Biochemical and biological characterization of the protective Leishmania pifanoi amastigote antigen P-8.

The Leishmania pifanoi amastigote antigen P-8 has been previously shown to induce protective immunity in a murine model of cutaneous leishmaniasis (L. Soong, S. M. Duboise, P. Kima, and D. McMahon-Pratt, Infect. Immun. 63:3559-3566, 1995). As this antigen is of interest for further vaccine studies, the biochemical characterization of P-8 was undertaken. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot analysis, and gel filtration chromatography revealed that P-8 antigen consisted of two proteoglycolipid complexes. The P-8 epitope is associated with the L. pifanoi amastigote-specific glycolipid components found in the two complexes. The P-8 complex 1 (P-8c1) consists of a 56-kDa serine metalloproteinase, apolipoprotein E (derived from fetal bovine serum), and amastigote-specific glycolipids. The P-8 complex 2 (P-8c2) consists of a 31-kDa cysteine proteinase associated with amastigote glycolipids. Biochemical analyses suggest that the P-8 antigenic glycolipids may be distinct from previously described Leishmania glycolipids (glycosylinositol-phospholipids and sphingoglycolipids). Protective immunity studies revealed that P-8c1 (serine metalloproteinase-glycolipid complex) confers comparable protection against infection as immunopurified P-8. The isolated P-8c2 (cysteine proteinase-glycolipid complex) does not provide significant protection, nor does stimulation with P-8c2 result in significant T-cell activation in P-8- or P-8c2-vaccinated mice. Consequently, the P-8c1 complex appears to be the immunodominant component of P-8.

Internalization of Leishmania mexicana complex amastigotes via the Fc receptor is required to sustain infection in murine cutaneous leishmaniasis.

We show here that maintenance of Leishmania infections with Leishmania mexicana complex parasites (Leishmania amazonensis and Leishmania pifanoi) is impaired in the absence of circulating antibody. In these studies, we used mice genetically altered to contain no circulating antibody, with and without functional B cells. This experimental design allowed us to rule out a critical role for B cell antigen presentation in Leishmania pathogenesis. In addition, we show that mice lacking the common gamma chain of Fc receptors (FcgammaRI, FcepsilonRI, and FcgammaRIII) are similarly refractory to infection with these parasites. These observations establish a critical role for antibody in the pathogenesis associated with infection by members of the L. mexicana complex.

Surface-targeted lysosomal membrane glycoprotein-1 (Lamp-1) enhances lysosome exocytosis and cell invasion by Trypanosoma cruzi.

To gain entry into non-phagocytic cells, Trypanosoma cruzi trypomastigotes recruit lysosomes to the host cell surface. Lysosome fusion at the site of parasite entry leads to the formation of a parasitophorous vacuole with lysosomal properties. Here, we show that increased expression of the lysosomal membrane glycoprotein Lamp-1 at the cell surface renders CHO cells more susceptible to trypomastigote invasion in a microtubule-dependent fashion. Mutation of critical residues in the lysosome-targeting motif of Lamp-1 abolished the enhancement of T. cruzi invasion. This suggests that interactions dependent on Lamp-1 cytoplasmic tail motifs, and not the surface-exposed luminal domain, modulate T. cruzi entry. Measurements of Ca2+-triggered exocytosis of lysosomes in these cell lines revealed an enhancement of beta-hexosaminidase release in cells expressing wild-type Lamp-1 on the plasma membrane; this effect was not observed in cell lines transfected with Lamp-1 cytoplasmic tail mutants. These results also implicate Ca2+-regulated lysosome exocytosis in cell invasion by T. cruzi and indicate a role for the Lamp-1 cytosolic domain in promoting more efficient fusion of lysosomes with the plasma membrane.

T cell responses to crude and defined leishmanial antigens in patients from the lower Amazon region of Brazil infected with different species of Leishmania of the subgenera Leishmania and Viannia.

