Mucosal immunity in Toxoplasma gondii infection.

Toxoplasma gondii is an intracellular parasite that frequently infects a large spectrum of warm-blooded animals. This parasite induces abortion and establishes both chronic and silent infections, particularly in the brain. Parasite penetration into the host activates a strong anti-parasite immune response. In the present paper, we will discuss the interplay between innate and adaptive immunity that occurs within the infected intestine to clear the parasite and to maintain intestinal homeostasis despite the exacerbation of an inflammatory immune response.

Dynamics and responsiveness of T-lymphocytes in secondary lymphoid organs of rabbits developing immunity to Eimeria intestinalis.

Primary infection with Eimeria intestinalis confers very effective immunity against further infections in rabbits. This study was designed to determine the onset of the immune response in primary-infected rabbits and to characterise the immune status of protected rabbits. Variations in kinetics of CD4+ and CD8+ T-cell subpopulations were followed after primary infection at the intestinal sites of penetration (duodenum) and development (ileum), in mesenteric lymph nodes (MLN) and in the spleen. The response against the parasite was measured by specific lymphocyte proliferation in the spleen and MLN and by determining specific IgG titres in serum. The mucosal immune response was strong after primary infection and was characterised by (i) transient increase in the percentages of intestinal CD4+ lymphocytes and MLN CD8+ lymphocytes 14 days PI and (ii) strong increase in the percentages of intestinal CD8+ lymphocytes from 14 days PI persisting throughout further infections. Extensive infiltration of the lamina propria with CD8+ lymphocytes was observed 14 days PI. The specific proliferative response started between 7 and 14 days PI in MLN but remained undetectable in spleens for up to 21 days, in contrast to "immunised" rabbits. The fact that systemic immune responses were low after primary infection, in contrast to indicators of mucosal immune responsiveness, suggests that protection of rabbits against E. intestinalis infection is due to an effective mucosal immune response, and that systemic responses that increase after successive infections are only reflections of repeated encounters with parasite antigens.

Anti-SAG1 peptide antibodies inhibit the penetration of Toxoplasma gondii tachyzoites into enterocyte cell lines.

The initial attachment of Toxoplasma tachyzoites to the target host cell is an important event in the life-cycle of the parasite and a critical stage in infection. Previous studies have shown that polyclonal antibodies directed against the major surface antigen of Toxoplasma gondii (SAG1) inhibit the infection of enterocyte cell lines. Here, we demonstrate that antibodies raised against a central peptide (V41T) of SAG1 and the SAGI protein itself are able to inhibit the infection of various cell lines by the tachyzoites. Antibodies directed against SAG1 peptides were used to define a site on the SAGI antigen that interacts with the host cell. The epitope carried by V41T was identified on the tachyzoite surface by immunofluorescence. The peptide sequence seems to be conserved in all the members of the SAGI Related Sequence family (SRS). Using undifferentiated and differentiated Caco-2 cells, we found that tachyzoites enter preferentially via the basolateral side of the cell. These findings highlight the role of the SRS family members in the mediation of host cell invasion.

Murine ileitis after intracellular parasite infection is controlled by TGF-beta-producing intraepithelial lymphocytes.

BACKGROUND & AIMS: Acute inflammatory ileitis occurs in susceptible (C57BL/6) mice after oral infection with Toxoplasma gondii. Overproduction of interferon (IFN)-gamma and synthesis of nitric oxide mediate the inflammation. We evaluated the role of transforming growth factor (TGF)-beta produced by intraepithelial lymphocytes (IELs) in this process. METHODS: We analyzed the histologic and immunologic consequences of adoptive transfer of antigen-primed IELs into susceptible mice treated with anti-TGF-beta before oral challenge with T. gondii cysts. An in vitro coculture of enterocytes and IELs assessed the production of chemokines and cytokines in the presence of anti-TGF-beta. RESULTS: Antigen-primed IELs prevent acute ileitis in susceptible mice that is reversed with anti-TGF-beta. Resistant mice (CBA/J) develop ileitis after treatment with anti-TGF-beta. Antigen-primed IELs can induce systemic immunosuppression as measured by depressed IFN-gamma production. In vitro, primed IELs reduce the production of inflammatory chemokines by infected enterocytes and IFN-gamma by splenocytes. CONCLUSIONS: Regulation of the ileal inflammatory process resulting from T. gondii is dependent on TGF-beta-producing IELs. The IELs are an essential component in gut homeostasis after oral infection with this parasite.

