Vaccination of squirrel monkeys (Saimiri spp.) with nanoparticle-based Toxoplasma gondii antigens: new hope for captive susceptible species

Squirrel monkeys (Saimiri spp.), new world primates from South America, are very susceptible to toxoplasmosis. Numerous outbreaks of fatal toxoplasmosis in zoos have been identified around the world, resulting in acute respiratory distress and sudden death. To date, preventive hygiene measures or available treatments are not able to significantly reduce this mortality in zoos. Therefore, vaccination seems to be the best long-term solution to control acute toxoplasmosis. Recently, we developed a nasal vaccine composed of total extract of soluble proteins of Toxoplasma gondii associated with muco-adhesive maltodextrin-nanoparticles. The vaccine, which generated specific cellular immune responses, demonstrated efficacy against toxoplasmosis in murine and ovine experimental models. In collaboration with six French zoos, our vaccine was used as a last resort in 48 squirrel monkeys to prevent toxoplasmosis. The full protocol of vaccination includes two intranasal sprays followed by combined intranasal and s.c. administration. No local or systemic side-effects were observed irrespective of the route of administration. Blood samples were collected to study systemic humoral and cellular immune responses up to 1 year after the last vaccination. Vaccination induced a strong and lasting systemic cellular immune response mediated by specific IFN-γ secretion by peripheral blood mononuclear cells. Since the introduction of vaccination, no deaths of squirrel monkeys due to T. gondii has been observed for more than 4 years suggesting the promising usage of our vaccine. Moreover, to explain the high susceptibility of naive squirrel monkeys to toxoplasmosis, their innate immune sensors were investigated. It was observed that Toll-like and Nod-like receptors appear to be functional following T. gondii recognition suggesting that the extreme susceptibility to toxoplasmosis may not be linked to innate detection of the parasite.     

Toxoplasmosis: the first commercial vaccine

Infection with Toxoplasma gondii can have serious consequences in pregnant women and in immunocompromised individuals. For the farming industry it is a major cause of economic loss through neonatal mortality, particularly in sheep. In the following short review, David Buxton summarizes the main features of the complex immune response to the parasite and outlines how the first commercial vaccine was developed to control toxoplasmosis in pregnant sheep. Although the vaccine is inappropriate for use in human beings, the manner in which it induces immunity in sheep will prove vital to our understanding of the infection and the eventual development of a suitable vaccine to combat toxoplasmosis in people as well as in animals.     

Toxoplasma gondii: Protective effect of an intranasal SAG1 and MIC4 DNA vaccine in mice

Infections by the intracellular protozoan parasite Toxoplasma gondii are widely prevalent in humans and other animals which can cause severe or lethal toxoplasmosis. So the development of a more effective vaccine is needed urgently. A multiantigenic vaccine against toxoplasmosis was constructed in the present study, which contains two T. gondii antigens, SAG1 and MIC4 on the basis of previous immunological and immunization studies. The eukaryotic plasmid pcDNA3.1-SAG1-MIC4, pcDNA3.1-SAG1, pcDNA3.1-MIC4 were constructed first, which can express surface protein SAG1 and microneme protein MIC4 from different stages of T. gondii life cycle, and the expression ability of these DNA vaccine in HeLa cells were examined by Western blot. The efficacy of these plasmids with or without co-administration of a plasmid encoding cholera toxin A2/B as a genetic adjuvant by mucosal way to protect BALB/c mice against toxoplasmosis was evaluated. We found these vaccines were able to elicit a significant humoral and cellular immune response in vaccinated mice and they can increase survival rate and prolong the life of mice that were infected by T. gondii especially in the pcDNA3.1-SAG1-MIC4 group. Co-delivery of cholera toxin A2/B further enhanced the potency of multiantigenic DNA vaccine by intranasal route. These results encourage further research towards achieving vaccinal protection against the T. gondii in animals and humans.     

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 world's 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.     

