DNA vaccination
Encyclopedia
Organism
In biology, an organism is any contiguous living system . In at least some form, all organisms are capable of response to stimuli, reproduction, growth and development, and maintenance of homoeostasis as a stable whole.An organism may either be unicellular or, as in the case of humans, comprise...
against disease by injecting it with genetically engineered DNA
Naked DNA
Naked DNA is histone-free DNA that is passed from cell to cell during a gene transfer process called transformation or transfection. In transformation , purified or naked DNA is taken up by the recipient cell which will give the recipient cell a new characteristic or phenotype...
to produce an immunological response. Nucleic acid vaccines are still experimental, and have been applied to a number of viral
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
, bacterial and parasitic models of disease, as well as to several tumour models. DNA vaccines have a number of advantages over conventional vaccines, including the ability to induce a wider range of immune response types.
Vaccine
Vaccine
A vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe or its toxins...
s are among the greatest achievements of modern medicine – in industrial nations, they have eliminated naturally occurring cases of smallpox
Smallpox
Smallpox was an infectious disease unique to humans, caused by either of two virus variants, Variola major and Variola minor. The disease is also known by the Latin names Variola or Variola vera, which is a derivative of the Latin varius, meaning "spotted", or varus, meaning "pimple"...
, and nearly eliminated polio, while other diseases, such as typhus
Typhus
Epidemic typhus is a form of typhus so named because the disease often causes epidemics following wars and natural disasters...
, rotavirus
Rotavirus
Rotavirus is the most common cause of severe diarrhoea among infants and young children, and is one of several viruses that cause infections often called stomach flu, despite having no relation to influenza. It is a genus of double-stranded RNA virus in the family Reoviridae. By the age of five,...
, hepatitis
Hepatitis
Hepatitis is a medical condition defined by the inflammation of the liver and characterized by the presence of inflammatory cells in the tissue of the organ. The name is from the Greek hepar , the root being hepat- , meaning liver, and suffix -itis, meaning "inflammation"...
A and B and others are well controlled. Conventional vaccines, however, only cover a small number of diseases, and infections that lack effective vaccines kill millions of people every year, with AIDS
AIDS
Acquired immune deficiency syndrome or acquired immunodeficiency syndrome is a disease of the human immune system caused by the human immunodeficiency virus...
, hepatitis C
Hepatitis C
Hepatitis C is an infectious disease primarily affecting the liver, caused by the hepatitis C virus . The infection is often asymptomatic, but chronic infection can lead to scarring of the liver and ultimately to cirrhosis, which is generally apparent after many years...
and malaria
Malaria
Malaria is a mosquito-borne infectious disease of humans and other animals caused by eukaryotic protists of the genus Plasmodium. The disease results from the multiplication of Plasmodium parasites within red blood cells, causing symptoms that typically include fever and headache, in severe cases...
being particularly common.
First generation vaccines are whole-organism vaccines – either live and weakened
Attenuated virus
An attenuated vaccine is a vaccine created by reducing the virulence of a pathogen, but still keeping it viable . Attenuation takes an infectious agent and alters it so that it becomes harmless or less virulent. These vaccines contrast to those produced by "killing" the virus .-Examples:Examples of...
, or killed forms. Live, attenuated vaccines, such as smallpox and polio vaccines, are able to induce killer T-cell
Cytotoxic T cell
A cytotoxic T cell belongs to a sub-group of T lymphocytes that are capable of inducing the death of infected somatic or tumor cells; they kill cells that are infected with viruses , or are otherwise damaged or...
(TC or CTL) responses, helper T-cell (TH) responses and antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
immunity. However, there is a small risk that attenuated forms of a pathogen can revert to a dangerous form, and may still be able to cause disease in immunocompromised people (such as those with AIDS
AIDS
Acquired immune deficiency syndrome or acquired immunodeficiency syndrome is a disease of the human immune system caused by the human immunodeficiency virus...
). While killed vaccines do not have this risk, they cannot generate specific killer T cell responses, and may not work at all for some diseases. In order to minimise these risks, so-called second generation vaccines were developed. These are subunit vaccines, consisting of defined protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
antigen
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
s (such as tetanus
Tetanus
Tetanus is a medical condition characterized by a prolonged contraction of skeletal muscle fibers. The primary symptoms are caused by tetanospasmin, a neurotoxin produced by the Gram-positive, rod-shaped, obligate anaerobic bacterium Clostridium tetani...
or diphtheria
Diphtheria
Diphtheria is an upper respiratory tract illness caused by Corynebacterium diphtheriae, a facultative anaerobic, Gram-positive bacterium. It is characterized by sore throat, low fever, and an adherent membrane on the tonsils, pharynx, and/or nasal cavity...
toxoid
Toxoid
A toxoid is a bacterial toxin whose toxicity has been weakened or suppressed either by chemical or heat treatment, while other properties, typically immunogenicity, are maintained. In international medical literature the preparation also is known as Anatoxin or Anatoxine...