Intranasal immunization with SAG1 and nontoxic mutant heat-labile enterotoxins protects mice against Toxoplasma gondii.

Effective protection against intestinal pathogens requires both mucosal and systemic immune responses. Intranasal administration of antigens induces these responses but generally fails to trigger a strong protective immunity. Mucosal adjuvants can significantly enhance the immunogenicities of intranasally administered antigens. Cholera toxin (CT) and heat-labile enterotoxin (LT) are strong mucosal adjuvants with a variety of antigens. Moreover, the toxicities of CT and LT do not permit their use in humans. Two nontoxic mutant LTs, LTR72 and LTK63, were tested with Toxoplasma gondii SAG1 protein in intranasal vaccination of CBA/J mice. Vaccination with SAG1 plus LTR72 or LTK63 induced strong systemic (immunoglobulin G [IgG]) and mucosal (IgA) humoral responses. Splenocytes and mesenteric lymph node cells from mice immunized with LTR72 plus SAG1, but not those from mice immunized with LTK63 plus SAG1, responded to restimulation with a T. gondii lysate antigen in vitro. Gamma interferon and interleukin 2 (IL-2) production by splenocytes and IL-2 production by mesenteric lymph node cells were observed in vitro after antigen restimulation, underlying a Th1-like response. High-level protection as assessed by the decreased load of cerebral cysts after a challenge with the 76K strain of T. gondii was obtained in the group immunized with LTR72 plus SAG1 and LTK63 plus SAG1. They were as well protected as the mice immunized with the antigen plus native toxins. This is the first report showing protection against a parasite by using combinations of nontoxic mutant LTs and SAG1 antigen. These nontoxic mutant LTs are now attractive candidates for the development of mucosally delivered vaccines.

In vitro activity of the polyether ionophorous antibiotic monensin against the cyst form of Toxoplasma gondii.

Toxoplasma gondii. The experiments were conducted in vitro using 2 methods; cysts produced either in mice or in cell culture were exposed to monensin in vitro, and the infectivity of the parasites was then assessed in vivo or in vitro. The data obtained from these 2 systems of evaluation showed that monensin inhibits the infectivity and the viability of the bradyzoites. Its activity was time and concentration dependent. The first effects were observed at very low drug concentrations (i.e. 0.0001 microg/ml). Immunofluorescence and electron microscopy analysis showed significant cytological alterations of the monensin-treated bradyzoites: they were swollen, had a large number of vacuoles in their cytoplasm and were found lysed at higher concentrations in ionophore.

Induction of protective immunity against toxoplasmosis in mice by DNA immunization with a plasmid encoding Toxoplasma gondii GRA4 gene.

GRA4 is a dense granule protein of Toxoplasma gondii that is a candidate for vaccination against this parasite. We have inserted the entire coding sequence of GRA4 into an eukaryotic expression vector to determine whether DNA immunization can elicit protective immune response to T. gondii. Susceptible C57BL/6 mice were then vaccinated intramuscularly with GRA4 DNA and orally challenged with a lethal dose of 76 K T. gondii strain cysts. Immunization with pGRA4 resulted in a 62% survival of C57BL/6 infected mice. Mice immunized with GRA4 DNA developed high levels of serum anti-GRA4 immunoglobulin G antibodies as well as a cellular immune response, as assessed by splenocyte proliferation, in response to recombinant GRA4 protein restimulation in vitro. The cellular immune response was associated with IFN-gamma and IL-10 synthesis, suggesting a modulated Th1-type response. Splenocyte proliferation was strongly enhanced and protection slightly higher by inoculation with GRA4 DNA combined with a granulocyte-macrophage colony-stimulating factor expressing vector. This is the first report that demonstrates the establishment of a DNA vaccine-induced protective immunity against the acute phase of T. gondii infection.

Intranasal immunization with Toxoplasma gondii SAG1 induces protective cells into both NALT and GALT compartments.