A quantitative competitive PCR method to determine the parasite load in the brain of Toxoplasma gondii-infected mice

Efficacy of vaccine candidates against toxoplasmosis may be expressed in terms of reduction in cyst number in brains of animals vaccinated and then challenged with a cyst-forming strain of Toxoplasma gondii, compared to non-vaccinated animals. Cyst number generally has been determined by microscopic examination of brain homogenate samples, a technique which has a low sensitivity and is time-consuming. Here we describe a quantitative competitive PCR method, which allows quantifying T. gondii DNA in brain samples. The method uses a primer pair, which allows the amplification of a 301 bp fragment of the 35-fold repeated T. gondii B1 gene and an internal standard (non-homologous competitor) derived from phage lambda, which can be amplified using the same primers and whose size and G/C content are similar to that of the B1 target sequence. The method is sensitive (as few as 10 parasites can be quantified), reproducible, and is not affected by the presence of DNA extracted from mouse brain by means of a simple and rapid technique. It is suitable to quantify the parasite load in the brain of infected mice and to evaluate efficacy of toxoplasmosis vaccine candidates.     

Immunization against Toxoplasma gondii

The emergence of Toxoplasma gondii as a major opportunistic organism in immunocompromised individuals and the steady increase in economic losses due to animal toxoplasmosis have fuelled the interest in vaccine development. In this review, Fausto Araujo addresses aspects of vaccination against T. gondii with regard to the problem posed by this parasite to pregnant women and to severely immunocompromised individuals.     

Toxoplasma gondii α-amylase deletion mutant is a promising vaccine against acute and chronic toxoplasmosis

Individuals with inhibited immunity may develop lethal toxoplasmosis; thus, a safe and effective vaccine is urged to be developed. Toxoplasma gondii (T. gondii) α-amylase (α-AMY) is one of the enzymes responsible for starch digestion. In the present study, we first generated a ME49Δα-amy mutant and discovered that loss of α-AMY robustly grew in vitro but contributed to significant virulence attenuation in vivo. Therefore, we established a mouse model to explore the protective immunity of Δα-amy mutant against acute and chronic toxoplasmosis. The results indicated that the survival rates of short-term or long-term immunized mice re-infected with the tachyzoites of multiple T. gondii strains were nearly 100%. ME49Δα-amy not only could provide protective immunity against tachyzoites infection but also could resist the infection of tissue cysts. Furthermore, we detected that ME49Δα-amy vaccination could effectively eliminate the proliferation of parasites in mice and prevent the formation of cysts. The significant increases of Th1-type cytokines, Th2-type cytokines and specific total IgG and IgG subclasses (IgG2a and IgG1) confirmed efficiency of a combination of cellular and humoral immunity against infection. In conclusion, ME49Δα-amy attenuated strain can produce strong immune responses to provide efficient protection against toxoplasmosis, which signifies that ME49Δα-amy mutant may be a potential vaccine candidate.     

Molecular vaccines against parasites

Prophylactic vaccines can be expected to be one of the major practical outputs of parasitology research. Various groups within Australia have pursued the vaccine objective for several years, with particular emphasis on blood-stage falciparum malaria in man, intestinal helminths of sheep and cattle, cutaneous myiasis (blowfly strike) in sheep, cysticercosis in sheep and cattle, bovine babesiosis, and cattle ticks. Other vaccine programmes are concerned with giardiasis, filariasis, toxoplasmosis, fascioliasis, coccidiosis in poultry, cutaneous leishmaniasis and schistosomiasis japonica. For many years, the only available vaccine against a parasite in Australia has been the attenuated Babesia bovis vaccine produced by the Tick Fever Research Centre of the Queensland Department of Primary Industries. Strategies for achieving molecular vaccines are generally similar within the various research groups. They involve analysis of the immunology and immunochemistry of a model or in-vitro system; development of functional monoclonal antibodies; analysis of antibody specificities in clinically and/or functionally defined polyclonal sera; screening of cDNA or genomic expression libraries; peptide synthesis; identification of an appropriate vaccination schedule involving adjuvants or new recombinant DNA-based antigen delivery systems. Outlined below are five of the major vaccine programmes.     

