VIRAL VACCINES
In order to develop a successful vaccine, certain characteristics of the
viral infection must be known. One of these is the site at which the virus
enters the body.
Three major sites may be defined:
Infection via mucosal surfaces of the respiratory tract and gastrointestinal tract.
Virus families in this group are: rhinoviruses; coronaviruses;
parainfluenzaviruses; respiratory syncytial viruses; rotaviruses
etc
Infection via mucosal surfaces followed by spread systemically via the
blood and/or neurones to target organs. Virus families in this group are
hepatitis B virus; flaviviruses; bunyaviruses
IgA-mediated local immunity is very important
Thus, we need to know:
Viral antigen(s) that elicit neutralizing antibody
Cell surface antigen(s) that elicit neutralizing antibody
The site of replication of the virus
Types of vaccines
There are four basic types of vaccine today
Killed vaccines: These are preparations of the normal (wild type) infectious,
pathogenic virus that has been rendered non-pathogenic, usually by chemical
treatment such as with formalin that cross-links viral proteins.
Attenuated vaccines: These are live virus particles that grow in the vaccine
recipient but do not cause disease because the vaccine virus has been altered
(mutated) to a non-pathogenic form.
Sub-unit vaccines: These are purified components of the virus, such as a surface
antigen.
DNA vaccines: The protective antigen is made in the vaccine recipient to
elicit an immune response
Problems in vaccine development
•Antigenic drift and shift . This is especially true for RNA viruses and those with
segmented genomes
•Large animal reservoirs. If these occur, re-infection after elimination from the
human population may occur
•Integration of viral DNA. Vaccines will not work on latent virions unless they
express antigens on cell surface. In addition, if the vaccine virus integrates into
host cell chromosomes, it may cause problems
•Transmission from cell to cell via syncytia - This is a problem for potential AIDS
vaccines since the virus may spread from cell to cell without the virus entering
the circulation.
•Recombination and mutation of the vaccine virus in an attenuated vaccine.
Despite these problems, anti-viral vaccines have, in some cases, been
spectacularly successful .
A successful vaccine is the polio vaccine which may lead to the elimination of this
disease from the human population soon.
This vaccine comes in two forms. The Salk vaccine is a killed vaccine, while that
developed by Albert Sabin is a live attenuated vaccine. Polio is presently
restricted to parts of Africa (Nigeria and surrounding countries and south Asia
India, Pakistan and Afghanistan).
POLIO
Vaccines
There are two types of polio vaccine, both of which were developed in the 1950s.
The first, developed by Jonas Salk, is a formalin-killed preparation of normal wild
type polio virus. This is grown in monkey kidney cells and the vaccine is given by
injection. It elicits good humoral (IgG) immunity and prevents transport of the
virus to the neurons where it would otherwise cause paralytic polio. This vaccine
is the only one used in some Scandinavian countries where it completely wiped
out the disease.
A second vaccine was developed by Albert Sabin. This is a live attenuated vaccine
that was produced empirically by serial passage of the virus in cell culture. This
resulted in the selection of a mutated virus that grew well in culture and in the
human gut where the wild type virus grows.
It cannot, however, migrate to the neurones and it elicits both humoral and cellmediated immunity. It is given orally, a route that is taken by the virus in a normal
infection since the virus is passed from human to human by the oral-fecal route.
This became the preferred vaccine of it ease of administration (often on a sugar
lump), the fact that the vaccine virus replicates in the gut and only one
administration is needed to get good immunity (though repeated administration
was usually used). In addition, the immunity that results from the Sabin vaccine
lasts much longer than that by the Salk vaccine, making fewer boosters
necessary. Since it elicits mucosal immunity (IgA) in the gut, the Sabin vaccine
has the potential to wipe out wild type virus.
The attenuated Sabin vaccine, however, came with a problem: back mutation.
This may result from recombination between wild type virus and the vaccine
strain. Virulent virus is frequently isolated from recipients of the Sabin vaccine.
