GENE VACCINES

By :Akeel Akab sarhan

INTRODUCTION

DNA vaccination is a technique for protecting anorganism against disease by injecting it with geneticallyengineered DNA to produce an immunological response.

The direct injection of genetic material into a living hostcauses a small amount of its cells to produce theintroduced gene products.

This inappropriate gene expression within the host hasimportant immunological consequences, resulting in thespecific immune activation of the host against the genedelivered antigen .

In this way ,DNA vaccine provide immunity against thatpathogen.

Nucleic acid vaccines are;

experimentally proved

applied to a number of viral, bacterial and parasiticdiseases, tumor models.

DNA vaccines have a number of advantages over conventional vaccines, including the ability to induce a wider range of immune response types

TYPES OF VACCINES

1 FIRST GENERATION VACCINES

Are whole-organism vaccines – either live and weakened, or killed

forms.

1. Live, attenuated vaccines, such as smallpox and polio vaccines,

are able to induce killer T-cell (TC or CTL) responses, helper T-cell

(TH) responses and antibody 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).

2 . Killed, but previously virulent, micro-organisms that

have been destroyed with chemicals, heat, radioactivity

or antibiotics. Eg. influenza vaccine, cholera

vaccine, polio vaccine, hepatitis A vaccine, and rabies

vaccine

killed vaccines

do not have this risk, they cannot generate specific

killer T cell responses, and

may not work at all for some diseases.

Attenuated(live, attenuated microorganisms eg.Yersinia

pestis is used for plague immunization)

2 SECOND GENERATION VACCINES

were developed to minimize the risks of the live attenuated

vaccines.

protein antigens (such as tetanus or diphtheria toxoid)

recombinant 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.

3 Third generation vaccines

DNA vaccines are third generation vaccines, and are made up of a small, circular piece of bacterial DNA

(called a plasmid)

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 recognized as foreign, when they are processed by the host cells and

displayed on their surface, implies; the immune system is alerted, which

then triggers a range of immune responses.

HOW DNA VACCINES ARE MADE?

Plasmid vectors for use in vaccination DNA vaccines are composed of bacterial plasmids.

Expression plasmids used in DNA-based vaccination normally contain two units:

The antigen expression unit composed of promoter/enhancer sequences, followed by antigen-encoding and polyadenylation sequences and the production unit composed of bacterial sequences necessary for plasmid amplification and selection .

The construction of bacterial plasmids with vaccine inserts is accomplished using recombinant DNA technology.

Once constructed, the vaccine plasmid is transformed into bacteria, where bacterial growth produces multiple plasmid copies.

The plasmid DNA is then purified from the bacteria, by separating the circular plasmid from the much larger bacterial DNA and other bacterial impurities.

This purified DNA acts as the vaccine .

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 transcription and translation of the gene (or complementary DNA) of interest

Because the plasmid is the “vehicle” from which the immunogen is expressed, optimizing vector design for maximal protein expression is essential.

Ways of enhancing protein expression is by :

1. optimizing the codon usage of pathogenic mRNAs for eukaryotic cells.

2. altering the gene sequence of the immunogen to reflect the codons.

Vector design

MECHANISMS OF ACTION A plasmid vector that expresses the protein of

interest (e.g. viral protein) under the control of an appropriate promoter is

injected into the skin or muscle of the the host.

After uptake of the plasmid,

protein is produced endogenously (Antigenic Protein is presented by cell in which it is produced)and

intracellularly processed into small antigenic peptides by the host proteases.

The peptides then enter in to

lumen of the endoplasmic reticulum (E.R.) by transporters.

In the E.R., peptides bind to MHC class I molecules.

Subsequent CD8+ cytotoxic T cells (CTL) are

stimulated and

evoke cell-mediated immunity.

The foreign protein can also be presented by

MHC class II pathway by APCs

These CD4+ cells are able to

recognize the peptides formed from exogenous proteins

degraded to peptide fragments and loaded onto MHC class II molecules.

Depending on the type of CD4+ cell that binds to the complex,

B cells are stimulated and

antibody production is stimulated.

So in addition to mounting an attack against

the free-floating proteins,

the immune system attacks and

eliminates cells that have been colonized by a pathogen.