Intranasal (i.n.) immunization with the SAG1 protein of Toxoplasma gondii plus cholera toxin (CT) provides protective immunity. The aim of this study was to analyze the cellular activation of several mucosal compartments after i.n. immunization. Cervical and mesenteric lymph node (CLN and MLN, respectively) lymphoid cell and intraepithelial lymphocyte (IEL) passive transfer experiments were performed with CBA/J mice immunized i.n. with SAG1 plus CT. CLN and MLN cells and IEL isolated 42 days after immunization conferred protective immunity on naive recipient mice challenged with strain 76K T. gondii, as assessed by the reduction in the number of brain cysts. There were proliferative specific responses in nose-associated lymphoid tissue and the CLN and MLN cells from mice immunized with SAG1 plus CT, but no cytokine was detectable. Thus, protective immunity is associated with a specific cellular response in the nasal and mesenteric compartments after i.n. immunization.

Nasal immunization of mice with human papillomavirus type 16 (HPV-16) virus-like particles or with the HPV-16 L1 gene elicits specific cytotoxic T lymphocytes in vaginal draining lymph nodes.

Human papillomavirus type 16 (HPV-16) infects the genital tract and is closely associated with the development of cervical cancer. HPV-16 initiates infection at the genital mucosal surface; thus, mucosal immune responses are likely to contribute to defense against HPV-16 infection. However, little information is available regarding the induction of immune responses in the genital tract mucosa. In this study, we evaluated the potential of intranasally administered papillomavirus vaccines to elicit both systemic and vaginal immune responses. HPV-16 virus-like particles (VLPs) produced by self-assembly of L1 protein and the HPV-16 L1 gene cloned into a mammalian expression vector were used as vaccines. Intranasally administered VLPs induced serum immunoglobulin G (IgG) and vaginal IgA secretory antibodies. Very weak serum IgG and vaginal IgA responses were found after DNA immunization. Both splenic and vaginal lymphocytes could be activated by intranasal immunization with VLPs and the HPV-16 L1 gene. Activated CD4(+) Th1-like T cells were shown to synthesize gamma interferon, and activated CD8(+) T cells were demonstrated to be cytotoxic.

Protection of kids against Cryptosporidium parvum infection after immunization of dams with CP15-DNA.

In this study the effectiveness of a DNA vaccine to confer protection against cryptosporidiosis, an enteric infection of lifestock and humans, was evaluated. A vaccination protocol using a recombinant plasmid encoding the 15 kDa surface sporozoite protein of Cryptosporidium parvum was developed in adult pregnant goats. The present study reports that nasal immunization of pregnant goats with CP15-DNA led to a transfer of immunity to offspring conferring protection against C. parvum infection. Kids from CP15-DNA-vaccinated dams shed significantly fewer oocysts and over a shorter period than did kids from unvaccinated goats. The low level of parasite development in protected kids did not affect their growth whereas unprotected kids grew much slowly. There was still a significant difference in the weights of protected and unprotected kids after complete recovery. Anti-CP15 antibodies were present in serum and colostrum from vaccinated goats. Nevertheless, the precise immune mechanism of protection has still to be determined. This vaccine should reduce the economic losses due to cryptosporidiosis in ruminants, specially in small ruminants (calves, lambs, kids). It has also the potential to reduce environmental contamination by reducing oocyst shedding.

Intraepithelial lymphocytes traffic to the intestine and enhance resistance to Toxoplasma gondii oral infection.

Toxoplasma gondii Ag-primed intraepithelial lymphocytes (IEL) from the mouse intestine have been shown to be protective against an lethal parasite challenge when adoptively transferred into recipient mice. In the present study, we observed that Ag-primed IEL traffic to the intestine of naive mice following i.v. administration. Primed and CD8beta+ IEL were the most efficient cells at homing to the host organ. In congenic mice, IEL migrated from intestine within several hours posttransfer. On Ag reexposure, the primed IEL return to the intestine where they enhance resistance as determined by reduction in the number of brain cysts. Treatment of recipient mice with anti-alpha4 and anti-alphaE Abs partially inhibited IEL intestinal homing. The Ab treatment dramatically impaired resistance to a subsequent oral infection. These finding indicate that lymphocyte homing is an important parameter in establishing long term immunity to recurrent infection with this parasite.

Gut-derived intraepithelial lymphocytes induce long term immunity against Toxoplasma gondii.

