Marcin M. Grzybowski

Evaluation of three recombinant multi-antigenic vaccines composed of surface and secretory antigens of Toxoplasma gondii in murine models of experimental toxoplasmosis.

The great clinical and economical impact of Toxoplasma gondii infections makes the development of an effective vaccine for controlling toxoplasmosis an extremely important aim. In the presented study, we evaluate the protective and immunogenic properties of three recombinant subunit vaccines composed of rROP2+rGRA4+rSAG1, rROP2+rROP4+rGRA4 and rROP2+rROP4+rSAG1 proteins of T. gondii in an experimental toxoplasmosis model in the C3H/HeJ and C57BL/6 mouse strains. All three recombinant vaccines induced partial protection as measured by the reduction of brain cyst burden following challenge with five tissue cysts of the low virulence DX T. gondii strain. The level of protection was dependent on the antigen composition of the vaccine and the genetic background of the laboratory animals. The strongest protection against chronic toxoplasmosis was induced in both C3H/HeJ and C57BL/6 mice by the mixture of rhoptry proteins rROP2 and rROP4 combined with tachyzoite major protein rSAG1. The average parasite burden in these groups of mice was reduced by 71% and 90%, respectively, compared to non-vaccinated mice. The observed protective effect was related to the vaccine-induced cellular and humoral immune responses, as measured by the antigen-induced release of the Th1 cytokines IFN-γ and IL-2, the antigen-stimulated proliferation of spleen cells of vaccinated animals in comparison to control animals and the development of systemic antigen-specific IgG1 and IgG2a (C3H/HeJ) or IgG2c (C57BL/6) antibodies. Our studies show that recombinant rROP2, rROP4, rGRA4 and rSAG1 antigens may be promising candidates for a subunit vaccine against toxoplasmosis. Additionally, we demonstrate that the ideal composition of vaccine antigens can be equally effective in mice with different genetic backgrounds and variable levels of innate resistance to toxoplasmosis, resulting in strong protection against T. gondii invasion.

Toxoplasma gondii: The vaccine potential of three trivalent antigen-cocktails composed of recombinant ROP2, ROP4, GRA4 and SAG1 proteins against chronic toxoplasmosis in BALB/c mice

Toxoplasmosis is one of the worlds most widespread zoonoses caused by protozoan parasite Toxoplasma gondii. The development of an effective vaccine for controlling toxoplasmosis is an extremely important issue due to the serious clinical and veterinary outcomes of this parasitosis. The objective of this study was evaluation of vaccine potential of three trivalent subunit recombinant vaccines composed of rROP2+rGRA4+rSAG1, rROP2+rROP4+rGRA4 and rROP2+rROP4+rSAG1 against chronic toxoplasmosis in BALB/c (H-2(d)) mice. All tested vaccines provided a partial protection against challenge with tissue cysts of the low virulence DX T. gondii strain, but the strongest level of protection was induced by the mixtures of both rhoptry proteins (rROP2 and rROP4) administered with the dense granule rGRA4 antigen or the main surface rSAG1 protein. The average parasite burden in these groups of vaccinated BALB/c mice was reduced by 84% and 77%, respectively, compared to the control PBS-injected animals. The vaccine-induced protection was correlated with the development of cellular and humoral immune responses demonstrated by the antigen-specific in vitro proliferation of spleen cells, the specific antigen-induced in vitro synthesis of Th1-type cytokines, IFN-γ and IL-2, and the generation of the high titers of systemic antigen-specific IgG1 and IgG2a antibodies. This study completed and confirmed our earlier investigations in C3H/HeJ (H-2(k)) and C57BL/6 (H-2(b)) mouse strains on the utility of the tested trivalent recombinant antigen-cocktails as potential vaccines against chronic toxoplasmosis and showed that particularly rROP2+rROP4+rGRA4 and rROP2+rROP4+rSAG1 protein-combinations are very effective in the development of a high level of protection irrespective of the genetic backgrounds and innate resistance to toxoplasmosis of the laboratory mice. It makes these two mixtures of recombinant antigens very promising for further experiments.