) or recombinant
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
protein components (such as the hepatitis B surface antigen). These, too, are able to generate TH and antibody responses, but not killer T cell responses.
DNA vaccines are third generation vaccines, and are made up of a small, circular piece of bacterial DNA (called a plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
) that has been genetically engineered to produce one or two specific proteins (antigens) from a pathogen. 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. These DNA vaccines developed from “failed” gene therapy
Gene therapy
Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development...
experiments. The first demonstration of a plasmid-induced immune response was when mice
MICE
-Fiction:*Mice , alien species in The Hitchhiker's Guide to the Galaxy*The Mice -Acronyms:* "Meetings, Incentives, Conferencing, Exhibitions", facilities terminology for events...
inoculated with a plasmid expressing human growth hormone elicited antibodies instead of altering growth.
Current use
Thus far, few experimental trials have evoked a response strong enough to protect against disease, and the usefulness of the technique, while tantalizing, remains to be conclusively proven in human trials. However, in June 2006 positive results were announced for a bird flu DNA vaccine and a veterinary DNA vaccine to protect horses from West Nile virusWest Nile virus
West Nile virus is a virus of the family Flaviviridae. Part of the Japanese encephalitis antigenic complex of viruses, it is found in both tropical and temperate regions. It mainly infects birds, but is known to infect humans, horses, dogs, cats, bats, chipmunks, skunks, squirrels, domestic...
has been approved. In August 2007, a preliminary study in DNA vaccination against multiple sclerosis
Multiple sclerosis
Multiple sclerosis is an inflammatory disease in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring as well as a broad spectrum of signs and symptoms...
was reported as being effective.
Advantages and disadvantages of DNA vaccines
| Advantages | Disadvantages |
|---|---|
|
|
Vector design
DNA vaccines elicit the best immune response when highly active expression vectors are used. These are plasmids which usually consist of a strong viral promoter to drive the in vivo transcriptionTranscription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
and translation
Translation
Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. Whereas interpreting undoubtedly antedates writing, translation began only after the appearance of written literature; there exist partial translations of the Sumerian Epic of...
of the gene (or complementary DNA
Complementary DNA
In genetics, complementary DNA is DNA synthesized from a messenger RNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes...
) of interest. Intron A may sometimes be included to improve mRNA stability and hence increase protein expression. Plasmids also include a strong polyadenylation
Polyadenylation
Polyadenylation is the addition of a poly tail to an RNA molecule. The poly tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA for translation...
/transcriptional termination signal, such as bovine growth hormone
Growth hormone
Growth hormone is a peptide hormone that stimulates growth, cell reproduction and regeneration in humans and other animals. Growth hormone is a 191-amino acid, single-chain polypeptide that is synthesized, stored, and secreted by the somatotroph cells within the lateral wings of the anterior...
or rabbit beta-globulin polyadenylation sequences. Multicistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein.
Because the plasmid is the “vehicle” from which the immunogen is expressed, optimising vector design for maximal protein expression is essential. One way of enhancing protein expression is by optimising the codon usage of pathogenic mRNAs for eukaryotic cells. Pathogens often have different AT contents than the species being immunized, so altering the gene sequence of the immunogen to reflect the codon
Genetic code
The genetic code is the set of rules by which information encoded in genetic material is translated into proteins by living cells....
s more commonly used in the target species may improve its expression.
Another consideration is the choice of promoter. The SV40
SV40
SV40 is an abbreviation for Simian vacuolating virus 40 or Simian virus 40, a polyomavirus that is found in both monkeys and humans...
promoter was conventionally used until research showed that vectors driven by the Rous Sarcoma Virus
Rous sarcoma virus
Rous sarcoma virus is a retrovirus and is the first oncovirus to have been described: it causes sarcoma in chickens.As with all retroviruses, it reverse transcribes its RNA genome into cDNA before integration into the host DNA.-History:...
(RSV) promoter had much higher expression rates. More recently, expression rates have been further increased by the use of the cytomegalovirus
Cytomegalovirus
Cytomegalovirus is a viral genus of the viral group known as Herpesviridae or herpesviruses. It is typically abbreviated as CMV: The species that infects humans is commonly known as human CMV or human herpesvirus-5 , and is the most studied of all cytomegaloviruses...
(CMV) immediate early promoter. Inclusion of the Mason-Pfizer monkey virus (MPV)-CTE with/without rev increased envelope expression. Furthermore the CTE+rev construct was significantly more immunogenic then CTE alone vector.
