Toxoplasma gondii: evaluation of an intranasal vaccine using recombinant proteins against brain cyst formation in BALB/c mice

The purpose of this work was to evaluate protective activity against brain cyst formation in BALB/c mice intranasally vaccinated with recombinant proteins from Toxoplasma gondii. The recombinant proteins rROP2, rGRA5 and rGRA7 were used in vaccine preparation. Thirty-three female mice were divided into three groups, these animals received two doses by intranasal route at days 0 and 21 as follows; group 1 (G1, n=11) received 12.5 microg of each recombinant protein plus 0.5 microg of cholera toxin, group 2 (G2, n=11) received phosphate buffer saline (PBS) plus 0.5 microg of cholera toxin, and group 3 (G3, n=11) received PBS only. At challenge day (day 33) three animals from each group were euthanatized for IgA measure from intestine. Mice were infected orally with 50 cysts from the VEG strain at day 33. At challenge day the G1 animals had high immunoglobulin A levels, however, they only showed IgG antibody titers against rROP2 and rGRA7. Animals from G1 also exhibited strong resistance to cyst formation compared with the control group (G3, P<0.05). However, we did not observe differences in protection against brain cyst formation between G1 and G2 (P>0.1). These results indicate that intranasal immunization in BALB/c mice with recombinant proteins rROP2, rGRA5 and rGRA7 associated with cholera toxin induced partial protection, when compared with G3, against tissue cyst formation after oral infection with tissue cysts from T. gondii.     

Toxoplasma gondii: protective immunity against experimental toxoplasmosis induced by a DNA vaccine encoding the perforin-like protein 1

Toxoplasma gondii is an important zoonotic parasite infecting about one third of the world population, causing congenital infections and eye disease. T. gondii perforin-like protein 1 (TgPLP1) is believed to be involved in the acute virulence of T. gondii in mice, and is therefore of interest as a vaccine candidate. In this study, we constructed a DNA vaccine expressing TgPLP1, and evaluated the immune response in Kunming mice. The gene sequence encoding TgPLP1 was inserted into the eukaryotic expression vector pVAX I, and Kunming mice were immunized intramuscularly with the plasmid. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally with 1 × 10³ tachyzoites of the virulent T. gondii RH strain. The results showed that pVAX/TgPLP1 alone or with pVAX/IL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/IL-18 significantly increased the production of IFN-γ and IL-2. Further, challenge experiments showed that co-immunization of pVAX/TgPLP1 with pVAX/IL-18 significantly (P < 0.05) increased survival time (12.7 ± 1.2 days) of immunized mice, compared with pVAX/TgPLP1 alone (11.3 ± 0.9 days). These results demonstrate that TgPLP1 is a potential vaccine candidate against toxoplasmosis, worth further evaluation in other animal hosts. IL-18 could enhance the immune effect of TgPLP1, prolonging the survival time of immunized mice.     

Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ～98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer-sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.     

Mechanisms of vaccine-induced protective immunity against Coxiella burnetii infection in BALB/c mice

To elucidate the mechanisms of vaccine-induced protective immunity against Coxiella burnetii infection, we compared the protective efficacy and immunogenicity between formalin-inactivated phase I vaccine (PI-V) and phase II vaccine (PII-V) in BALB/c mice. PI-V generated significant protection while PII-V did not confer measurable protection. Analysis of cytokine and subclass Ab responses indicated that both PI-V and PII-V were able to induce a Th1-dominant immune response but did not identify the component of host response that distinguished their ability to induce protective immunity. Interestingly, immunoblot analysis identified a difference between PI-V and PII-V vaccinates in antigenic recognition by specific Ab isotypes. The observation that PI-LPS elicited significant protection but PII-LPS did not confer measurable protection suggests PI-LPS may play a key role in PI-V-induced protection. Adoptive transfer of either immune sera or splenocytes mediated significant protection in naive BALB/c mice, supporting the notion that both humoral and cellular immunity are important for development of protective immunity. However, the evidence that immune sera and B cells were unable to control infection while T cells conferred significant protection in SCID mice supports the hypothesis that T cell-mediated immunity is critical for host defense against C. burnetii infection. This report presents novel evidence to highlight the importance of PI-LPS and Abs in protective immunity and has important implications for the design of new generation vaccines against Q fever.     