Intraepithelial lymphocytes (IEL) of the intestine represent an important barrier in the prevention of infection against orally acquired pathogens. Adoptive transfer of Ag-primed IEL into a naive host can protect against challenge. Using a murine model, we demonstrate in two genetically distinct mouse strains (C57BL/6 and CBA/J) that protective IEL can be isolated at specific times after oral infection with cysts containing bradyzoites. Adoptive transfer of IEL obtained from the intestine of infected mice at these specific times can provide long term protection, as determined by mortality and cyst number against challenge. The protective IEL appear to be CD8+, TCR-alpha/beta and are at least partially dependent upon the presence of TCR-gamma/delta T cells in the host. Endogenous production of the pivotal cytokine, IFN-gamma, is essential for host immunity. These findings demonstrate that gut-derived IEL represent a potentially important mechanism to provide long term immunity to the host.

Murine dendritic cells pulsed in vitro with Toxoplasma gondii antigens induce protective immunity in vivo.

The activation of a predominant T-helper-cell subset plays a critical role in disease resolution. In the case of Toxoplasma gondii, the available evidence indicates that CD4(+) protective cells belong to the Th1 subset. The aim of this study was to determine whether T. gondii antigens (in T. gondii sonicate [TSo]) presented by splenic dendritic cells (DC) were able to induce a specific immune response in vivo and to protect CBA/J mice orally challenged with T. gondii cysts. CBA/J mice immunized with TSo-pulsed DC exhibited significantly fewer cysts in their brains after oral infection with T. gondii 76K than control mice did. Protected mice developed a strong humoral response in vivo, with especially high levels of anti-TSo immunoglobulin G2a antibodies in serum. T. gondii antigens such as SAG1 (surface protein), SAG2 (surface protein), MIC1 (microneme protein), ROP2 through ROP4 (rhoptry proteins), and MIC2 (microneme protein) were recognized predominantly. Furthermore, DC loaded with TSo, which synthesized high levels of interleukin-12 (IL-12), triggered a strong cellular response in vivo, as assessed by the proliferation of lymph node cells in response to TSo restimulation in vitro. Cellular proliferation was associated with gamma interferon and IL-2 production. Taken together, these results indicate that immunization of CBA/J mice with TSo-pulsed DC can induce both humoral and Th1-like cellular immune responses and affords partial resistance against the establishment of chronic toxoplasmosis.

Mapping of B epitopes in GRA4, a dense granule antigen of Toxoplasma gondii and protection studies using recombinant proteins administered by the oral route.

GRA4, a dense granule protein of Toxoplasma gondii elicits both mucosal and systemic immune responses following oral infection of mice with cysts. We studied the antigenicity and immunogenicity of truncated and soluble forms of GRA4 expressed as glutathione S-transferase fusion proteins in Escherichia coli. Protein C (amino-acids 297-345) was particularly well recognized by serum IgG antibodies, milk IgA antibodies and intestinal IgA antibodies from T. gondii infected mice and by serum IgG antibodies from T. gondii infected humans and T. gondii infected sheep. One major B epitope was localized within the last 11 C-terminal residues of GRA4. A second epitope, recognized with lower frequency, was mapped within the region 318-334. In contrast, the N domain of GRA4 (amino acids 25-276) was poorly recognized. Oral immunization of C57BL/6 mice with N, C or NC (amino acids 25-276 fused to 297-345) in association with cholera toxin induced a significant production of serum anti-GRA4 IgG antibodies but a weak and inconsistent intestinal anti-GRA4 IgG antibody response and afforded partial resistance to oral infection with T. gondii. These results provide new molecular and immunological understanding of GRA4 and indicate that it is a potential candidate for oral vaccination against T. gondii.

Some Opportunistic Parasitic Infections in AIDS: Candidiasis, Pneumocystosis, Cryptosporidiosis, Toxoplasmosis.

Almost 80% of patients with AIDS die from infections other than human immunodeficiency virus (HIV). These infections usually occur late in the course of disease when CD4(+) T-cell count has fallen below 200 permm(3) cells per milliliter. Most of these infections are caused by organisms that do not normally afflict healthy individuals and are thus considered to be opportunistic. In this article, Lloyd Kasper and Dominique Buzoni-Gatel review the host-parasite interaction for four important pathogens: Candida albicans and Pneumocystis carinii (usually non-invasive pathogens), Cryptosporidium parvum (invades the cells but remains localized in the gut) and Toxoplasma gondii (penetrates through the gut to cause systemic infection). These organisms, which generally cause limited or even insignificant clinical evidence of infection in the normal host, were chosen because of their high prevalence in AIDS patients and because they exhibit different invasive abilities. The reason why individuals with AIDS are susceptible to this particular group of pathogens is uncertain.