The residual cases in countries that use the attenuated live virus vaccine resulted
from mutation of the vaccine strain to virulence. About half of these cases were in
vaccinees and half in contacts of vaccinees. Paralytic polio arises in 1 in 100
cases of infection by wild type virus and 1 in about 3 million vaccinations as a
result of back reversion of the vaccine to virulence.
The vaccinee who has received killed Salk vaccine still allows wild type virus to
replicate in his/her gastro-intestinal tract, since the major immune response to the
injected killed vaccine is circulatory IgG. As noted above, this vaccine is
protective against paralytic polio since, although the wild type virus can still
replicate in the vaccinee's gut, it cannot move to the nervous system where the
symptoms of polio are manifested.
An additional problem of using a live attenuated vaccine is that preparations may
contain other pathogens from the cells on which the virus was grown. This was
certainly a problem initially because the monkey cells used to produce the polio
vaccine were infected with simian virus 40 (SV40) and this was also in the
vaccine.
SV40 is a polyoma virus and has the potential to cause cancer. It appears,
however, not to have caused problems in vaccinees who inadvertently received it.
Of course, there can also be similar problems with the killed vaccine if it is
improperly inactivated. This has also occurred.
Current recommendations concerning polio vaccines
Once the only polio cases in the US were vaccine-associated, the previous
policy of using the Sabin vaccine only was reevaluated. At first, both vaccines
were recommended with the killed vaccine first and then the attenuated vaccine.
The killed vaccine would stop the revertants of the live vaccine giving trouble by
moving to the nervous system. Thus, in 1997 the following protocol was
recommended:
To reduce the vaccine associated cases (8 to 10 per year), the CDC Advisory
Committee on Immunization Practices (ACIP) has recommended (January 1997) a
regimen of two doses of the injectable killed (inactivated: Salk) vaccine followed
by two doses of the oral attenuated vaccine on a schedule of 2 months of age
(inactivated), 4 months (inactivated), 12-18 months (oral) and 4-6 years (oral).
It is thought that the new schedule will eliminate most of the cases of vaccineassociated disease. This regimen has already been adopted by several
European countries.
The regimen of polio vaccination was subsequently amended again in 2000: To
eliminate the risk for Vaccine-Associated Paralytic Poliomyelitis, the ACIP
recommended an all-inactivated poliovirus vaccine (IPV) schedule for routine
childhood polio vaccination in the United States. As of January 1, 2000, all
children should receive four doses of IPV at ages 2 months, 4 months, 6-18
months, and 4-6 years.
Attenuated Vaccines
Attenuation is usually achieved by passage of the virus in foreign host such as
embryonated eggs or tissue culture cells with the hope to be less virulent for the
original host.
To produce the Sabin polio vaccine, attenuation was only achieved with high
inocula and rapid passage in primary monkey kidney cells. The virus population
became overgrown with a less virulent strain (for humans) that could grow well in
non-nervous (kidney) tissue but not in the central nervous system.
Molecular basis of attenuation
We do not know the basis of attenuation in most cases since attenuation was
achieved empirically. The empirical foreign-cell passage method causes many
mutations in a virus and it is difficult to determine which are the important
mutations. Many attenuated viruses are temperature-sensitive (that is, they grow
better at 35 - 37 degrees than 40 degrees) or cold adapted (they may grow at
temperatures as low as 25 degrees).
In the type 1 polio virus attenuated vaccine strain, there are 57 nucleotide
changes in the genome, resulting in 21 amino acid changes . One third of the
mutations are in the VP1 gene (this gene is only 12% of genome).
Recently, an attenuated nasal vaccine for influenza has been developped . This
contains cold-adapted vaccine strains of the influenza virus that have been grown
in tissue culture at progressively lower temperatures. After a dozen or more of
these passages, the virus grows well only at around 25° and in vivo growth is
restricted to the upper respiratory tract.