Toxoplasma gondii: chimeric Dr fimbriae as a recombinant vaccine against toxoplasmosis

Toxoplasma gondii is responsible for fetopathy in farm animals and humans and severe disease in immunocompromised individuals (i.e. AIDS patients). Effective vaccines, inducing protective and long-lasting immunity to this global parasite, are still desired. In the work, we evaluated the immunogenic and immunoprotective activity of Escherichia coli chimeric Dr fimbriae bearing selected antigenic epitopes of three T. gondii antigens (SAG1, GRA1 and MAG1), in comparison with conventional recombinant antigens obtained in E. coli expression system. Our data demonstrate a very high protective efficacy of recombinant antigens supplemented with Freund's adjuvants, whereas chimeric Dr fimbriae as a vaccine proved non-protective. The recombinant antigen vaccine induced a strong specific antibody response and prevented the brain cysts formation by 89%. The results are promising and should be confirmed in further study on farm animals by use of less aggressive than Freund's adjuvant preparations.     

A recombinant pseudorabies virus expressing TgSAG1 protects against challenge with the virulent Toxoplasma gondii RH strain and pseudorabies in BALB/c mice

The major immunodominant surface antigen 1 (TgSAG1) of invasive tachyzoites is a vaccine candidate antigen for Toxoplasma gondii. In this study, we developed a recombinant pseudorabies virus (PRV) expressing TgSAG1 (rPRV/SAG1) based on the PRV vaccine strain Bartha K-61 by homologous recombination, in which partial PK and gG genes were deleted. The growth assay of rPRV/SAG1 showed that the recombinant virus can replicate in vitro as efficiently as PRV Bartha K-61, demonstrating that insertion of the TgSAG1 gene in the PK and gG locus of PRV does not affect the replication of PRV. All mice vaccinated with rPRV/SAG1 developed a high level of specific antibody responses against T. gondii lysate antigen (TLA), a strong increase of the splenocyte proliferative response, and significant levels of IFN-gamma and IL-2 production. And the immunization of mice with rPRV/SAG1 elicited strong cytotoxic T lymphocyte (CTL) responses in vitro. These results demonstrate that rPRV/SAG1 could induce significant humoral and cellular Th1 immune responses. Moreover, rPVR/SAG1 immunization induced partial protection (60%) against a lethal challenge with the highly virulent T. gondii RH strain, and neutralizing antibodies against PRV in a BALB/c mouse model. These results suggest that expression of protective antigens of T. gondii in PRV Bartha K-61 is a novel approach towards the development of a vaccine against both animal toxoplasmosis and pseudorabies.     

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.     

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.     

Combination of CpG-oligodeoxynucleotides with recombinant ROP2 or GRA4 proteins induces protective immunity against Toxoplasma gondii infection

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN) have been characterized as Th1-promoting immunopotentiators, an adjuvant activity desirable for vaccination against intracellular parasites like Toxoplasma gondii. In an attempt to find new antigen–adjuvant combinations that enhance the immunogenicity of antigen candidates for toxoplasma vaccines, we analyzed the extent of protection in mice immunized with ROP2 and GRA4 recombinant proteins when co-administered with CpG-ODN. Both GRA4 + CpG-ODN and ROP2 + CpG-ODN formulations were shown to induce a strong humoral Th1-biased response characterized by a high IgG_2a to IgG₁ antibody ratio. Both vaccination regimens led to increased secretion of IFN-γ and IL-10, and negligible amounts of IL-4, upon specific re-stimulation of spleen cells from these groups of mice. After a non-lethal challenge with tissue cysts of a moderately virulent strain, only the brains from mice vaccinated with ROP2 or GRA4 in combination with CpG-ODN showed a significant reduction (63% and 62%, respectively) in their parasite load compared to the controls. The rate of protection obtained with GRA4 + ROP2 + CpG-ODN resulted equivalent (66%) to those achieved with the single antigens plus CpG-ODN. Taken together, these results indicate that CpG-ODN is an important candidate adjuvant for use in potential multicomponent anti-T. gondii vaccines for animals and humans.     

Immunization with MIC1 and MIC4 induces protective immunity against Toxoplasma gondii

Host cell invasion by Toxoplasma gondii is tightly coupled to the apical release of micronemal proteins (MIC). In this work, we evaluated the protective effect encountered in C57BL/6 mice immunized with MIC1 and MIC4 purified from soluble tachyzoite antigens by affinity to immobilized lactose. The immunized mice presented high serum levels of IgG1 and IgG2b specific antibodies. MIC1/4-stimulated spleen cells from immunized mice produced IL-2, IL-12, IFN-gamma, IL-10, but not IL-4, suggesting the induction of a polarized Th1 type immune response. When orally challenged with 40 cysts of the ME49 strain, the immunized mice had 68% fewer brain cysts than the control mice. Immunization was associated with 80% survival of the mice challenged with 80 cysts, contrasting with 100% mortality of the non-immunized mice in the acute phase. In this phase, there was much lower parasitism in the lungs and small intestine of the immunized mice, and they did not exhibit the early-stage signs of intestinal necrosis, which was clearly detected in the control mice. Our data demonstrate that MIC1 and MIC4 triggered a protective response against toxoplasmosis, and that these antigens are targets for the further development of a vaccine.     