Toxoplasma gondii: DNA vaccination with bradyzoite antigens induces protective immunity in mice against oral infection with parasite cysts

Infection of the host by Toxoplasma gondii leads to an acute systemic dissemination of tachyzoites, followed by a chronic phase, in which bradyzoites, enclosed in cysts, persist in the brain, the heart, and other tissues. Among putative vaccine candidates, the bradyzoite antigens BAG1 and MAG1 look promising since they are preferentially expressed during the chronic stage of the parasite. This work focused on studying the immunogenicity of bradyzoite antigens in a mouse model of chronic toxoplasmosis. A mixture of plasmids directing the cytoplasmic expression of MAG1 and BAG1 in mammalian cells was used to immunize mice. We show here that immunized mice developed, preferentially, specific anti-MAG1 and anti-BAG1 IgG2a subclass antibodies, indicating a shift towards a Th1-like response after DNA immunization. We then demonstrated that DNA immunization followed by challenge infection elicited effective protection in mice, suggesting that bradyzoite antigens should be considered in the design of vaccines against toxoplasmosis.     

Analysis of in vivo immune responses during Toxoplasma gondii infection using the technique of lymphatic cannulation

A major obstacle in the design of effective inactivated vaccines is the induction of an appropriate immune response in the recipient. Direct examination of immune responses induced in vivo following infection is possible using the technique of lymphatic cannulation. This allows a unique insight into the development of the immune response to particular pathogens and provides important information that could be exploited in the design of suitable vaccine delivery systems in order to induce and maintain appropriate protective immune responses. In this article, Elisabeth Innes and Jonathan Wastling review studies involving the use of lymphatic cannulation to examine immune responses induced in vivo during infection of sheep with the S48 strain of Toxoplasma gondii, which is currently used as a vaccine against toxoplasmosis for vetennary use only.     

Oral oocyst-induced mouse model of toxoplasmosis: effect of infection with Toxoplasma gondii strains of different genotypes, dose, and mouse strains (transgenic, out-bred, in-bred) on pathogenesis and mortality

Humans and other hosts acquire Toxoplasma gondii infection by ingesting tissue cysts in undercooked meat, or by food or drink contaminated with oocysts. Currently, there is no vaccine to prevent clinical disease due this parasite in humans, although, various T. gondii vaccine candidates are being developed. Mice are generally used to test the protective efficacy of vaccines because they are susceptible, reagents are available to measure immune parameters in mice, and they are easily managed in the laboratory. In the present study, pathogenesis of toxoplasmosis was studied in mice of different strains, including Human Leukocyte Antigen (HLA) transgenic mice infected with different doses of T. gondii strains of different genotypes derived from several countries. Based on many experiments, the decreasing order of infectivity and pathogenicity of oocysts was: C57BL/6 background interferon gamma gene knock out (KO), HLA-A*1101, HLA-A*0201, HLA-B*0702, Swiss Webster, C57/black, and BALB/c. Mice fed as few as 1 oocyst of Type I and several atypical strains died of acute toxoplasmosis within 21 days p.i. Some Type II, and III strains were less virulent. The model developed herein should prove to be extremely useful for testing vaccines because it is possible to accurately quantitate a challenge inoculum, test the response to different strains of T. gondii using the same preparations of oocysts which are stable for up to a year, and to have highly reproducible responses to the infection.     

Enhancement of protective immune response to recombinant Toxoplasma gondii ROP18 antigen by ginsenoside Re

Toxoplasma gondii, the etiological agent of toxoplasmosis, is an obligate intracellular protozoan parasite that infects a variety of mammals including humans. In an attempt to find new antigen-adjuvant combinations that enhance the immunogenicity of antigen candidates for toxoplasma vaccines, we analyzed the potent protection in mice immunized with recombinant protein ROP18 when co-administered with ginsenoside Re, a most important component isolated from Panax ginseng. All immunized mice produced specific anti-rROP18 immunoglobulins, with high levels of IgG antibody and a mixed IgG1/IgG2a response, with predominance of IgG1 production. The cellular and humoral immune responses were associated with the production of IFN-γ and IL-4 cytokines respectively. Vaccinated mice displayed a significantly increased survival time compared with control mice which died within 6 days of challenge with RH strain. Our data demonstrate that by addition of ginsenoside Re, the rROP18 triggered a stronger humoral and cellular response against T. gondii, and that Re is a promising vaccine adjuvant against toxoplasmosis, deserves further evaluation and development.     