Studies showed that influenza illness occurred in only :
7 percent of volunteers who received the intra-nasal influenza vaccine,
versus 13 percent injected with trivalent inactivated influenza vaccine and
45 percent of volunteers who were given placebo.
Both vaccine comparisons with placebo were statistically significant.
Advantages of attenuated vaccines
They activate all phases of immune system. They elicit humoral IgG and local IgA
They raise an immune response to all protective antigens.
Inactivation, such as by formaldehyde in the case of the Salk vaccine, may alter
antigenicity
They offer more durable immunity . Thus, they stimulate antibodies against
multiple epitopes which are similar to those elicited by the wild type virus
They cost less to produce
They give quick immunity in majority of vaccinees
In the cases of polio administration is easy
These vaccines are easily transported in the field
They can lead to elimination of wild type virus from the community
POLIOVIRUSES VACCINES
Secretory antibody (nasal and gut IgA) and serum antibody (serum IgG, IgM and
IgA) in response to killed polio vaccine (left) administered by intramuscular
injection and to live attenuated polio vaccine (right) administered orally
Disadvantages of Attenuated vaccine
1. Mutation. This may lead to reversion to virulence (this is a major disadvantage)
2. Spread to contacts of the vaccinee and this could be an advantage in
communities where vaccination is not 100%
3. Spread of the vaccine virus that may be mutated
4. Live viruses are a problem in immunodeficiency disease patients
Advantages of inactivated vaccine
1. They give sufficient humoral immunity if boosters are given
2. There is no mutation or reversion (This is a big advantage)
3. They can be used with immuno-deficient patients
Disadvantages of inactivated vaccines
1. Some vaccinees do not raise immunity
2. Boosters are needed
3. There is little mucosal / local immunity (IgA).
4. Higher cost
5. There have been failures in inactivation leading to immunization with virulent
virus.
NEW METHODS OF VACCINE PRODUCTION
Selection for mis-sense
Temperature-sensitive mutants in influenza A and RSV have been made by
mutation with 5-fluorouracil and then selected for temperature sensitivity. In the
case of influenza, the temperature-sensitive gene can be reassorted in the
laboratory to yield a virus strain with the coat of the strains circulating in the
population and the inner proteins of the attenuated strain. Cold adapted mutants
can also be produced in this way. It has been possible to obtain mis-sense
mutations in all six genes for non-surface proteins.
The attenuated influenza vaccine, called FluMist, uses a cold-sensitive mutant that
can be reassorted with any new virulent influenza strain that appears . The
reassorted virus will have the genes for the internal proteins from the attenuated
virus (and hence will be attenuated) but will display the surface proteins of the
new virulent antigenic variant. Because this is based on a live, attenuated virus,
the customization of the vaccine to each year's new flu variants is much more
rapid than the process of predicting what influenza strains will be important for
the coming flu season and combining these in a killed vaccine.
Synthetic peptides
Injected peptides which are much smaller than the original virus protein raise an
IgG response but there is a problem with poor antigenicity. This is because the
epitope may depend on the conformation of the virus as a whole.
Anti-idiotype vaccines
An antigen binding site in an antibody is a reflection of the three-dimensional
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 antiidiotype 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-antiidiotype 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
anti-viral antibodies.
S antigen elicit
Recombinant DNA techniques
Single gene approach (usually a surface glycoprotein of the virus)
A single gene (for a protective antigen) can be expressed in a foreign host.
Expression vectors are used to make large amounts of antigen to be used as a
vaccine. The gene could be expressed in and the protein purified from bacteria
using a fermentation process, although lack of post-translational processing by
the bacteria is a problem.
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.
An example of a vaccine in which a viral protein is expressed in and purified from
yeast is Gardasil, an anti-human papilloma virus vaccine that is very effective in
preventing cervical cancer.
The current hepatitis B vaccine is also this type.