Amazonian localized cutaneous leishmaniasis (LCL) is caused by parasites of the subgenera Leishmania and Viannia. Respectively, these parasites may cause diffuse cutaneous leishmaniasis (DCL) and mucocutaneous leishmaniasis (MCL). This, together with differing skin test responses, suggests some species-specificity in cell mediated immunity. In this study, T cell responses (proliferative and interferon-gamma) to crude and defined antigens were examined in paired samples pre and post chemotherapy. Untreated L. (L.) amazonensis LCL patients showed lower responses to crude leishmanial antigens than the L. (V.) spp. group. L. (V.) braziliensis antigen was a more potent stimulator of T cell responses than L. (L.) amazonensis antigen in all patient groups. Few positive responses were seen to the L. (L.) amazonensis glycoprotein GP46. A substantial proportion of LCL patients did respond to the L. (L.) pifanoi amastigote antigens A2, and the surface membrane glycoprotein P8. DCL patients were poor responders to all leishmanial antigens, except GP46. In contrast, MCL patients were good responders to all antigens except GP46 and A2. A significant rise in the response to P8 and A2 antigen was seen post treatment across all LCL and MCL patients, indicating that these antigens might provide suitable vaccine candidates.

Leishmania amastigote target antigens: the challenge of a stealthy intracellular parasite.

The development of a defined molecular vaccine against leishmaniasis involves the determination of candidate molecules that elicit protection against infection. As the amastigote stage is the developmental form found in the infected mammalian host, molecules specific to or upregulated in this stage represent potential antigenic vaccine targets. Diane McMahon-Pratt, Peter Kima and Lynn Soong summarize experiments which indicate that immunization with molecules upregulated in the amastigote stage can provide effective protection against infection. In the immunized host, both CD4(+) and CD8(+) T cells appear to be crucial to protection. Studies of antigen presentation of Leishmania-infected macrophages indicate that the amastigote stage can sequester endogenous leishmanial antigen from the major histocompatability complex (MHC) class II presentation pathway. However, evidence indicates that MHC class I presentation may be sustained in the infected macrophage. The effect of these findings on the design of a leishmanial vaccine are considered.

Presentation via the class I pathway by Leishmania amazonensis-infected macrophages of an endogenous leishmanial antigen to CD8+ T cells.

CD8+ T cells play a protective role in immunity to cutaneous leishmaniasis. However, it has been unclear how these cells execute this function, since results from several investigations attempting to demonstrate recognition of Leishmania-infected macrophages by CD8+ T cells have been contradictory. In this study, we report the generation of CD8+ T cell lines specific for GP46/M-2, a leishmanial Ag, previously shown to protectively immunize mice against a Leishmania amazonensis challenge. Using T cell cytolysis and IFN-gamma production to assess CD8+ T cell activation, we show that in addition to recognizing mammalian cells transfected with GP46/M-2, these CD8+ T cell lines also recognize macrophages infected with Leishmania amazonensis. MHC class I presentation of GP46/M-2 by infected macrophages can be blocked by treatment with brefeldin A and also by inhibitors of the cytosolic multicatalytic proteasome, N-acetyl-L-leucinyl-L-leucinal-L-norleucinal and N-acetyl-L-leucinyl-L-leucinylmethional. These results suggest that this leishmanial Ag is processed in the macrophage cytoplasm and is presented to CD8+ T cells via the classical pathway of MHC class I presentation. The relevance of these findings as they impact on our understanding of the biology of the parasite within the macrophage is discussed.

Leishmania-infected macrophages sequester endogenously synthesized parasite antigens from presentation to CD4+ T cells.

CD4+ T cell lines raised against the protective leishmanial antigens GP46 and P8 were used to study the presentation of endogenously synthesized Leishmania antigens by infected cells. Using two different sources of macrophages, the I4.07 macrophage cell line (H-2k) which constitutively expresses major histocompatibility complex (MHC) class II molecules, and elicited peritoneal exudate cells, we found that cells infected with Leishmania amastigotes presented little, if any endogenously synthesized parasite antigens to CD4+ T cells. In contrast, promastigote-infected macrophages did present endogenous parasite molecules to CD4+ T cells, although only for a limited time, with maximal presentation occurring within 24 h of infection and decreasing to minimal antigen presentation at 72 h post-infection. These observations suggest that once within the macrophage, Leishmania amastigote antigens are sequestered from the MHC class II pathway of antigen presentation. This allows live parasites to persist in infected hosts by evading the activation of CD4+ T cells, a major and critical anti-leishmanial component of the host immune system. Studies with drugs that modify fusion patterns of phagosomes suggest that the mechanism of this antigen sequestration includes targeted fusion of the parasitophorous vacuole with certain endocytic compartments.

