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1. GENE VACCINES
By :
Akeel Akab sarhan

2. INTRODUCTION
 DNA vaccination is a technique for protecting an
organism against disease by injecting it with genetically
engineered DNA to produce an immunological response.
 The direct injection of genetic material into a living host
causes a small amount of its cells to produce the
introduced gene products.
 This inappropriate gene expression within the host has
important immunological consequences, resulting in the
specific immune activation of the host against the gene
delivered antigen .
 In this way ,DNA vaccine provide immunity against that
pathogen.

3.  Nucleic acid vaccines are;
 experimentally proved
 applied to a number of viral, bacterial and parasitic
diseases, tumor models.
 DNA vaccines have a number of advantages over
conventional vaccines, including the ability to induce a
wider range of immune response types

4. 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).

5. 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)

6. 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.

7. 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.

8. 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 .

9. 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

10. 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.

11. 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.

12.  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.

13. 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.

14. Toxoplasma gondii
 is an obligate intracellular protozoan parasite that infects
all warm-blooded animals, including humans, and
causes toxoplasmosis.
 In primary human infections, various mild symptoms
may be observed, such as lymphadenopathy, low-grade
fever, sore throat, and lethargy.
 Immunosuppressed patients may exhibit severe
symptoms, including encephalitis, myocarditis,
pneumonitis, hepatitis, splenomegaly and multisystem
organ failure.

15.  In pregnant women, congenital infection can lead to
miscarriage, neonatal malformations, blindness or
severe cognitive impairment .
 In animals, toxoplasmosis is of great economic
importance worldwide because it causes abortions,
stillbirth, and neonatal loss in all types of livestock,
especially in sheep and goats.

16. 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 .

17. Antigens of T. gondii
Surface 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.

18. Immunity to T. gondii
When 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.

19. 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 mortality
after T. gondii infection. IFN-γ can protect against acute
toxoplasmosis without collaboration of lymphokines derived
from T cells.

20.  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) .

21.  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 .

22.  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 .

23.  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

24.  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.

25.  We have focused on the development of a DNA-based
vaccine because such vaccines have been shown to elicit
potent, long-lasting humoral and cell-mediated immunity,
as well as providing protection against viral, bacterial, and
parasitic infections .
 The most common method used to deliver DNA vaccines is
the intramuscular injection, which is known to induce a
Th1-type response , which is generally thought to protect
the host against T. gondii infection

26.  It has been shown that DNA vaccine-induced protective
immunity against the acute phase of T. gondii infection.
 Where as the entire coding sequence of GRA4 was inserted
into an eukaryotic expression vector to determine whether
DNA immunization can elicit protective immune response
to T. gondii.
 Results showed that Mice immunized with GRA4 DNA
developed high levels of serum anti-GRA4
immunoglobulin G antibodies as well as a cellular immune
response,

27.  Vercammen et al have shown that mice vaccinated
with plasmid encoding GRA1, GRA7, or ROP2 were
partially protected against a lethal oral challenge with
cysts of two different T. gondii strains.
 results showed that survival rates increased from 10%
in controls to at least 70% after vaccination in one case
and from 50% to at least 90% in the other.

28.  It has also been shown that DNA vaccination with
p1tPASAG1 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 animals
immunized with the plasmid produce anti-SAG1 antibodies
which 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 mice
immunized with empty plasmid, which is the greatest
efficacy of any vaccine against T. gondii produced so far.

29. 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.

30. 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 .

31. THANK YOU!