Additional modifications to improve expression rates have included the insertion of enhancer sequences, synthetic introns, adenovirus tripartite leader (TPL) sequences and modifications to the polyadenylation and transcriptional termination sequences. An example of DNA vaccine plasmid is pVAC, it uses SV40 promoter.
Vaccine insert design
Immunogens can be targeted to various cellular compartments in order to improve antibody or cytotoxic T-cell responses. Secreted or plasma membrane-bound antigens are more effective at inducing antibody responses than cytosolic antigens, while cytotoxic T-cell responses can be improved by targeting antigens for cytoplasmic degradation and subsequent entry into the major histocompatibility complexMajor histocompatibility complex
Major histocompatibility complex is a cell surface molecule encoded by a large gene family in all vertebrates. MHC molecules mediate interactions of leukocytes, also called white blood cells , which are immune cells, with other leukocytes or body cells...
(MHC) class I pathway. This is usually accomplished by the addition of N-terminal ubiquitin
Ubiquitin
Ubiquitin is a small regulatory protein that has been found in almost all tissues of eukaryotic organisms. Among other functions, it directs protein recycling.Ubiquitin can be attached to proteins and label them for destruction...
signals.
The conformation of the protein can also have an effect on antibody responses, with “ordered” structures (like viral particles) being more effective than unordered structures. Strings of minigenes (or MHC class I epitopes) from different pathogens are able to raise cytotoxic T-cell responses to a number of pathogens, especially if a TH epitope is also included.
Delivery methods
The two most popular approaches are injection of DNA in saline
Saline (medicine)
In medicine, saline is a general term referring to a sterile solution of sodium chloride in water but is only sterile when it is to be placed intravenously, otherwise, a saline solution is a salt water solution...
, using a standard hypodermic needle, and gene gun
Gene gun
A gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for injecting cells with genetic information. The payload is an elemental particle of a heavy metal coated with plasmid DNA...
delivery. A schematic outline of the construction of a DNA vaccine plasmid and its subsequent delivery by these two methods into a host is illustrated at Scientific American. Injection in saline is normally conducted intramuscularly (IM) in skeletal muscle
Skeletal muscle
Skeletal muscle is a form of striated muscle tissue existing under control of the somatic nervous system- i.e. it is voluntarily controlled. It is one of three major muscle types, the others being cardiac and smooth muscle...
, or intradermally
Dermis
The dermis is a layer of skin between the epidermis and subcutaneous tissues, and is composed of two layers, the papillary and reticular dermis...
(ID), with DNA being delivered to the extracellular spaces. This can be assisted by electroporation
Electroporation
Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field...
; by temporarily damaging muscle fibres with myotoxins such as bupivacaine
Bupivacaine
Bupivacaine is a local anaesthetic drug belonging to the amino amide group. AstraZeneca commonly markets it under various trade names, including Marcain, Marcaine, Sensorcaine and Vivacaine.-Indications:...
; or by using hypertonic solutions of saline or sucrose
Sucrose
Sucrose is the organic compound commonly known as table sugar and sometimes called saccharose. A white, odorless, crystalline powder with a sweet taste, it is best known for its role in human nutrition. The molecule is a disaccharide composed of glucose and fructose with the molecular formula...
. 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
Gold
Gold is a chemical element with the symbol Au and an atomic number of 79. Gold is a dense, soft, shiny, malleable and ductile metal. Pure gold has a bright yellow color and luster traditionally considered attractive, which it maintains without oxidizing in air or water. Chemically, gold is a...
or tungsten
Tungsten
Tungsten , also known as wolfram , is a chemical element with the chemical symbol W and atomic number 74.A hard, rare metal under standard conditions when uncombined, tungsten is found naturally on Earth only in chemical compounds. It was identified as a new element in 1781, and first isolated as...
microparticles into the target cells, using compressed helium
Helium
Helium is the chemical element with atomic number 2 and an atomic weight of 4.002602, which is represented by the symbol He. It is a colorless, odorless, tasteless, non-toxic, inert, monatomic gas that heads the noble gas group in the periodic table...
as an accelerant.
Alternative delivery methods have included aerosol
Aerosol
Technically, an aerosol is a suspension of fine solid particles or liquid droplets in a gas. Examples are clouds, and air pollution such as smog and smoke. In general conversation, aerosol usually refers to an aerosol spray can or the output of such a can...
instillation of naked DNA on mucosal surfaces, such as the nasal
Human nose
The visible part of the human nose is the protruding part of the face that bears the nostrils. The shape of the nose is determined by the ethmoid bone and the nasal septum, which consists mostly of cartilage and which separates the nostrils...
and lung
Human lung
The human lungs are the organs of respiration in humans. Humans have two lungs, with the left being divided into two lobes and the right into three lobes. Together, the lungs contain approximately of airways and 300 to 500 million alveoli, having a total surface area of about in...
mucosa, and topical administration of pDNA to the eye and vaginal mucosa. Mucosal surface delivery has also been achieved using cationic liposome
Liposome
Liposomes are artificially prepared vesicles made of lipid bilayer. Liposomes can be filled with drugs, and used to deliver drugs for cancer and other diseases. Liposomes are composite structures made of phospholipids and may contain small amounts of other molecules...
