates
an immune response against an unrecognised or poorly antigenic
tumour. Oncogene inactivation (be targeted at the level of the DNA,
RNA transcription or protein product) employed for dominantly inherited
monogenic diseases. Restoration of the tumour suppressor gene,
such as p53, can be sufficient to cause cellular apoptosis & arrest tumour
growth. Expression of p53 is synergistic with chemotherapeutic drugs
such as cisplastin & adjacent tumour cells that have not been transduced
are killed. Molecular chemotherapy is to transduce a gene coding for a
toxic product, killing a tumour cell  herpes simplex virus thymidine
kinase (HSV/TK)
Oncolytic viruses
• Containing genes to
initiate apoptosis
and/or to increase the
immune system’s
attack on the cancer.
Strategies used
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immunopotentiation,
oncogene inactivation,
tumor suppressor gene replacement,
molecular chemotherapy
& drug resistance genes.
Immunopotentiation
• Passive immunotherapy aims to increase
the pre-existing immune response to
the cancer whilst active immunotherapy
initiates an immune response against an
unrecognised or poorly antigenic tumour.
 A vaccinia virus vector called JX-594 has been
developed to deliver genes that activate the
epidermal growth factor receptor (EGFR)/Ras
pathway in cancer cells, resulting in cell lysis and
increased anticancer immunity.
Examples of oncolytic viruses
used in the therapy
Side effects
• Most of these oncolytic viruses carry
coding viral nucleic acids, which may
cause side effects owing to recombination
with the host chromosome or proviral
elements that are already in the host cell.
 Synthetic viral particles designed that lack
coding nucleic acids and that exclusively
package therapeutic proteins, which can be
released in a dose-dependent manner
Cancer sensing promoter
• Current objectives include
improvement of cell targeting
through vector & promoter
specificity & reducing the
immune response to the
current vectors.
Gene Therapy - Anti HIV
• a mutation in both copies of the CCR5
gene resistant to HIV infection