Oral vaccination with recombinant Lactobacillus plantarum encoding Trichinella spiralis inorganic pyrophosphatase elicited a protective immunity in BALB/c mice

BackgroundTrichinellosis is a serious zoonotic disease distributed around the world. It is needed to develop a safe, effective and feasible anti-Trichinella vaccine for prevention and control of trichinellosis. The aim of this study was to construct a recombinant Lactobacillus plantarum encoding Trichinella spiralis inorganic pyrophosphatase (TsPPase) and investigate its immune protective effects against T. spiralis infection.Methodology/Principal findingsThe growth of recombinant L. plantarum was not affected by TsPPase/pSIP409-pgsA′ plasmid, and the recombinant plasmid was inherited stably in bacteria. Western blot and immunofluorescence assay (IFA) indicated that the rTsPPase was expressed on the surface of recombinant L. plantarum. Oral vaccination with rTsPPase induced higher levels of specific serum IgG, IgG1, IgG2a and mucosal secretory IgA (sIgA) in BALB/c mice. ELISA analysis revealed that the levels of IFN-γ and IL-4 released from spleen, mesenteric lymph nodes and Peyer’s patches were evidently increased at 2–4 weeks following vaccination, compared to MRS (De Man, Rogosa, Sharpe) medium control group (P < 0.05). Immunization of mice with rTsPPase exhibited a 67.18, 54.78 and 51.91% reduction of intestinal infective larvae, adult worms and muscle larvae at 24 hours post infection (hpi), 6 days post infection (dpi) and 35 dpi, respectively (P < 0.05), and the larval molting and development was significantly inhibited by 45.45% at 24 hpi, compared to the MRS group.ConclusionsTsPPase plays a crucial role in T. spiralis molting and development, oral vaccination with rTsPPase induced a significant local mucosal sIgA response and systemic Th1/Th2 immune response, and immune protection against T. spiralis infection in BALB/c mice.     

香菇多糖对急性弓形虫感染小鼠Th应答调节作用的实验研究

目的 探讨香菇多糖(Lentinan,Lent)对急性弓形虫感染小鼠BALB/c Th1/Th2免疫应答调节效应的影响.方法 对RH强毒株感染的BALB/c小鼠进行不同时间点的香菇多糖预处理,动态观察用药后各组感染小鼠的生存率;在感染后第0、3、5、8和10天提取小鼠的脾细胞,ELISA法检测脾细胞培养上清中IFN-γ和IL-10的分泌水平.结果 感染前6 d 1 mg/kg LNT用药组与药物未处理组相比显著提高了生存率;显著增强了Th1免疫应答中关键细胞因子IFN-γ和Th2免疫应答中关键细胞因子IL-10的分泌水平.结论 对急性弓形虫感染的BALB/c小鼠采用香菇多糖预处理之后能有效激发Th1/Th2型免疫应答的有效建立,对于抵抗弓形虫感染具有重要意义.     

Molecular cloning of ribosomal P protein in Toxoplasma gondii and the availability to detect antibody against recombinant protein in toxoplasmosis patients

Among the panel of monoclonal antibodies (mAb) against Toxoplasma gondii, mAb of Tg621 (Tg621) clone blotted 38 kDa protein which localized in the cytoplasm of tachyzoites by immunofluorescence microscopy. The protein was not released into the parasitophorous vacuole during or after invasion. The cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg621. The full length cDNA sequence was completed with 5'-RACE as 1,592 bp, which contained open reading frame of 942 bp. The deduced amino acid sequence of Tg621 consisted of a polypeptide of 313 amino acids, with significant homology to ribosomal P proteins (RPP) of other organisms especially high to those of apicomplexan species. The expressed and purified TgRPP was assayed in western blot with the sera of toxoplasmosis patients and normal sera, which resulted in the 74.0% of positive reactions in toxoplasmosis patients whereas 8.3% in normal group. Therefore, the antibody formation against TgRPP in toxoplasmosis patients was regarded as specific for T. gondii infection and suggested a potential autoantibody.     

Protection against toxoplasmosis in mice immunized with different antigens of Toxoplasma gondii incorporated into liposomes.