-DNA preparations, biodegradable microspheres, attenuated Shigella
Shigella
Shigella is a genus of Gram-negative, nonspore forming, non-motile, rod-shaped bacteria closely related to Escherichia coli and Salmonella. The causative agent of human shigellosis, Shigella causes disease in primates, but not in other mammals. It is only naturally found in humans and apes. During...
or Listeria vectors for oral administration to the intestinal mucosa, and recombinant adenovirus vectors.
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, although quantities as low as 16 ng have been reported. 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 (such as the basal lamina
Basal lamina
The basal lamina is a layer of extracellular matrix secreted by the epithelial cells, on which the epithelium sits. It is often confused with the basement membrane, and sometimes used inconsistently in the literature, see below....
and large amounts of connective tissue
Connective tissue
"Connective tissue" is a fibrous tissue. It is one of the four traditional classes of tissues . Connective Tissue is found throughout the body.In fact the whole framework of the skeleton and the different specialized connective tissues from the crown of the head to the toes determine the form of...
, to mention a few) before it is taken up by the cells, while gene gun deliveries bombard DNA directly into the cells, resulting in less “wastage”.
Another approach to DNA vaccination is expression library immunization (ELI). Using this technique, potentially all the genes from a pathogen can be delivered at one time, which may be useful for pathogens which are difficult to attenuate or culture. ELI can be used to identify which of the pathogen’s genes induce a protective response. This has been tested with Mycoplasma
Mycoplasma
Mycoplasma refers to a genus of bacteria that lack a cell wall. Without a cell wall, they are unaffected by many common antibiotics such as penicillin or other beta-lactam antibiotics that target cell wall synthesis. They can be parasitic or saprotrophic. Several species are pathogenic in humans,...
pulmonis, a murine lung pathogen with a relatively small genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
, and it was found that even partial expression libraries can induce protection from subsequent challenge.
| Method of Delivery | Formulation of DNA | Target Tissue | Amount of DNA | |
|---|---|---|---|---|
| Parenteral | Injection (hypodermic needle) | Aqueous solution in saline | IM (skeletal); ID; (IV, subcutaneous and intraperitoneal with variable success) | Large amounts (approximately 100-200 μg) |
| Gene Gun | DNA-coated gold beads | ED (abdominal skin); vaginal mucosa; surgically exposed muscle and other organs | Small amounts (as little as 16 ng) | |
| Pneumatic (Jet) Injection | Aqueous solution | ED | Very high (as much as 300 μg) | |
| Topical application | Aqueous solution | Ocular; intravaginal | Small amounts (up to 100 μg) | |
| Cytofectin-mediated | Liposomes (cationic); microspheres; recombinant adenovirus vectors; attenuated Shigella Shigella Shigella is a genus of Gram-negative, nonspore forming, non-motile, rod-shaped bacteria closely related to Escherichia coli and Salmonella. The causative agent of human shigellosis, Shigella causes disease in primates, but not in other mammals. It is only naturally found in humans and apes. During... vector; aerosolised cationic lipid Lipid Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others... formulations |
IM; IV (to transfect tissues systemically); intraperitoneal; oral immunization to the intestinal mucosa; nasal/lung mucosal membranes | variable | |
| Method of Delivery | Advantage | Disadvantage |
|---|---|---|
| Intramuscular or Intradermal injection |
|
|
| Gene Gun |
|
|
| Jet injection |
|
|
| Liposome-mediated delivery |
|
|
Helper T-Cell responses
DNA immunization is able to raise a range of TH responses, including lymphoproliferation and the generation of a variety of cytokineCytokine
Cytokines are small cell-signaling protein molecules that are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication...
profiles. A major advantage of DNA vaccines is the ease with which they can be manipulated to bias the type of T-cell help towards a TH1 or TH2 response. Each type of response has distinctive patterns of lymphokine
Lymphokine
Lymphokines are a subset of cytokines that are produced by a type of immune cell known as a lymphocyte. They are protein mediators typically produced by T cells to direct the immune system response by signalling between its cells...
and chemokine expression, specific types of immunoglobulins expressed, patterns of lymphocyte trafficking, and types of innate immune responses generated.