Adoptive transfer of gut intraepithelial lymphocytes protects against murine infection with Toxoplasma gondii.

Intraepithelial lymphocytes (IEL) of the gut represent a primary immune barrier against infection by orally acquired pathogens. Naturally acquired infection with Toxoplasma gondii induces the proliferation of CD8+ T cells in both the gut and spleen. Gut-derived CD8alpha/beta+ IEL exhibit MHC-restricted cytotoxicity against parasite-infected enterocytes and macrophages. In a murine model, we demonstrate that the adoptive transfer of IEL obtained from inbred mice at day 11 postinfection is able to protect against a virulent challenge in syngenic recipients. In CBA mice, the parasite cyst load within the brain of the recipients receiving primed IEL was reduced by 90%. In BALB/c and C57BL/6 mice, a 50% decrease in mortality was observed following adoptive transfer of primed IEL. To determine the T cell subset responsible for protective immunity, a purified CD8alpha/beta+ IEL population was isolated from infected mice at day 11 postinfection. These cells were able to protect naive mice by adoptive transfer against a lethal parasite challenge. RNA analysis by reverse-transcriptase PCR revealed that primed CD8alpha/beta+ IEL produce significant message for IFN-gamma, an essential cytokine for host protection against toxoplasmosis. Administration of anti-IFN-gamma at the time of adoptive transfer of primed IEL abrogated protection. The adoptive transfer of these protective IEL was not restricted to the Ld class I locus. These data demonstrate that IFN-gamma-producing IEL may be an important primary barrier against acute and perhaps recurrent infection with T. gondii.

Cell mediated immunity induced in mice by HPV 16 L1 virus-like particles.

Recombinant human papillomavirus (HPV) type 16 L1 virus-like particles (VLPs) expressed in the baculovirus system were used to investigate the cellular immune response to human papillomavirus type 16. The cell-mediated immune response was evaluated through immunization of mice with HPV 16 L1 virus-like particles using a lymphoproliferation assay and cytokine production and cytometric analysis of lymphocyte subsets. A significant proliferative response was observed which was associated with secretion of both interferon-gamma and interleukin-2. FACS analysis of splenic lymphocytes revealed that CD8+ T-cells were increased in the immunized mice. These results demonstrate that HPV 16 L1 VLPs induce a T-cell response characterized by a Th1 profile and confirm that the HPV 16 VLP is a reasonable candidate for vaccine development.

Differences in immunological response to a T. gondii protein (SAG1) derived peptide between two strains of mice: effect on protection in T. gondii infection.

This study presents the analysis of the immunogenicity, antigenicity and protective effects of a peptide derived from the major surface antigen of Toxoplasma gondii, SAG1. This synthetic peptide carrying three predicted H-2k restricted T cell epitopes was used to immunize mice. The protective effect of the peptide was evaluated in CBA/J and C57BL/6 mice using the decrease in brain cyst load as evidence of protection. Immunization of C57BL/6 mice yielded high antibody titres but had no protective effect after oral challenge. Immunized CBA/J, mice which responded with a lower titre, showed a 35% reduction in cyst burden after oral challenge. Both strains yielded antibodies which recognized the cognate SAG1 protein on immunoblot assay. Using the BIAcore, system, it was shown that at lower titres the CBA/J mouse sera recognized the native SAG1 protein more effectively than the C57BL/6 mouse sera, yielding much higher anti-peptide titres. Lymphoproliferation assays using the peptide experimentally confirmed the predicted T-cell epitopes and showed that they were also recognized by cells of T. gondii infected mice. The anti-peptide subclass analysis suggested a Th1 orientation in CBA/J mice, whereas a Th2 orientation was observed in C57BL/6 mice. Finally, fine analysis of sequences recognized under MHC class I indicated the existence of a T-cell epitope in the H-2k haplotype (CBA/J mice) but not in the H-2b haplotype (C57BL/6 mice). This study provides a structural basis to the understanding of the vaccination response to one of the T. gondii antigens in different strains of mice.