A similar anti-human papilloma vaccine, Cervarix, is made by expressing viral
genes recombined into a bacculovirus and expressed in insect cells.
Cloning of a gene into another virus
By cloning the gene for a protective antigen into another harmless virus, we
present the antigen just as the original virus does. In addition, 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 in this way as vaccinia will accept several foreign genes.
However, the use of vaccinia against smallpox has shown rare but serious
complications in immuno-compromised patients and alternatives have been
sought.
Adjuvants
Certain substances, when administered simultaneously with a specific antigen,
will enhance the immune response to that antigen. Such compounds are routinely
included in inactivated or purified antigen vaccines.
Adjuvants in common use:
1. Aluminium salts
First safe and effective compound to be used in human vaccines.
It promotes a good antibody response, but poor cell mediated immunity.
2. Liposomes and Immunostimulating complexes (ISCOMS)
3. Complete Freunds adjuvant is an emulsion of Mycobacteria, oil and water
Too toxic for man
Induces a good cell mediated immune response.
4. Incomplete Freund's adjuvant as above, but without Mycobacteria.
Possible modes of action:
By trapping antigen in the tissues, thus allowing maximal exposure to specific T
and B lymphocytes.
By activating antigen-presenting cells to secrete cytokines that enhance the
recruitment of antigen-specific T and B cells to the site of inoculation.
Viral Vaccines in general use
Measles
Live attenuated virus grown in chick embryo fibroblasts, first introduced in the
1960's. Its extensive use has led to the virtual eradication of measles in developed
world. In developed countries, the vaccine is administered to all children in the
second year of life (at about 15 months). However, in developing countries, where
measles is still widespread, children tend to become infected early (in the first
year), which frequently results in severe disease. It is therefore important to
administer the vaccine as early as possible (between six months and one year). If
the vaccine is administered too early, however, there is a poor take rate due to the
interference by maternal antibody. For this reason, when vaccine is administered
before the age of one year, a booster dose is recommended at 15 months.
Mumps
Live attenuated virus developed in the 1960's. In developed countries it is
administered together with measles and rubella at 15 months in the MMR vaccine.
Rubella
Live attenuated virus. Rubella causes a mild febrile illness in children, but if
infection occurs during pregnancy, the foetus may develop severe congenital
abnormalities. The vaccine is administered to all children in their second year of
life ,in an attempt to eradicate infection.Immunization of all children is the current
practice.
Polio
Two highly effective vaccines containing all 3 strains of poliovirus are in general
use:
The killed virus vaccine (Salk, 1954).
The live attenuated oral polio vaccine (Sabin, 1957) has been adopted in most
parts of the world; its chief advantages being: low cost, the fact that it induces
mucosal immunity and the possibility that, in poorly immunized communities,
vaccine strains might replace circulating wild strains and improve herd
immunity. Against this is the risk of reversion to virulence (especially of types 2
and 3) and the fact that the vaccine is sensitive to storage under adverse
conditions.
The inactivated Salk vaccine is recommended for children who are
immunosuppressed.
Hepatitis B
Two vaccines are in current use: a serum derived vaccine and a recombinant
vaccine. Both contain purified preparations of the hepatitis B surface protein.
The serum derived vaccine is prepared from hepatitis B surface protein, purified
from the serum of hepatitis B carriers. This protein is synthesised in vast excess
by infected hepatocytes and secreted into the blood of infected individuals. A
vaccine trial performed on homosexual men in the USA has shown that, following
three intra-muscular doses at 0, 1 and 6 months, the vaccine is at least 95%
protective.
A second vaccine, produced by recombinant DNA technology, has since become
available. Previously, vaccine administration was restricted to individuals who
were at high risk of exposure to hepatitis B. However, hepatitis B has been
targetted for eradication , and since 1995 the vaccine has been included in the
universal childhood immunization schedule.
Three doses are given; at 6, 10, and 14 weeks of age.