Raising of different types of T-cell help
The type of T-cell help raised is influenced by the method of delivery and the type of immunogen expressed, as well as the targeting of different lymphoid compartments. Generally, saline needle injections (either IM or ID) tend to induce TH1 responses, while gene gun delivery raises TH2 responses. This is true for intracellular and plasma membrane-bound antigens, but not for secreted antigens, which seem to generate TH2 responses, regardless of the method of delivery.Generally the type of T-cell help raised is stable over time, and does not change when challenged or after subsequent immunizations which would normally have raised the opposite type of response in a naïve animal. However, Mor et al.. (1995) immunized and boosted mice with pDNA encoding the circumsporozoite protein of the mouse malaria
Malaria
Malaria is a mosquito-borne infectious disease of humans and other animals caused by eukaryotic protists of the genus Plasmodium. The disease results from the multiplication of Plasmodium parasites within red blood cells, causing symptoms that typically include fever and headache, in severe cases...
l parasite Plasmodium yoelii
Plasmodium yoelii
Plasmodium yoelii is a parasite of the genus Plasmodium subgenus Vinckeia.Like all Plasmodium species P. yoelii has both vertebrate and insect hosts. The vertebrate hosts for this parasite are mammals.- Notes :...
(PyCSP) and found that the initial TH2 response changed, after boosting, to a TH1 response.
Mechanistic basis for different types of T-Cell help
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 ILInterleukin
Interleukins are a group of cytokines that were first seen to be expressed by white blood cells . The term interleukin derives from "as a means of communication", and "deriving from the fact that many of these proteins are produced by leukocytes and act on leukocytes"...
-12 (which supports TH1 cell development) or IL-4 (which supports TH2 responses). pDNA injected by needle is endocytosed
Endocytosis
Endocytosis is a process by which cells absorb molecules by engulfing them. It is used by all cells of the body because most substances important to them are large polar molecules that cannot pass through the hydrophobic plasma or cell membrane...
into the dendritic cell, which is then stimulated to differentiate for TH1 cytokine
Cytokine
Cytokines are small cell-signaling protein molecules that are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication...
production, while the gene gun bombards the DNA directly into the cell, thus bypassing TH1 stimulation.
Practical uses of polarised T-Cell help
This polarisation in T-cell help is useful in influencing allergic responses and autoimmune diseases. In autoimmune diseases, the goal would be to shift the self-destructive TH1 response (with its associated cytotoxic T cell activity) to a non-destructive TH2 response. This has been successfully applied in predisease priming for the desired type of response in preclinical models and somewhat successful in shifting the response for an already established disease.Cytotoxic T-cell responses
One of the greatest advantages of DNA vaccines is that they are able to induce cytotoxic T lymphocytes (CTL) without the inherent risk associated with live vaccines. CTL responses can be raised against immunodominant and immunorecessive CTL epitopes, as well as subdominant CTL epitopes, in a manner which appears to mimic natural infectionInfection
An infection is the colonization of a host organism by parasite species. Infecting parasites seek to use the host's resources to reproduce, often resulting in disease...
. 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
MHC class I
MHC class I molecules are one of two primary classes of major histocompatibility complex molecules and are found on every nucleated cell of the body...
molecules (Restifo et al., 1995). These peptides are derived from endogenous cytosolic proteins which are degraded and delivered to the nascent MHC class I molecule within the endoplasmic reticulum
Endoplasmic reticulum
The endoplasmic reticulum is an organelle of cells in eukaryotic organisms that forms an interconnected network of tubules, vesicles, and cisternae...
(ER). Targeting gene products directly to the ER (by the addition of an amino-terminal insertion sequence) should thus enhance CTL responses. This has been successfully demonstrated using recombinant vaccinia
Vaccinia
Vaccinia virus is a large, complex, enveloped virus belonging to the poxvirus family. It has a linear, double-stranded DNA genome approximately 190 kbp in length, and which encodes for approximately 250 genes. The dimensions of the virion are roughly 360 × 270 × 250 nm, with a mass of...
viruses expressing influenza
Influenza
Influenza, commonly referred to as the flu, is an infectious disease caused by RNA viruses of the family Orthomyxoviridae , that affects birds and mammals...
proteins, but the principle should be applicable to DNA vaccines too. Targeting antigens for intracellular degradation (and thus entry into the MHC class I pathway) by the addition of ubiquitin
Ubiquitin
Ubiquitin is a small regulatory protein that has been found in almost all tissues of eukaryotic organisms. Among other functions, it directs protein recycling.Ubiquitin can be attached to proteins and label them for destruction...
signal sequences, or mutation of other signal sequences, has also been shown to be effective at increasing CTL responses.