The vaccine works like a live vaccine, but without the

risk.

Toxoplasma gondii is an obligate intracellular protozoan parasite that infects

all warm-blooded animals, including humans, andcauses toxoplasmosis.

In primary human infections, various mild symptomsmay be observed, such as lymphadenopathy, low-gradefever, sore throat, and lethargy.

Immunosuppressed patients may exhibit severesymptoms, including encephalitis, myocarditis,pneumonitis, hepatitis, splenomegaly and multisystemorgan failure.

In pregnant women, congenital infection can lead tomiscarriage, neonatal malformations, blindness orsevere cognitive impairment .

In animals, toxoplasmosis is of great economicimportance worldwide because it causes abortions,stillbirth, and neonatal loss in all types of livestock,especially in sheep and goats.

In an attempt to overcome these problems, current research is investigating subunit, recombinant and DNA vaccines, but they do not provide complete protection against T. gondii infection .

Antigens of T. gondiiSurface membrane antigens and antigens released from secretory organelles.

There are several surface antigens SAG1 (Surface AntiGen 1) is used for the

major tachyzoite surface protein .Also, SAG2 and SAG3

Other minor antigens found on the surface are SRS1 (SAG1-related sequence 1)

or SRS2 .

As a member of the Phylum Apicomplexa, T. gondii contains an apical

complex consisting of organelles at the anterior part. These organelles

include polar rings, conoid, rhoptries, micronemes and dense granules.

Antigens released from the dense granules are named GRA1 , GRA2 , GRA4

and GRA7

ROP1 ; and ROP2 are secreted from rhoptries.

Immunity to T. gondiiWhen infected with T. gondii, immunocompetent generally

develop immune responses that effectively control the infection

and protect against reinfection .

Since T. gondii is an intracellular parasite, the infection is mainly

controlled by cellular immune mechanisms. However,

antibodies also play a role in regulation of the infection.

The cytokine gamma interferon (IFN-γ) has been identified as a

major mediator of resistance against T. gondii . IFN-γ is

predominantly released by T cells and natural killer (NK) cells.

It activates macrophages to destroy intracellular parasites and

cytotoxic T cells to destroy T. gondii infected cells.

It has been suggested that in case of chronic toxoplasmosis, IFN-γ

protects against recurrent disease by preventing cyst rupture.

When the mice were given a monoclonal antibody (mAb) against

IFN-γ, encephalitis was induced and tachyzoites and T. gondii

antigen were observed around the periphery of the tissue cysts,

indicating cyst disruption .

Giving anti-IFN-γ mAb to mice also led to increased mortalityafter T. gondii infection. IFN-γ can protect against acutetoxoplasmosis without collaboration of lymphokines derivedfrom T cells.

This has been shown in athymic mice, which lack T cells.

When recombinant IFN-γ (rIFN-γ) was given every other day

after infection, the mice survived, but after cessation of giving

rIFN-γ the mice died .

Suzuki and Remington (1988) showed that passive transfer of T

cells from immune mice to naïve recipients conferred

protection. Among T cell subsets, the cytotoxic CD8+ T

lymphocytes were found to be the most important against T.

gondii infection . Adoptive transfer of CD8+ cells increased

survival time and reduced brain cysts in challenged mice. It was

also shown that these cytotoxic CD8+ T cells produced IFN-γ

and interleukin-2 (IL-2) .

The killing effect of CD8+ cells has been demonstrated in vitro by lysis of

T. gondii infected macrophages .

Besides the cytotoxic T cells, the helper T cells are also important against

toxoplasmosis. At least in mice, they are generally subdivided into T helper

1 (Th1) and T helper 2 (Th2) subpopulations according to the cytokines they

produce. The Th1 cells secrete IFN-γ, IL-2 and beta tumor necrosis factor

(TNF-β whereas the Th2 cells produce IL-4, IL-5, IL-10 and IL-13

Immunity against toxoplasmosis is associated with a Th1 type of response

The synergistic effects and relative importance of CD4+ and CD8+ T cells

have been demonstrated by depletion of one or both subsets. Depletion of

CD4+ T cells in naïve mice led to increased susceptibility and increased

cyst burden and mortality after inoculation with an a virulent strain of T.

gondii .