As with any killed viral vaccines, a booster will be required at some interval (not
yet determined, but about 5 years) to provide protection in later life from hepatitis
B infection as a venereal disease.
Hepatitis A
A vaccine for hepatitis A has been developed from formalin-inactivated , cell
culture-derived virus.
Two doses, administered one month apart, appear to induce high levels of
neutralising antibodies. The vaccine is recommended for travellers to third world
countries, and indeed all adults who are not immune to hepatitis A.
Yellow Fever
The 17D strain is a live attenuated vaccine developed in 1937. It is a highly
effective vaccine which is administered to residents in the tropics and travellers
to endemic areas. A single dose induces protective immunity to travellers and
booster doses, every 10 years, are recommended for residents in endemic areas.
Rabies
No safe attenuated strain of rabies virus has yet been developed for humans.
Vaccines in current use include:
The neurotissue vaccine - here the virus is grown in the spinal cords of rabbits,
and then inactivated with beta-propiolactone. There is a high incidence of
neurological complications following administration of this vaccine due to a
hypersensitivity reaction to the myelin in the preparation and largely it has been
replaced by
A human diploid cell culture-derived vaccine (also inactivated) which is much
safer.
There are two situations where vaccine is given:
a) Post-exposure prophylaxis, following the bite of a rabid animal:
A course of 5-6 intramuscular injections, starting on the day of exposure.
Hyperimmune rabies globulin may also administered on the day of exposure.
b) Pre-exposure prophylaxis is used for protection of those whose occupation
puts them at risk of infection with rabies; for example, vets, abbatoir and
laboratory workers.
This schedule is 2 doses one month apart ,and a booster dose one year later.
Further boosters every 2-3 years should be given if risk of exposure continues.
Influenza
Repeated infections with influenza virus are common due to rapid antigenic
variation of the viral envelope glycoproteins. Antibodies to the viral
neuraminidase and haemagglutinin proteins protect the host from
infection. However, because of the rapid antigenic variation, new vaccines,
containing antigens derived from influenza strains currently circulating in the
community, are produced every year.
Surveillance of influenza strains now allows the inclusion of appropriate antigens
for each season.The vaccines consist of partially purified envelope proteins of
inactivated current influenza A and B strains.
Individuals who are at risk of developing severe, life threatening disease if
infected with influenza should receive vaccine. People at risk include the elderly,
immunocompromised individuals, and patients with cardiac disease. In these
patients, protection from disease is only partial, but the severity of infection is
reduced.
Varicella-Zoster virus
A live attenuated strain of varicella zoster virus has been developed. It is not
licensed in South Africa for general use, but is used in some oncology units to
protect immuno-compromised children who have not been exposed to wild-type
varicella zoster virus. Such patients may develop severe, life threatening
infections if infected with the wild type virus.
DNA VACCINES
The Third Vaccine Revolution
These vaccines are based on the deliberate introduction of a DNA plasmid into the
vaccinee. The plasmid carries a protein-coding gene that transfects cells in vivo at
very low efficiency and expresses an antigen that causes an immune response.
These are often called DNA vaccines but would better be DNA-based
immunization since it is not the purpose to raise antibodies against the DNA
molecules themselves but to get the protein expressed by cells of the vaccinee.
These DNA vaccines developed from “failed” gene therapy experiments. The
first demonstration of a plasmid-induced immune response was when mice
inoculated with a plasmid expressing human growth hormone elicited antibodies
instead of altering growth.
Usually, muscle cells do this since the plasmid is given intramuscularly. It should
be noted that the plasmid does not replicate in the cells of the vaccinee, only
protein is produced.
It has also be shown that DNA can be introduced into tissues by bombarding the
skin with DNA-coated gold particles. It is possible to introduce 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 vaccinee.