CTL responses can also be enhanced by co-inoculation with co-stimulatory molecules such as B7-1 or B7-2 for DNA vaccines against influenza nucleoprotein, or GM-CSF for DNA vaccines against the murine malaria model P. yoelii. Co-inoculation with plasmids encoding co-stimulatory molecules IL-12 and TCA3 have also been shown to increase CTL activity against HIV-1 and influenza nucleoprotein antigens.
Humoral (antibody) response
Kinetics of antibody response
Humoral responses after a single DNA injection can be much longer-lived than after a single injection with a recombinant protein. 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. Antibody-secreting cells migrate to the bone marrowBone marrow
Bone marrow is the flexible tissue found in the interior of bones. In humans, bone marrow in large bones produces new blood cells. On average, bone marrow constitutes 4% of the total body mass of humans; in adults weighing 65 kg , bone marrow accounts for approximately 2.6 kg...
and spleen
Spleen
The spleen is an organ found in virtually all vertebrate animals with important roles in regard to red blood cells and the immune system. In humans, it is located in the left upper quadrant of the abdomen. It removes old red blood cells and holds a reserve of blood in case of hemorrhagic shock...
for long-term antibody production, and are generally localised there after one year.
Comparisons of antibody responses generated by natural (viral) infection, immunization with recombinant protein and immunization with pDNA are summarised in Table 4. 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, which supports both primary and secondary phases of antibody response.
| Method of Immunization | |||
|---|---|---|---|
| DNA Vaccine | Recombinant protein | Natural Infection | |
| Amount of inducing antigen | ng | μg | ? (ng-μg) |
| Duration of Ag presentation | several weeks | < 1 week | several weeks |
| Kinetics of Ab response | slow rise | rapid rise | rapid rise |
| Number of inoculations to obtain high avidity IgG and migration of ASC to bone marrow | one | two | one |
| Ab isotype (murine models) | C’-dependent or C’-independent | C’-dependent | C’-independent |
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. As mentioned previously, DNA immunization can be used to bias the TH profile of the immune response, and thus the antibody isotype, which is not possible with either natural infection or recombinant protein immunization. Antibody responses generated by DNA are useful not just in vaccination but as a preparative tool, too. For example, polyclonal and monoclonal antibodies can be generated for use as reagents.
DNA Uptake Mechanism
When DNA uptake and subsequent expression was first demonstrated in vivo in muscleMuscle
Muscle is a contractile tissue of animals and is derived from the mesodermal layer of embryonic germ cells. Muscle cells contain contractile filaments that move past each other and change the size of the cell. They are classified as skeletal, cardiac, or smooth muscles. Their function is to...
cells, it was thought that these cells were unique in this ability because of their extensive network of T-tubules. Using electron microscopy, it was proposed that DNA uptake was facilitated by caveolae
Caveolae
In biology, caveolae , which are a special type of lipid raft, are small invaginations of the plasma membrane in many vertebrate cell types, especially in endothelial cells and adipocytes....
(or, non-clathrin coated pits). However, subsequent research revealed that other cells (such as keratinocytes, fibroblasts and epithelial Langerhans 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.
Two theories are currently popular – that in vivo uptake of DNA occurs non-specifically, in a method similar to phago- or pinocytosis
Pinocytosis
In cellular biology, pinocytosis is a form of endocytosis in which small particles are brought into the cell—forming an invagination, and then suspended within small vesicles that subsequently fuse with lysosomes to hydrolyze, or to break down, the particles...
, or through specific receptors. These might include a 30kDa surface receptor
Immune receptor
An immune receptor is a receptor, usually on a cell membrane, which binds to a substance and causes a response in the immune system.-Types:...
, or macrophage
Macrophage
Macrophages are cells produced by the differentiation of monocytes in tissues. Human macrophages are about in diameter. Monocytes and macrophages are phagocytes. Macrophages function in both non-specific defense as well as help initiate specific defense mechanisms of vertebrate animals...
scavenger receptors. The 30kDa surface receptor binds very specifically to 4500-bp genomic DNA fragments (which are then internalised) and is found on professional APCs and T-cells. Macrophage scavenger receptors bind to a variety of macromolecules, including polyribonucleotides, and are thus also candidates for DNA uptake. Receptor mediated DNA uptake could be facilitated by the presence of polyguanylate sequences
Guanine
Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine . In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with...
. Further research into this mechanism might seem pointless, considering that gene gun delivery systems, cationic liposome packaging, and other delivery methods bypass this entry method, but understanding it might be useful in reducing costs (e.g. by reducing the requirement for cytofectins), which will be important in the food animals industry.