Disruption of CD40-CD40 ligand interactions results in an enhanced susceptibility to Leishmania amazonensis infection.

To study the role of CD40 ligand (CD40L) in the host immune responses against intracellular pathogens, we infected CD40L knockout (CD40L-/-) mice with Leishmania amazonensis. Although wild-type mice were susceptible to infection and developed progressive ulcerative lesions, tissue parasite burdens in CD40L-/- mice were significantly higher. This heightened susceptibility to infection was associated with an impaired T cell and macrophage activation and altered inflammatory response, as reflected by low levels of IFN gamma, lymphotoxin-tumor necrosis factor (LT-TNF), and nitric oxide (NO) production. Furthermore, CD40L-/- mice failed to generate a protective immune response after immunization. These results indicate an essential role of cognate CD40-CD40L interactions in the generation of cellular immune responses against an intracellular parasite.

Leishmania pifanoi amastigote antigens protect mice against cutaneous leishmaniasis.

In the search for a leishmaniasis vaccine, extensive studies have been carried out with promastigote (insect stage) molecules. Information in this regard on amastigote (mammalian host stage) molecules is limited. To investigate host immune responses to Leishmania amastigote antigens, we purified three stage-specific antigens (A2, P4, and P8) from in vitro-cultivated amastigotes of Leishmania pifanoi by using immunoaffinity chromatography. We found that with Corynebacterium parvum as an adjuvant, three intraperitoneal injections of 5 micrograms of P4 or P8 antigen provided partial to complete protection of BALB/c mice challenged with 10(5) to 10(7) L. pifanoi promastigotes. These immunized mice developed significantly smaller or no lesions and exhibited a 39- to 1.6 x 10(5)-fold reduction of lesion parasite burden after 15 to 20 weeks of infection. In addition, P8 immunization resulted in complete protection against L. amazonensis infection of CBA/J mice and partial protection of BALB/c mice, suggesting that this antigen provided cross-species protection of mice with different H-2 haplotypes. At different stages during infection, vaccinated mice exhibited profound proliferative responses to parasite antigens and increased levels of gamma interferon production, suggesting that a Th1 cell-mediated immune response is associated with the resistance in these mice. Taken together, the data in this report indicate the vaccine potential of amastigote-derived antigens.

Immunity to erythrocytic stages of malarial parasites.

In those individuals who live in endemic areas, immunity to malaria is slow to develop and stage-specific. The nature and antigenic specificity of this response, which may involve components of both cell-mediated and humoral immunity, is not well understood. Rodent models provide useful systems to explore the spectrum of host responses that may contribute to resolution of erythrocytic-stage infection or possibly to pathogenesis. Moreover, these models allow identification of plasmodial molecules that can induce different types of host responses. Two different mouse model systems, Plasmodium yoelii yoelii and P. chabaudi adami are presented. These have been selected because resolution of infection by P. yoelii yoelii has been shown to require B cell-dependent mechanisms, while control of acute P. chabaudi adami infection can be achieved by T cell-dependent mechanisms. A monoclonal antibody that provides passive protection to P. yoelii challenge infection has been shown to recognize the cysteine-rich, carboxyl-terminal region of the merozoite surface protein-1. This region, obtained in an appropriate configuration from recombinant Escherichia coli, can induce significant protective immune responses in naive mice. In contrast, cell-mediated immune mechanisms make a major contribution to resolution of asexual-stage P. chabaudi adami infection. An empirical approach using continuous flow electrophoresis has identified several low molecular weight plasmodial proteins that can induce partial protective responses in susceptible hosts. These observations are briefly discussed with respect to human malaria.

Proteins with molecular masses of 25 to 40 kilodaltons elicit optimal protective responses against Plasmodium chabaudi adami infection.

