Vaccines against Toxoplasma gondii: challenges

• T. gondii is an obligate intracellular parasite, cellular immunity has been considered the

major response to eliminate the parasite within the host

• Cellular immune response plays a major role, mostly by both CD4+ and CD8+ cells

and specifically due to their ability to secrete cytokines.

• IFN-γ is the key mediator in resistance against both early and late stages of the infection

• Cytokines secreted in the immune response to T. gondii include upregulated factors

(IFNγ, IL-2, TNFα, IL-1, IL-7, IL-12, IL-15) and downregulated factors (IL-4, IL-6,

IL-10).

• Humoral mediated immune responses by antibodies can provide protective immunity

against toxoplasmosis

Immunogenicity against T. gondii

• Inactivated, killed antigen vaccines of T. gondii, can probably not be sufficiently efficacious in any of the infection models used.

• Live attenuated vaccines are capable of mobilizing a MHC class I-restricted CD8+ T-cell response and have major response for clear intracellular infections

• Most Toxoplasma strain candidates for Live attenuated vaccines : RH, S48 strains

• Use of live, attenuated vaccines in humans is difficult to usage due to side effects and risks for breakthrough infection.

• S48 strain Toxovax® vaccine: The only commercial vaccine available\

The attenuated live tachyzoites of strain S48 (Toxovax) has been licensed for veterinary use to limit the incidence of abortion in sheep.

This vaccine is not suitable for human: 1- fear of reverting to a wild-type virulence

2- because a live attenuated vaccine may be pathogenic in individuals with a compromised immune systems

3- accidental infections in farmers have been reported.

Vaccine types against T.gondii

• DNA vaccines are attractive due to cost-effectiveness, thermal stability, homogeneity, long-lasting immunity and induction of a wide range of cellular and humoral immune responses.

• DNA vaccines have the particular capacity to induce CD4+ T-lymphocyte and CD8+ cytotoxic T-lymphocyte (CTL) responses against the antigen of interest

• As protection against T. gondii has been associated with a Th1 response, this vaccination strategy has received considerable attention.

• The core issue for DNA vaccine is safety. DNA vaccines have the possibility to integrate partial or complete plasmid sequences into

the host genome, causing insertion mutations with activation of oncogenes or inactivation of tumor suppressor genes.

Protein and Recombinant Subunit Vaccines

Advantages including:

strong B-cell responses

long-term memory

presence of specific immunogenic antigens

safety

Protein vaccines incorporate a highly purified antigen as the vaccine

component.

Due to this, protein and subunit vaccines are extremely safe and chances

of side effects occurring in recipients are low

Prime-boost strategies

Interestingly, it has been proven that heterologous prime-boost

strategies, containing plasmid DNA and recombinant

protein vaccine, develop effective immunization against T.

gondii infection.

• In the study of Min et al (2012) , A DNA prime-protein boost

vaccination based on GRA7 of T. gondii could induce both

humoral and cellular immune responses associated with high

levels of total IgG, IgG2a isotype and IFN-g and significantly

increase survival rate (60%), compared with controls of which all

died within 8 days of challenge

Short peptides containing B- and T-cell

Advantages: simultaneous stimulation of targeting epitopes,

strong antigenicity, elimination of deleterious components and

optimization of immune protection.

• Immunization with multiple antigenic peptide (MAP) vaccine against T.

gondii including B-cell and T-cell epitopes can induce an effective

immune response.

• For example: multi- epitopes derived from SAG1, GRA4 and GRA1

showed significantly increased humoral and cellular immunes responses

and prolonged survival time in comparison with unvaccinated controls.

• Bioinformatics tools are needed to design these types of vaccines.

Factors Restricting the Development of T. gondii Vaccine

• There is no standard protocol for the evaluation of vaccines against T. gondii

• There are safety concerns about use of some vaccines, such as DNA vaccine

• Lack of efficacious protective antigen candidates

• Selection of animal models

• Selection of T. gondii isolate

• Selection of adjuvants

• The antigen delivery

• Route of infection

• vaccine schedule (dosage, route of administration)

• Selection of target antigens is a crucial step to design effective vaccines

• The combination of multiple antigens in a single formulation could

significantly enhance cellular immune responses and protection, as compared

to the single gene vaccines. Vaccination with stage-specific antigens leads to

stage limited protection.