Protective effect of vaccination with Toxoplasma lysate antigen and CpG as an adjuvant against Toxoplasma gondii in susceptible C57BL/6 mice

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems to both humans and livestock and of great economic impact worldwide. Oligodeoxynucleotides (ODN) which contain immunostimulatory CG motifs (CpG ODN) can promote Th1 responses, an adjuvant activity that is desirable for vaccination against intracellular pathogens. We investigated the feasibility of using CpG as an adjuvant combined with Toxoplasma lysate antigen (TLA) as a vaccine against toxoplasmosis. Genetically susceptible C57BL/6 mice were vaccinated with TLA with or without CpG ODN as an adjuvant and then challenged with 85 cysts of the moderately virulent RRA (Beverley) strain of T. gondii. Prior to challenge infection, immunization with TLA plus CpG ODN directed cellular and humoral immunity toward a Th1 pattern, characterized by enhanced INF gamma production by splenic cells in response to TLA, and enhanced production of toxoplasma-specific IgG and IgG (2a) antibodies. Consequently, CpG/TLA-treated mice showed prolonged survival and 64% reduction in brain parasite burden compared to non-CpG/TLA treated group. Our results suggest that CpG ODN would provide a stable and effective adjuvant for use in vaccination against toxoplasmosis.     

Toxoplasma MIC2 is a major determinant of invasion and virulence

Like its apicomplexan kin, the obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and uses a unique form of gliding motility. The recent identification of several transmembrane adhesive complexes, potentially capable of gripping external receptors and the sub-membrane actinomyosin motor, suggests that the parasite has multiple options for host-cell recognition and invasion. To test whether the transmembrane adhesin MIC2, together with its partner protein M2AP, participates in a major invasion pathway, we utilized a conditional expression system to introduce an anhydrotetracycline-responsive mic2 construct, allowing us to then knockout the endogenous mic2 gene. Conditional suppression of MIC2 provided the first opportunity to directly determine the role of this protein in infection. Reduced MIC2 expression resulted in mistrafficking of M2AP, markedly defective host-cell attachment and invasion, the loss of helical gliding motility, and the inability to support lethal infection in a murine model of acute toxoplasmosis. Survival of mice infected with MIC2-deficient parasites correlated with lower parasite burden in infected tissues, an attenuated inflammatory immune response, and induction of long-term protective immunity. Our findings demonstrate that the MIC2 protein complex is a major virulence determinant for Toxoplasma infection and that MIC2-deficient parasites constitute an effective live-attenuated vaccine for experimental toxoplasmosis.     

Congenital toxoplasmosis: epidemiologic features and control.

Toxoplasmosis is caused by the parasite Toxoplasma gondii. It is acquired from undercooked meat or from food or fomites contaminated by cat feces. The disease can be transmitted to the fetus only during maternal parasitemia, which is associated with primary infection. Extrapolation from current data suggests that there are 140 to 1400 cases of congenital toxoplasmosis per year in Canada and that 70 to 280 of the infants are severely affected at birth; many of the others suffer sequelae later in life. Serologic diagnosis of primary infection in the mother is quite sensitive and specific. Diagnosis in the infant is more difficult and may take several months. Prenatal treatment of the woman and postnatal treatment of the infant are hampered by the lack of proven efficacy as well as ethical and compliance problems. Preventive serologic screening and prophylaxis have the same drawbacks. Educating young women to avoid infection is an inexpensive, low-risk intervention that would be the preferred preventive strategy if it could be shown to be effective. Immunization may prove to be the most cost-effective method of preventing congenital toxoplasmosis if a safe and effective vaccine is developed.     