The presence of the CD4+ T cell has been shown to be crucial for resolution of acute infection in the Plasmodium chabaudi adami murine malaria model. This model is, therefore, suitable for the isolation of malaria antigens that are capable of activating protective T cells. In light of this, we set out to identify P. chabaudi adami molecules that activate protective responses in this model. Denatured P. chabaudi adami proteins were isolated by continuous-flow electrophoresis on the basis of their apparent molecular masses and then sequentially assessed for the ability to protect mice in immunization experiments. We report here that low-molecular-mass P. chabaudi adami polypeptides in the range from 25 to 40 kDa are most effective at immunizing mice against a challenge infection with viable P. chabaudi adami. The method used to obtain these proteins could also be applied to identify molecules that activate protective cell-mediated responses in other infectious disease models.

Occurrence of a 6S intermediate form of the progesterone receptor that is sensitive to ribonuclease.

Steroid receptors exist in cytosol as 9S, non-DNA-binding species and as 4S (transformed) species that bind to DNA or nuclei. Labeling the progesterone receptor from rabbit uterine cytosol with [3H]progesterone in the presence of 10 mM sodium molybdate revealed a 9S species on sucrose gradient centrifugation. Without molybdate, the receptor sedimented as an intermediate species of 6S, which converted to 4S in 0.3 M NaCl. The 6S species could also be generated from the 4S species by dialysis. Dilution of the same 4S species gave only partial re-aggregation with 50% of the receptor remaining as 4S. Dialysis appeared to retain the association of a macromolecular aggregation factor present in cytosol. Serum did not seem to be the source of the aggregation factor, as perfusion of the uterine vasculature before excision did not affect the S value of the receptor. We tested whether RNA was involved by treating receptor with RNase A (100 micrograms/400 microliters cytosol). While the molybdate-stabilized cytosol receptor (9S) was unaffected, RNase A partially (50%) converted the 6S form of receptor to 4S. RNase A also partially converted the re-aggregated form back to 4S. Protease inhibitors had no effect on this action of RNase. Formation of receptor-ribonucleotide protein particles may play a role in steroid action in the cell.

Evidence for a single steroid-binding protein in the rabbit progesterone receptor.

The rabbit uterine progesterone receptor copurifies as two molecular weight (Mr) forms of about 105,000 and 78,000. To investigate whether these are different proteins, we have used protease digestion, reversible denaturation, and photoaffinity labeling in studies on the steroid-binding domain of the receptor. Digestion of the Mr 105,000 and 78,000 forms, photoaffinity labeled with [3H]R5020, with Staphylococcus aureus V8 protease revealed identical peptide fragments of Mr 43,000, 39,000, and 27,000-30,000. When receptor in cytosol was denatured, separated by electrophoresis, and then reconstituted, [3H]progesterone bound specifically to a single form at about Mr 105,000. After partial purification, the reversible denaturation procedure revealed both the larger and the smaller progesterone-binding species similar to the photoaffinity-labeled species in this preparation. Receptor in uterine cytosol prepared under mild conditions appeared as a predominant large molecular weight form on photoaffinity labeling with [17 alpha-methyl-3H]R5020, [6,7-3H]R5020, or [3H]RU27987. Further purification of this cytosol showed the generation of a smaller labeled species. These results from three different approaches reinforce the view that the rabbit progesterone receptor contains a single steroid-binding protein.

Characterization of calciphorin, the low molecular weight calcium ionophore, from rat liver mitochondria.

Calciphorin, the putative mitochondrial calcium ionophore from rat liver mitochondria, exhibits the inherent properties of the mitochondrial calcium transport system and is similar to the calf heart preparation reported earlier. The protein has a strong selectivity for Ca2+, and has a Kd for Ca2+ of 56.5 +/- 6.6 microM and 13.9 +/- 2.1 microM in organic extraction and flow dialysis experiments, respectively. Reduction of the contaminating lipids from 23 +/- 6.5 to 1.73 +/- 0.4 moles per mole protein does not alter the affinities, Ca2+/protein stoichiometry or selectivity for Ca2+.