The plasmid DNA vaccine (above) carries the genetic code for a piece of
pathogen. The plasmid vector is taken up into cells and transcribed in the nucleus
(1). The single stranded mRNA (2) is translated into protein in the cytoplasm. The
DNA vaccine-derived protein antigen (3) is then degraded by proteosomes into
intracellular peptides (4). The vaccine derived-peptide binds MHC class I
molecules (5). Peptide antigen/MHC I complexes are presented on the cell surface
(6), binding cytotoxic CD 8+ lymphocytes, and inducing a cell-mediated immune
response.
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. This means that we can
respond to changes in the infectious agent
By using the plasmid in the vaccinee 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.
This makes a far better antigen than, for example, 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 so that 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
Because of the way the antigen is presented, there is a cell-mediated response
that may be directed against any antigen in the pathogen.
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)
Initial studies
When they have been well-characterized, the immune responses are broad-based
and mimic the situation seen in a normal infection by the homologous virus. The
immune response can be remarkably long-lasting and even more so after one
booster injection of plasmid. Cytotoxic T lymphocyte (CTL) responses are also
well produced as might be expected since the immune system is seeing what is a
model of an infected cell.
One important demonstration using a DNA vaccine has been the induction of
cytotoxic cellular immunity to a conserved internal protein of influenza A to
determine if it might be possible to overcome the annual variation (antigenic drift
and shift) of the virus. CTLs were derived in mice against the conserved flu
nucleoprotein and this was effective at protecting the mice against disease, even
when they were challenged with a lethal dose of a virulent heterologous virus with
a different surface hemagglutinin. Because transfer of anti-nucleoprotein
antibodies to untreated mice does not protect them from disease, the protective
effect of the vaccine must have been cell-mediated.
The current influenza vaccine is an inactivated preparation containing antigens
from the flu strains that are predicted to infect during the next flu season. If such
a prediction goes away, the vaccine is of little use. It is the surface antigens that
change as a result of reassortment of the virus in the animal (duck) reservoir . The
vaccine is injected intramuscularly and elicits an IgG response (humoral antibody
in the circulation). The vaccine is protective because enough of the IgG gets
across the mucosa of the lungs where it can bind and neutralize incoming virus
by binding to surface antigens.
If a plasmid-based DNA vaccine is used, both humoral and cytotoxic T
lymphocytes are produced, which recognize antigens presented by plasmidinfected cells. The CTLs are produced because the infected muscle cells present
flu antigens in association with MHC class I molecules. If the antigen presented is
the nucleoprotein (which is a conserved protein), this overcomes the problem of
antigenic variation. Such an approach could revolutionize the influenza vaccine.
Other studies have used a mix of plasmids encoding both nucleoprotein and
surface antigens.
The vaccine DNA is injected into the cells of the body, where the "inner
machinery" of the host cells "reads" the DNA and converts it into pathogenic
proteins. Because these proteins are recognised as foreign, when they are
processed by the host cells and displayed on their surface, the immune system is
alerted, which then triggers a range of immune responses.
Vector design
These are plasmids which usually consist of a strong viral promotor to drive the
in vivo transcription and translation of the gene of interest. Plasmids also
include a strong polyadenylation/transcriptional termination signal.
Multicistronic vectors are sometimes constructed to express more than one
immunogen. Another consideration is the choice of promoter.
Expression rates have been increased by the use of the cytomegalovirus (CMV)
immediate early promoter.
Delivery methods
DNA vaccines have been introduced into animal tissues by a number of different
methods. The two most popular approaches are injection of DNA in saline , using
a standard hypodermic needle, and gene gun delivery. Injection in saline is
normally conducted intramuscularly (IM) in skeletal muscle, with DNA being
delivered to the extracellular spaces. Immune responses to this method of
delivery can be affected by many factors, including needle type, needle alignment,
speed of injection, volume of injection, muscle type, and age, sex and
physiological condition of the animal being injected.
Gene gun delivery, the other commonly used method of delivery, ballistically
accelerates plasmid DNA (pDNA) that has been adsorbed onto gold
microparticles into the target cells, using compressed helium as an accelerant.