In chronically infected mice depletion of either CD4+ or CD8+ T cells did

not cause any reactivation, while depletion of both subsets resulted in

activation of the infection .

Protective immunity can be induced by vaccination with an a virulent non-

persistent strain of T. gondii.

In vaccinated mice, the administration of both anti-CD4+ and anti-CD8+

antibody or anti-IFN-γ completely abrogates the resistance to the infection,

whereas giving anti-CD4+ does not affect immunity. Giving anti-CD8+

antibody partially reduces resistance to the infection .

Interestingly, an increase of NK cells, another important source of IFN-γ,

was observed during T. gondii infection .

Although cell-mediated immune responses play the essential role in

immunity against toxoplasmosis, antibodies also contribute. For example,

specific antibodies against SAG1 have been found to inhibit invasion of

human fibroblast cells by tachyzoites. This was confirmed in another study

showing that both monoclonal and polyclonal antibodies against rSAG1

partially inhibited the adhesion and/or invasion of host cells by T. gondii

tachyzoites .

In natural infection, antibodies might neutralize extracellular tachyzoites

through opsonisation or complement activation

Development of vaccines against Toxoplasma gondii infection

in humans is of high priority, given the high burden of disease

in some areas of the world.

Regardless of the vaccine construct,

the vaccines have not been able to induce protective immunity

when the organism is challenged with T. gondii, either directly or

via a vector.

Only a few live, attenuated T. gondii strains used for

immunization have been able to confer protective immunity,

which is measured by a lack of tissue cysts after challenge.

We have focused on the development of a DNA-basedvaccine because such vaccines have been shown to elicitpotent, long-lasting humoral and cell-mediated immunity,as well as providing protection against viral, bacterial, andparasitic infections .

The most common method used to deliver DNA vaccines isthe intramuscular injection, which is known to induce aTh1-type response , which is generally thought to protectthe host against T. gondii infection

It has been shown that DNA vaccine-induced protectiveimmunity against the acute phase of T. gondii infection.

Where as the entire coding sequence of GRA4 was insertedinto an eukaryotic expression vector to determine whetherDNA immunization can elicit protective immune responseto T. gondii.

Results showed that Mice immunized with GRA4 DNAdeveloped high levels of serum anti-GRA4immunoglobulin G antibodies as well as a cellular immuneresponse,

Vercammen et al have shown that mice vaccinatedwith plasmid encoding GRA1, GRA7, or ROP2 werepartially protected against a lethal oral challenge withcysts of two different T. gondii strains.

results showed that survival rates increased from 10%in controls to at least 70% after vaccination in one caseand from 50% to at least 90% in the other.

It has also been shown that DNA vaccination withp1tPASAG1 give effective protection in mice against T.gondii infection .

A plasmid expressing the SAG1 surface antigen of T. gondii,p1tPASAG1, were constructed and showed that animalsimmunized with the plasmid produce anti-SAG1 antibodieswhich recognize the native SAG1.

Mice immunized with p1tPASAG1 showed 80 to 100%protection against challenge with the non-cyst-producing,virulent RH isolate, compared to an 80% mortality in miceimmunized with empty plasmid, which is the greatestefficacy of any vaccine against T. gondii produced so far.

In another attempt to achieve complete protection against toxoplasmosis, MIC3 has been shown to be a good candidate vaccine

which could be combined with other relevant and previously described candidates, such as SAG1 and GRA4.

This response produced by plasmid encoding the immature form of the MIC3 protein was increased by the co-administration of a plasmid encoding the granulocyte-macrophage colony-stimulating factor (pGM-CSF).

Similarly, a specific and significant cellular immune response was obtained in mice immunized with pMIC3i, and this response was markedly enhanced by pGM-CSF coadministration.

This was confirmed by the production of large amounts of IgG.

Recently , immunization of mice with a DNA cocktail containing plasmids encoding the SAG1 and ROP2 genes resulted in a Th1-type response, and specific T-cell proliferation and IFN-γ production.

Also, significant long-lasting protection was observed after challenge infection with the virulent RH strain .

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