Leishmania amazonensis: inhibition of 3'-nucleotidase activity by Cu2+ ions

Toxoplasma gondii is an ubiquitous intracellular parasite, causative agent of toxoplasmosis, and a worldwide zoonosis for which an effective vaccine is needed. A group of proteins secreted by tachyzoites during host-cell invasion was isolated from the interaction medium. It induced the permeability of the cells as assessed by alpha-sarcin and consequently facilitated the entry of the parasite into the cells. SDS-PAGE of the purified proteins showed a pattern of four proteins of 67, 42, 32 and 27 kDa. MRC-5 cells incubated with the total protein and the different electroeluted bands endured a high cellular death in presence of alpha-sarcin. BALb/C mice immunized with the group of proteins had a mixed Th1/Th2 response and were protected upon challenge with the parasites.     

Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice

Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection.     

The History of Toxoplasma gondii—The First 100 Years

ABSTRACT. In this paper the history of Toxoplasma gondii and toxoplasmosis is reviewed. This protozoan parasite was first discovered in 1908 and named a year later. Its medical importance remained unknown until 1939 when T. gondii was identified in tissues of a congenitally infected infant, and veterinary importance became known when it was found to cause abortion storms in sheep in 1957. The discovery of a T. gondii specific antibody test, Sabin–Feldman dye test in 1948 led to the recognition that T. gondii is a common parasite of warm‐blooded hosts with a worldwide distribution. Its life cycle was not discovered until 1970 when it was found that felids are its definitive host and an environmentally resistant stage (oocyst) is excreted in feces of infected cats. The recent discovery of its common infection in certain marine wildlife (sea otters) indicates contamination of our seas with T. gondii oocysts washed from land. Hygeine remains the best preventive measure because currently there is no vaccine to prevent toxoplasmosis in humans.     

New insights in toxoplasmosis immunology during pregnancy. Perspective for vaccine prevention

Toxoplasma gondii is one of the few pathogens that can cross the placenta. Frequency and severity of transmission vary with gestational age. While acquired toxoplasmosis is already well explored, the control of maternal-foetal transmission of the parasite remains almost unknown. This is partly due to inherent inadequacies of animal models. This review summarises the studies which have been undertaken and shows that the mouse is a valuable model despite obvious differences to the human case. The paramount role of the cellular immune response during primary infection has been consistently shown. Surprisingly, IFN-g has a dual role in this process. While its beneficial effects in the control of toxoplasmosis are well known, it also seems to have transmission-enhancing effects within the placenta and can also directly harm the developing foetus. This shows the importance of designing vaccines which protects both mother and foetus. Therefore, it is useful to study the mechanisms of natural resistance against transmission during a secondary infection. In this setting, the process is more complicated, involving cellular, but also humoral components of the immune system. In summary, even if the whole process is far from being elucidated, important insights have been gained so far which will help us to undertake rational vaccine research.     

Identification of potential apicoplast associated therapeutic targets in human and animal pathogen Toxoplasma gondii ME49

Toxoplasma gondii ME49 is an obligatory intracellular apicomplexa parasite that causes toxoplasmosis in humans, domesticated and wild animals. Waterborne outbreaks of acute toxoplasmosis worldwide reinforce the transmission of Toxoplasma gondii ME49 to humans through contaminated water and may have a greater epidemiological impact than previously believed. In the quest for drug and vaccine target identification subtractive genomics involving subtraction between the host and pathogen genome has been implemented for enlisting essential pathogen specific proteins. Using this approach, our analysis on both human and Toxoplasma gondii ME49 reveals that out of 7987 protein coding sequences of the pathogen, 950 represent essential non human-homologous proteins. Subcellular localization prediction & comparative-biochemical pathway analysis of these essential proteins gives a list of apicoplast-associated proteins having unique pathogen-specific metabolic pathway. These apicoplast-associated enzymes involved in fatty acid biosynthesis pathway of Toxoplasma gondii ME49, may be used as potential drug targets, as the pathway is vital for the protozoan's survival. Structure prediction of drug target proteins was done using fold based recognition method. Screening of the functional inhibitors against these novel targets may result in discovery of novel therapeutic compounds that can be effective against Toxoplasma gondii ME49. ABBREVIATIONS: DEG - Database of Essential Gene, KEGG - Kyoto Encyclopaedia of Genes and Genomes, KAAS - KEGG Automated Annotation Server, PFP - Protein Function Prediction, COG - Cluster of Orthologous Genes.