The method of delivery determines the dose of DNA required to raise an effective
immune response. Saline injections require variable amounts of DNA, from 10 μg1 mg, whereas gene gun deliveries require 100 to 1000 times less DNA than
intramuscular saline injection to raise an effective immune response. Generally,
0.2 μg – 20 μg are required These quantities vary from species to species, with
mice, for example, requiring approximately 10 times less DNA than primates.
Saline injections require more DNA because the DNA is delivered to the
extracellular spaces of the target tissue (normally muscle), where it has to
overcome physical barriers before it is taken up by the cells, while gene gun
deliveries bombard DNA directly into the cells, resulting in less “wastage”.
The Helios Gene Gun is a new way for in vivo transformation of cells or
organisms.
This gun uses Biolistic ® particle bombardment where DNA- or RNAcoated gold particles are loaded into the gun and you pull the trigger. A
low pressure helium pulse delivers the coated gold particles into virtually
any target cell or tissue.
The gene gun is part of a method called bioballistic method, and under
certain conditions, DNA (or RNA) become “sticky,” adhering to
biologically inert particles such as metal atoms (usually tungsten or gold).
By accelerating this DNA-particle complex in a partial vacuum and placing
the target tissue within the acceleration path, DNA is effectively
introduced .
Raising of different types of T-cell help
Generally the type of T-cell help raised is stable over time, and does not change
when challenged or after subsequent immunizations.
It is not understood how these different methods of DNA immunization, or the
forms of antigen expressed, raise different profiles of T-cell help. It was thought
that the relatively large amounts of DNA used in IM injection were responsible for
the induction of TH1 responses. However, evidence has shown no differences in
TH type due to dose.
It has been postulated that the type of T-cell help raised is determined by the
differentiated state of antigen presenting cells.
Dendritic cells can differentiate to secrete IL-12 (which supports TH1 cell
development) or IL-4 (which supports TH2 responses).
pDNA injected by needle is endocytosed into the dendritic cell, which is then
stimulated to differentiate for TH1 cytokine production, while the gene gun
bombards the DNA directly into the cell, thus bypassing TH1 stimulation.
Cytotoxic T-cell responses
One of the greatest advantages of DNA vaccines is that they are able to induce
cytotoxic T lymphocytes (CTL) in a manner which appears to mimic natural
infection . This may prove to be a useful tool in assessing CTL epitopes of an
antigen, and their role in providing immunity.
Cytotoxic T-cells recognise small peptides (8-10 amino acids) complexed to MHC
class I molecules . These peptides are derived from endogenous cytosolic
proteins which are degraded and delivered to the nascent MHC class I molecule
within the endoplasmic reticulum (ER).
Co-inoculation with plasmids encoding co-stimulatory molecules IL-12 have also
been shown to increase CTL activity against HIV-1 and influenza nucleoprotein
antigens.
Kinetics of antibody response
Antibody responses against hepatitis B virus (HBV) envelope protein (HBsAg)
have been sustained for up to 74 weeks without boost, while life-long
maintenance of protective response to influenza haemagglutinin has been
demonstrated in mice after gene gun delivery.
DNA-raised antibody responses rise much more slowly than when natural
infection or recombinant protein immunization occurs. It can take as long as 12
weeks to reach peak titres in mice, although boosting can increase the rate of
antibody production. This slow response is probably due to the low levels of
antigen expressed over several weeks.
Additionally, the titres of specific antibodies raised by DNA vaccination are lower
than those obtained after vaccination with a recombinant protein. However, DNA
immunization-induced antibodies show greater affinity to native epitopes than
recombinant protein-induced antibodies. In other words, DNA immunization
induces a qualitatively superior response. Antibody can be induced after just one
vaccination with DNA, whereas recombinant protein vaccinations generally
require a boost.