• T. gondii has a complex life cycle with three morphologically distinct

infectious stages, tachyzoites, bradyzoites and oocyst.

• Combination of antigens such as SAG1, MIC3, ROP2, ROP4, GRA1, GRA4,

GRA7, BAG1 and SAG2 showed effective and durable protection against all

stages of the parasite life cycle.

Lack of efficacious protective antigen candidates

Summery of important antigens are available in one, two or three stage

(tachyzoite, bradyzoite, and sporozoite) of T. gondii

• One of the effective and promising strategies in the last decade has

been the utilization of live attenuated microorganisms including viruses,

bacteria, and parasites as vectors for the delivery of Toxoplasma antigens in

experimental models.

• These vectors display advantages such as the ability to activate antigen

presenting cells, ability to deliver large or multiple antigens and having high

immunogenicity at mucosal and systemic levels

• Most live attenuated vectors used in studies are Salmonella and Pseudorabies

(PRV)

• Adjuvants can enhance the immunogencity of the antigenic vaccine

components against T. gondii infection through several strategies: including enhancing the time of DNA expression, facilitating the uptake of antigen by host cells or antigen-presenting cells (APCs) and protecting antigen from degradation.

• Also, Adjuvants can determine the type of immune responses (Th1, Th2).

• There is an urgent need for the use of nontoxic adjuvants for human vaccines such as Aluminum compounds, CPG-ODN, liposomes, and MPL.

• Subunit vaccines tend to lack immunogenicity and therefore require the use of appropriate adjuvants to enhance their potency

• Proper selection of animal models influences the outcome of vaccination

protocols against toxoplasmosis

• BALB/c and C3H/HeJ mice are resistant to acute and chronic toxoplasmosis,

whereas CBA/J mice are strongly resistant to the acute phase but susceptible to

the chronic infection; and C57BL6 mice are highly susceptible to T. gondii

infection

• The sex of the mice can also affect the susceptibility to toxoplasmosis. Male

mice demonstrated more resistance to mortality of Toxoplasma infection.

• Rats are the best animal models for congenital and ocular toxoplasmosis.

• Sheep and ginea pigs are proper animal models for the evaluation of

cellular and humoral immunity in vaccination against T. gondii

• The virulence of T. gondii is strongly dependent on the genotype of parasite.

• T. gondii consists of three main clonal lineages (types I, II and III) and many

other Toxoplasma genotypes emerging in human and animal reservoirs

worldwide .

• Type I strain (RH) is highly virulent and suitable for the evaluation of acute

toxoplasmosis, while Type II strains (ME49, 76 K and Beverley) and Type III

strains (VEG and C56) are significantly less virulent and appropriate for chronic

toxoplasmosis and cyst formation

• The strategy of antigen delivery is also one of the most critical aspects of optimizing vaccine immunogenicity

• Immunization in mice is induced by intramuscular (i.m.), subcutaneous (s.c.), intraperitoneal (i.p.), intranasal (i.n.) and intraoral (i.o.) routes.

• Intramuscular injection is used to deliver a vaccine deep into the muscles, which allows the vaccine to be absorbed into the bloodstream quickly.

• Subcutaneous injection is administered into the skin, which probably increases the exposure rate of vaccine to the antigen presenting cells (APC),

• Intranasal immunization is also an effective way for inducing mucosal and systemic immune responses

Directions for Future Research

• Future studies should focus on devising effective vaccines, to select target

antigens with the development of bioinformatics, genomics and proteomics

• Vaccines against toxoplasmosis should have different antigens expressed

in all stages of the parasite associated with adequate adjuvant and

appropriate delivery strategy.

• Future studies should address the issues of standardizing immunization

protocol and evaluation criteria.

• Future studies should not ignore the mechanisms of pathogenic, immune and

host cell invasion associated with T. gondii as well as vaccine action against

toxoplasmosis.

• To date, there are no effective chemical drugs to treat T. gondii infection, so

further research would develop therapeutic vaccines against the parasite.

Directions for Future Research

• Currently, numerous atypical strains have been reported throughout the globe

and several of these are estimated to possess greater pathogenicity than those

reported from the clonal types. Based on this notion, vaccines conferring cross-

strain protective immunity should also be investigated.

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Thank you for attention