Different toxoplasma antigens were entrapped within liposomes and evaluated, in this form, for their ability to protect Swiss mice against toxoplasma infection: soluble tachyzoite antigen (L/TAg), tissue cyst (L/CAg), tachyzoite plus tissue cyst (L/TCAg) or purified antigen of tachyzoite (L/pTAg). The protein used in L/pTAg was purified from tachyzoites using a stage-specific monoclonal antibody which reacted at a molecular weight of 32 kD in SDS PAGE and silver stain using reduced condition. To compare the immuno-adjuvant action of liposomes and of Freund's Complete Adjuvant (FCA), another group of mice was immunized with soluble tachyzoite antigen (STAg) emulsified in FCA (FCA/TAg). Control groups were inoculated with (STAg) alone, phosphate-buffered saline (PBS), FCA with PBS (FCA/PBS) and empty liposomes (L/PBS). Mice were inoculated subcutaneously with these antigens six, four and two weeks before a challenge with 80 tissue cysts of the P strain of Toxoplasma gondii orally. All mice immunized with or without adjuvant showed a humoral response, as measured by Elisa. However, no correlation was found between antibody titer and protection against the challenge. All mice immunized with L/pTAg or L/TCAg survived (100), whereas 80% and 90% of mice from groups which received respectively PBS or FCA/PBS and L/PBS died. All mice immunized with antigens entrapped within liposomes (L/TAg, L/CAg, L/TCAg and L/pTAg) showed low numbers of intracerebral cysts.     

IL-17 and IL-22 elicited by a DNA vaccine encoding ROP13 associated with protection against Toxoplasma gondii in BALB/c mice

Toxoplasma gondii, an intracellular parasitic protozoan, is capable of infecting man and all warm-blooded animals. Cell-mediated immunity is vital in mounting protective responses against T. gondii infection. Recent studies have shown that T-helper (Th) 17 responses may play a key role in parasite control. In this current study, we constructed a DNA vaccine encoding T. gondii ROP13 in a pcDNA vector. Groups of BALB/c mice were immunized intramuscularly with pcROP13 or controls and challenged with the RH strain of T. gondii. The results showed that immunization with pcROP13 could elicit an antibody response against T. gondii. The expression of the canonical Th17 cytokines, interleukin (IL)-17 and IL-22, were significantly increased after immunization with pcROP13 compared with control groups ( p < 0.05). Furthermore, vaccination resulted in a significant decrease in parasite load ( p < 0.05). The induction of Th17 related cytokines, using a ROP13 DNA vaccine, against T. gondii should be considered as a potential vaccine approach for the control of toxoplasmosis.     

Temporal development of protective cell-mediated and humoral immunity in BALB/c mice infected with Brucella abortus

In BALB/c mice infected i.v. with attenuated strain 19 of Brucella abortus, the organism replicates to high numbers in the spleen and reaches peak concentrations at 2 wk postinfection (p.i.). The infection is then progressively cleared so that by 8 wk p.i. numbers of bacteria have decreased 10,000 fold or more. Passive transfer assays were performed with T cell-enriched spleen cells and serum of donor mice infected 2, 3, 4, 5, 6, or 8 wk previously. Antibodies conferred significant protection to recipients at and after 3 wk p.i., whereas protection by T cells was not evident until 4 wk p.i. The combined transfer of serum and cells enhanced protection over that provided by serum or cells alone when transfers were made before, but not after, challenge infection. Protection conferred by T cell-enriched spleen cells of 6-wk donors was unaffected by the presence of equal quantities of cells from 3-wk donors, but was abrogated by the removal of both CD4 and CD8 T cell subsets. Experiments with purified CD4 and CD8 subsets revealed that cell-mediated protection resided at equivalent levels in both subsets. Daily treatment of mice with Cyclosporin A for 4 wk after infection caused some increase in numbers of brucellae in spleens and livers. Although immune responses of treated animals were markedly suppressed, there was little effect of treatment on numbers of macrophages in the spleen, on enhanced killing of Listeria monocytogenes in the spleen, or on the nature and intensity of splenic and hepatic inflammatory responses. These data indicate that acquired resistance to infection with B. abortus in mice is the result of independent, and probably also interactive, effects of antibodies and T effector cells of both CD4 and CD8 phenotypes. The initial decline in bacterial numbers in the spleen, which occurred in the absence of detectable cell-mediated immunity in that organ, could probably be ascribed principally to effects of antibodies and to nonimmune stimuli responsible for increased formation, attraction, and activation of macrophages.     