DNA Uptake Mechanism
When DNA uptake and subsequent expression was first demonstrated in vivo in
muscle cells, it was thought that these cells were unique in this ability because of
their extensive network of T-tubules. However, subsequent research revealed that
other cells such as keratinocytes , fibroblasts and epithelial cells could also
internalize DNA. This phenomenon has not been the subject of much research, so
the actual mechanism of DNA uptake is not known.
After gene gun inoculation to the skin, transfected Langerhans cells migrate to
the draining lymphe node to present antigen. After IM and ID injections, dendritic
cells have also been found to present antigen in the draining lymph node and
transfected macrophages have been found in the peripheral blood.
IM and ID delivery of DNA initiate immune responses differently. In the skin,
keratinocytes, fibroblasts and Langerhans cells take up and express antigen, and
are responsible for inducing a primary antibody response. Transfected
Langerhans cells migrate out of the skin (within 12 hours) to the draining lymph
node where they prime secondary B- and T-cell responses.
IM inoculated DNA “washes” into the draining lymph node within minutes, where
distal dendritic cells are transfected and then initiate an immune response.
Transfected myocytes seem to act as a “reservoir” of antigen for trafficking APCs
Modulation of the immune response
Cytokine modulation
The ability of DNA vaccines to polarise TH1 or TH2 profiles, and generate CTL
and/or antibody when required, is a great advantage in this regard. This can be
accomplished by modifications to the form of antigen expressed (i.e. intracellular
vs. secreted), the method and route of delivery, and the dose of DNA delivered.
However, it can also be accomplished by the co-administration of plasmid DNA
encoding immune regulatory molecules, i.e. cytokines, lymphokines or costimulatory molecules. These “genetic adjuvants” can be administered a number
of ways:
In general, co-administration of pro-inflammatory agents (such as various
interleukins , and GM-CSF) plus TH2 inducing cytokines increase antibody
responses, whereas pro-inflammatory agents and TH1 inducing cytokines
decrease humoral responses and increase cytotoxic responses (which is more
important in viral protection, for example). This concept has been successfully
applied in topical administration of pDNA encoding IL-10. The advantages of using
genetic adjuvants are their low cost and simplicity of administration, as well as
avoidance of unstable recombinant cytokines and potentially toxic,
“conventional” adjuvants (such as alum). However, the potential toxicity of
prolonged cytokine expression has not been established, and in many
commercially important animal species, cytokine genes still need to be identified
and isolated.
Immunostimulatory CpG motifs
Bacterially derived DNA has been found to trigger innate immune defence
mechanisms, the activation of dendritic cells, and the production of TH1
cytokines. This is due to recognition of certain CpG dinucleotide sequences
which are immunostimulatory. CpG stimulatory (CpG-S) sequences occur twenty
times more frequently in bacterially derived DNA than in eukaryotes. This is
because eukaryotes exhibit “CpG suppression” – i.e. CpG dinucleotide pairs
occur much less frequently than expected. Additionally, CpG-S sequences are
hypomethylated. This occurs frequently in bacterial DNA, while CpG motifs
occurring in eukaryotes are usually methylated at the cytosine nucleotide.
In contrast, nucleotide sequences which inhibit the activation of an immune
response (termed CpG neutralising, or CpG-N) are over represented in eukaryotic
genomes. The optimal immunostimulatory sequence has been found to be an
unmethylated CpG dinucleotide flanked by two 5’ purines and two 3’
pyrimidines.Additionally, flanking regions outside this immunostimulatory
hexamer must be guanine-rich to ensure binding and uptake into target cells.
CpG-S sequences induce polyclonal B-cell activation and the upregulation of
cytokine expression and secretion. Stimulated macrophages secrete IL-12, IL-18,
TNF-α, IFN-α, IFN-β and IFN-γ, while stimulated B-cells secrete IL-6 and some IL12.
Most of the evidence for the existence of immunostimulatory CpG sequences
comes from murine studies. Clearly, extrapolation of this data to other species
should be done with caution – different species may require different flanking
sequences.