Antigen presentation by bone marrow-derived cells
Chimera (genetics)
A chimera or chimaera is a single organism that is composed of two or more different populations of genetically distinct cells that originated from different zygotes involved in sexual reproduction. If the different cells have emerged from the same zygote, the organism is called a mosaic...
mice have shown that antigen is presented by bone-marrow derived cells, which include dendritic cells, macrophages and specialised B-cells called professional antigen presenting cells (APC) Iwasaki et al., 1997). After gene gun inoculation to the skin, transfected Langerhans cells migrate to the draining lymph node
Lymph node
A lymph node is a small ball or an oval-shaped organ of the immune system, distributed widely throughout the body including the armpit and stomach/gut and linked by lymphatic vessels. Lymph nodes are garrisons of B, T, and other immune cells. Lymph nodes are found all through the body, and act as...
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.
Besides direct transfection of dendritic cells or macrophages, cross priming is also known to occur following IM, ID and gene gun deliveries of DNA. Cross priming occurs when a bone marrow-derived cell presents peptides from proteins synthesised in another cell in the context of MHC class 1. This can prime cytotoxic T-cell responses and seems to be important for a full primary immune response.
Role of the target site
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. In skeletal muscle, on the other hand, striated muscle cells are most frequently transfected, but seem to be unimportant in mounting an immune response. Instead, 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 professional APCs.Maintenance of immune response
DNA vaccination generates an effective immune memory via the display of antigen-antibody complexes on follicular dendritic cells (FDC), which are potent B-cell stimulators. T-cells can be stimulated by similar, germinal centre dendritic cells. FDC are able to generate an immune memory because antibodies production “overlaps” long-term expression of antigen, allowing antigen-antibody immunocomplexes to form and be displayed by FDC.Interferons
Both helper and cytotoxic T-cells can control viral infections by secreting interferons. Cytotoxic T cells usually kill virally infected cells. However, they can also be stimulated to secrete antiviral cytokines such as INF-γ and TNF-α, which don’t kill the cell but place severe limitations on viral infection by down-regulating the expression of viral components. DNA vaccinations can thus be used to curb viral infections by non-destructive IFN-mediated control. This has been demonstrated for the hepatitis B virus. IFN-γ is also critically important in controlling malaria infections, and should be taken into consideration when developing anti-malarial DNA vaccines.Cytokine modulation
For a vaccine to be effective, it must induce an appropriate immune response for a given pathogen, and the ability of DNA vaccines to polarise T-cell help towards 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:- as a mixture of 2 separate plasmids, one encoding the immunogen and the other encoding the cytokine;
- as a single bi- or polycistronic vector, separated by spacer regions; or
- as a plasmid-encoded chimeraFusion proteinFusion proteins or chimeric proteins are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins...
, or fusion protein.
In general, co-administration of pro-inflammatory agents (such as various interleukins, tumor necrosis factor
Tumor necrosis factor
Tumor necrosis factor is a cytokine involved in systemic inflammation and is a member of a group of cytokines that stimulate the acute phase reaction...
, 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). Co-stimulatory molecules like B7-1, B7-2 and CD40L are also sometimes used.
This concept has been successfully applied in topical administration of pDNA encoding IL-10. Plasmid encoded B7-1 (a ligand on APCs) has successfully enhanced the immune response in anti-tumour models, and mixing plasmids encoding GM-CSF and the circumsporozoite protein of P. yoelii (PyCSP) has enhanced protection against subsequent challenge (whereas plasmid-encoded PyCSP alone did not). It was proposed that GM-CSF may cause dendritic cells to present antigen more efficiently, and enhance IL-2 production and TH cell activation, thus driving the increased immune response. This can be further enhanced by first priming with a pPyCSP and pGM-CSF mixture, and later boosting with a recombinant poxvirus expressing PyCSP. However, co-injection of plasmids encoding GM-CSF (or IFN-γ, or IL-2) and a fusion protein of P. chabaudi merozoite surface protein 1 (C-terminus)-hepatitis B virus surface protein (PcMSP1-HBs) actually abolished protection against challenge, compared to protection acquired by delivery of pPcMSP1-HBs alone.
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, calcium phosphate, monophosphoryl lipid A, cholera
Cholera
Cholera is an infection of the small intestine that is caused by the bacterium Vibrio cholerae. The main symptoms are profuse watery diarrhea and vomiting. Transmission occurs primarily by drinking or eating water or food that has been contaminated by the diarrhea of an infected person or the feces...
toxin, cationic and mannan-coated liposomes, QS21, carboxymethylcellulose and ubenimix). 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. In addition, various plasmid encoded cytokines modulate the immune system differently according to the time of delivery. For example, some cytokine plasmid DNAs are best delivered after the immunogen pDNA, because pre- or co-delivery can actually decrease specific responses, and increase non-specific responses.
Immunostimulatory CpG motifs
Plasmid DNA itself appears to have an adjuvant effect on the immune system. 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 all 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.The innate system works synergistically with the adaptive immune system to mount a response against the DNA encoded protein. 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.