The role of host-derived heat-shock protein in immunity against Toxoplasma gondii infection

Heat-shock proteins (HSPs) are evolutionarily highly conserved polypeptides synthesized by cells to preserve cellular functions under a variety of stressful conditions, including infections. In infections, both host cells and pathogens express HSPs, although the role of these molecules in the host-pathogen relationship is elusive. Here, Hajime Hisaeda and Kunisuke Himeno show that a correlation exists between the 65 kDa HSP molecule (HSP65) and protection against Toxoplasma gondii infection, suggesting that this protein contributes to the host defense system. These findings may help in the understanding of the complicated host-parasite relationship.     

Class Ia MHC-deficient BALB/c mice generate CD8+ T cell-mediated protective immunity against Listeria monocytogenes infection

CD8(+) T cells are required for protective immunity against intracellular pathogens such as Listeria monocytogenes. In this study, we used class Ia MHC-deficient mice, which have a severe reduction in circulating CD8(+) T cells, to determine the protective capacity of class Ib MHC-restricted T cells during L. monocytogenes infection. The K(b-/-)D(b-/-) mutation was backcrossed onto a C.B10 (BALB/c congenic at H-2 locus with C57BL/10) background, because BALB/c mice are more susceptible to Listeria infection than other commonly studied mouse strains such as C57BL/6. C.B10 K(b-/-)D(b-/-) mice immunized with a sublethal dose of L. monocytogenes were fully protected against a subsequent lethal infection. Adoptive transfer of Listeria-immune splenocyte subsets into naive K(b-/-)D(b-/-) mice indicated that CD8(+) T cells were the major component of this protective immune response. A CD8(+) T cell line isolated from the spleen of a Listeria-infected class Ia MHC-deficient mouse was shown to specifically recognize Listeria-infected cells in vitro, as determined by IFN-gamma secretion and cytotoxicity assays. Adoptive transfer of this T cell line alone resulted in significant protection against L. monocytogenes challenge. These results suggest that even a limited number of class Ib MHC-restricted T cells are sufficient to generate the rapid recall response required for protection against secondary infection with L. monocytogenes.     

Trichinella spiralis: characterization of phage-displayed specific epitopes and their protective immunity in BALB/c mice

Trichinellosis is a global zoonosis mainly caused by Trichinella spiralis. We have previously reported that a novel Ts87 gene from the cDNA library of adult T. spiralis was cloned and expressed in a prokaryotic expression system. Vaccination with recombinant Ts87 protein (rTs87) induced a muscle larvae burden reduction in BALB/c mice by 29% in response to T. spiralis infection. In the present study, we screened a random phage-displayed peptide library using monoclonal antibody 5A3 which recognized Ts87 protein. Four positive phage clones were selected to subcutaneously immunize BALB/c mice without adjuvant. Two phage clones could effectively stimulate specific antibodies against rTs87. Mice vaccinated with these two combined phage clones showed a 28.7% worm burden reduction as compared to the control group. Therefore, the identified phage clones displayed peptides representing specific epitopes of Ts87 protein and could be considered as potential vaccine candidates for T. spiralis.     

Early diagnosis of congenital toxoplasmosis in newborn infants using IgG subclasses against two Toxoplasma gondii recombinant proteins

The aim of this work was to evaluate the utility of ELISA-based testing of total IgG (IgGt) antibodies and its subclasses (IgG1, IgG2, IgG3 and IgG4) against soluble (STAg) and recombinant (rSAG1 and rMIC3) antigens of Toxoplasma gondii for diagnosing congenital toxoplasmosis. Sera from 217 newborns initially testing positive for specific IgM in filter paper dried blood spots were tested for specific IgM and IgG by ELFA-VIDAS. Congenital toxoplasmosis was confirmed in 175 and ruled out in 42 infants. The validity of the ELISA tests was determined using the persistence of IgG antibodies (ELFA-VIDAS kit) at the end of 12 months, which is considered the reference test for the diagnosis of congenital toxoplasmosis. The frequency of positivity with IgGt against STAg, rSAG1 and rMIC3 was found in 97.2%, 96.3% and 80.2%, respectively, of the newborns with confirmed congenital toxoplasmosis. IgG1 reacted with all three antigens, while IgG3 and IgG4 reacted preferentially with rMIC3. Higher mean values of reactivity (sample optical density/cut-off) were found for all subclasses when using rMIC3. All of the antigens showed high sensitivity and low specificity in detecting anti-T. gondii IgGt and IgG1 and low sensitivity and high specificity in detecting IgG3 and IgG4. In conclusion, the combined detection of IgG antibody subclasses against recombinant toxoplasmic antigens may be useful for the early diagnosis of congenital toxoplasmosis.