Veterinary vaccines against toxoplasmosis

Toxoplasma gondii is a cosmopolitan protozoan parasite that infects a wide range of mammal and bird species. Common infection leads to high economic (e.g., abortions in sheep) and human (e.g., congenital toxoplasmosis or neurotoxoplasmosis in humans) losses. With one exception (Toxovax for sheep), there are no vaccines to prevent human or animal toxoplasmosis. The paper presents the current state and challenges in the development of a vaccine against toxoplasmosis, designed for farm animals either bred for consumption or commonly kept on farms and involved in parasite transmission. So far, the trials have mostly revolved around conventional vaccines and, compared with the research using laboratory animals (mainly mice), they have not been very numerous. However, the results obtained are promising and could be a good starting point for developing an effective vaccine to prevent toxoplasmosis.

Towards vaccine against toxoplasmosis: evaluation of the immunogenic and protective activity of recombinant ROP5 and ROP18 Toxoplasma gondii proteins

Toxoplasmosis is one of the most common parasitic infections worldwide. An effective vaccine against human and animal toxoplasmosis is still needed to control this parasitosis. The polymorphic rhoptry proteins, ROP5 and ROP18, secreted by Toxoplasma gondii during the invasion of the host cell have been recently considered as promising vaccine antigens, as they appear to be the major determinants of T. gondii virulence in mice. The goal of this study was to evaluate their immunogenic and immunoprotective activity after their administration (separately or both recombinant proteins together) with the poly I:C as an adjuvant. Immunization of BALB/c and C3H/HeOuJ mice generated both cellular and humoral specific immune responses with some predominance of IgG1 antibodies. The spleen cells derived from vaccinated animals reacted to the parasite’s native antigens. Furthermore, the immunization led to a partial protection against acute and chronic toxoplasmosis. These findings confirm the previous assumptions about ROP5 and ROP18 antigens as valuable components of a subunit vaccine against toxoplasmosis.

Phytoecdysteroids as modulators of the Toxoplasma gondii growth rate in human and mouse cells

BackgroundSearching for new effective drugs against human and animal toxoplasmosis we decided to test the anti-Toxoplasma potential of phytoecdysteroids (α-ecdysone and 20-hydroxyecdysone) characterized by the pleiotropic activity on mammalian organisms including the enhancement of host’s anti-parasitic defence. This objective was accomplished by the in vitro evaluation of T. gondii growth in phytoecdysteroid-treated immunocompetent cells of selected hosts: humans and two strains of inbred mice with genetically determined different susceptibility to toxoplasmosis.MethodsPeripheral mononuclear blood cells were isolated from Toxoplasma-positive and Toxoplasma-negative women (N = 43) and men (N = 21). Non-infected mice (C57BL/6, N = 10 and BALB/c, N = 14) and mice (BALB/c, N = 10) challenged intraperitoneally with 5 tissue cysts of the T. gondii DX strain were also used in this study as a source of splenocytes. The effects of phytoecdysteroids on the viability of human PBMC and mouse splenocytes were evaluated using the MTT assay. The influence of phytoecdysteroids on PBMCs, splenocytes and T. gondii proliferation was measured using radioactivity tests (the level of 3[H] uracil incorporation by toxoplasms or 3[H] thymidine by PBMCs and splenocytes), which was confirmed by quantitative Real-Time PCR. Statistical analysis was performed using SigmaStat 3.5 (Systat Software GmbH). The best-fit IC50 curves were plotted using GraphPad Prism 6.0 (GraphPad Software, Inc.).ResultsOur results showed that phytoecdysteroids promote the multiplication of Toxoplasma in cultures of human or murine immune cells, in contrast to another apicomplexan parasite, Babesia gibsoni. Additionally, the tested phytoecdysteroids did not stimulate the in vitro secretion of the essential protective cytokines (IFN-γ, IL-2 and IL-10), neither by human nor by murine immune cells involved in an effective intracellular killing of the parasite.ConclusionsJudging by the effect of phytoecdysteroids on the T. gondii proliferation, demonstrated for the first time in this study, it seems that these compounds should not be taken into consideration as potential medications to treat toxoplasmosis. Phytoecdysteroids included in the food are most likely not harmful for human or animal health but certain nutrients containing ecdysteroids at high concentrations could promote T. gondii proliferation in chronically infected and immunocompromised individuals. In order to assess the real impact of ecdysteroids on the course of natural T. gondii invasion, in vivo research should be undertaken because it cannot be ruled out that the in vivo effect will be different than the in vitro one. However, taking into account the possible stimulating effect of ecdysteroids on some opportunistic parasites (such as Toxoplasma or Strongyloides) further studies are necessary and should focus on the mechanisms of their action, which directly or indirectly enhance the parasite growth. Since ecdysteroids are considered as potential drugs, it is essential to determine their effect on various parasitic pathogens, which may infect the host at the same time, especially in immunocompromised individuals.

