VIRAL VACCINESIn 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 gastro-

intestinal tract. Virus families in this group are: rhinoviruses; coronaviruses;

parainfluenzaviruses; respiratory syncytial viruses; rotavirusesetc 

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 antibodyThe 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 cell-mediated 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 vaccine-associated 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.

Wild poliovirus (WPV) cases

                       

Attenuated VaccinesAttenuation 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 produceThey 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 VACCINESSecretory 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 given2. 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 immunity2. 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 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.

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 amountsDNA 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 interestThere is no protein component and so there will be no immune response against

the vector itselfBecause of the way the antigen is presented, there is a cell-mediated response

that may be directed against any antigen in the pathogen.

Possible ProblemsPotential integration of plasmid into host genome leading to insertional

mutagenesisInduction 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 plasmid-

infected 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 μg-

1 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 RNA-coated 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 responseCytokine 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 co-stimulatory 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 IL-

12.

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.