Manipulation of CpG-S and CpG-N sequences in the plasmid backbone of DNA vaccines can ensure the success of the immune response to the encoded antigen, and drive the immune response toward a TH1 phenotype. This is useful if a pathogen requires a TH response for protection. CpG-S sequences have also been used as external adjuvants for both DNA and recombinant protein vaccination with variable success rates. Other organisms with hypomethylated CpG motifs have also demonstrated the stimulation of polyclonal B-cell expansion. However, the mechanism behind this may be more complicated than simple methylation – hypomethylated murine DNA has not been found to mount an immune response.
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, as binding specificities of scavenger receptors differ between species. Additionally, species such as ruminants may be insensitive to immunostimulatory sequences due to the large gastrointestinal load they exhibit. Further research may be useful in the optimisation of DNA vaccination, especially in the food animal industry.
Alternative boosts
DNA-primed immune responses can be boosted by the administration of recombinant protein or recombinant poxviruses. “Prime-boost” strategies with recombinant protein have successfully increased both neutralising antibody titre, and antibody avidity and persistence, for weak immunogens, such as HIV-1 envelope protein. Recombinant virus boosts have been shown to be very efficient at boosting DNA-primed CTL responses. Priming with DNA focuses the immune response on the required immunogen, while boosting with the recombinant virus provides a larger amount of expressed antigen, leading to a large increase in specific CTL responses.Prime-boost strategies have been successful in inducing protection against malarial challenge in a number of studies. Primed mice with plasmid DNA encoding Plasmodium yoelii circumsporozoite surface protein (PyCSP), then boosted with a recombinant vaccinia virus expressing the same protein had significantly higher levels of antibody, CTL activity and IFN-γ, and hence higher levels of protection, than mice immunized and boosted with plasmid DNA alone. This can be further enhanced by priming with a mixture of plasmids encoding PyCSP and murine GM-CSF, before boosting with recombinant vaccinia virus. An effective prime-boost strategy for the simian
Simian
The simians are the "higher primates" familiar to most people: the Old World monkeys and apes, including humans, , and the New World monkeys or platyrrhines. Simians tend to be larger than the "lower primates" or prosimians.- Classification and evolution :The simians are split into three groups...
malarial model P. knowlesi has also been demonstrated. Rhesus monkeys were primed with a multicomponent, multistage DNA vaccine encoding two liver-stage antigens - the circumsporozoite surface protein (PkCSP) and sporozoite surface protein 2 (PkSSP2) - and two blood stage antigens - the apical merozoite surface protein 1 (PkAMA1) and merozoite surface protein 1 (PkMSP1p42). They were then boosted with a recombinant canarypox virus encoding all four antigens (ALVAC-4). Immunized monkeys developed antibodies against sporozoites and infected erythrocytes, and IFN-γ-secreting T-cell responses against peptides from PkCSP. Partial protection against sporozoite challenge was achieved, and mean parasitemia was significantly reduced, compared to control monkeys. These models, while not ideal for extrapolation to P. falciparum in humans, will be important in pre-clinical trials.
Formulations of DNA
The efficiency of DNA immunization can be improved by stabilising DNA against degradation, and increasing the efficiency of delivery of DNA into antigen presenting cells. This has been demonstrated by coating biodegradable cationic microparticles (such as poly(lactide-co-glycolide) formulated with cetyltrimethylammonium bromide) with DNA. Such DNA-coated microparticles can be as effective at raising CTL as recombinant vaccinia viruses, especially when mixed with alum. Particles 300 nm in diameter appear to be most efficient for uptake by antigen presenting cells.Alphavirus vectors
Recombinant alphavirus-based vectors have also been used to improve DNA vaccination efficiency. The gene encoding the antigen of interest is inserted into the alphavirus replicon, replacing structural genes but leaving non-structural replicase genes intact. The Sindbis virus and Semliki Forest virus have been used to build recombinant alphavirus replicons. Unlike conventional DNA vaccinations, however, alphavirus vectors kill transfected cells, and are only transiently expressed. Also, alphavirus replicase genes are expressed in addition to the vaccine insert. It is not clear how alphavirus replicons raise an immune response, but it is thought that this may be due to the high levels of protein expressed by this vector, replicon-induced cytokine responses, or replicon-induced apoptosis leading to enhanced antigen uptake by dendritic cells.External links
- PowderMed pdf report
- DyNAVacS, an Integrative Tool for Optimised DNA Vaccine Design from the Institute of Genomics and Integrative BiologyInstitute of Genomics and Integrative BiologyInstitute of Genomics and Integrative Biology is a scientific research institute devoted primarily to integrated biological research. It is a part of Council of Scientific and Industrial Research , India....
.