Human toxoplasmosis: a comparative evaluation of the diagnostic potential of recombinant Toxoplasma gondii ROP5 and ROP18 antigens.

Toxoplasmosis is one of the most common parasitic diseases worldwide and it poses a serious challenge regarding prevention, diagnosis and therapy. The commonly used diagnostic methods are mostly based on the detection of specific antibodies in sera. Since they are not always accurate enough and do not allow precise definition of the phase of the Toxoplasma gondii infection, there is an urgent need to find specific molecular markers of acute or chronic infection stages. This study provides a comparative assessment of recombinant ROP5 and ROP18 T. gondii proteins in the serodiagnosis of human toxoplasmosis. We found that both ROP5 and ROP18 proteins allowed the detection of specific IgM and IgG antibodies with a relatively low sensitivity; however, ROP18 IgM ELISA proved to be more sensitive than the SAG1 assay. This study also points to a relatively weak potential of the corresponding native ROP5 and ROP18 kinases in the generation of a strong antibody response in humans.

Mycobacterium tuberculosis AtsG (Rv0296c), GlmU (Rv1018c) and SahH (Rv3248c) Proteins Function as the Human IL-8-Binding Effectors and Contribute to Pathogen Entry into Human Neutrophils

Mycobacterium tuberculosis is an extremely successful intracellular pathogen that has evolved a broad spectrum of pathogenic mechanisms that enable its manipulation of host defense elements and its survival in the hostile environment inside phagocytes. Cellular influx into the site of mycobacterial entry is mediated by a variety of chemokines, including interleukin-8 (IL-8), and the innate cytokine network is critical for the development of an adaptive immune response and infection control. Using affinity chromatography, liquid chromatography electrospray ionization tandem mass spectrometry and surface plasmon resonance techniques, we identified M. tuberculosis AtsG arylsulphatase, bifunctional glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyl transferase (GlmU) and S-adenosyl-L-homocysteine hydrolase (SahH) as the pathogen proteins that bind to human IL-8. The interactions of all of the identified proteins (AtsG, GlmU and SahH) with IL-8 were characterized by high binding affinity with KD values of 6.83x10-6 M, 5.24x10-6 M and 7.14x10-10 M, respectively. Furthermore, the construction of Mtb mutant strains overproducing AtsG, GlmU or SahH allowed determination of the contribution of these proteins to mycobacterial entry into human neutrophils. The significantly increased number of intracellularly located bacilli of the overproducing M. tuberculosis mutant strains compared with those of “wild-type” M. tuberculosis and the binding interaction of AtsG, GlmU and SahH proteins with human IL-8 may indicate that these proteins participate in the modulation of the early events of infection with tubercle bacilli and could affect pathogen attachment to target cells.