Evaluation of immune responses and protection induced by A2 and nucleoside hydrolase (NH) DNA vaccines against Leishmania chagasi and Leishmania amazonensis experimental infections

Several antigens have been tested as vaccine candidates against Leishmania infections but controversial results have been reported when different antigens are co-administered in combined vaccination protocols. Immunization with A2 or nucleoside hydrolase (NH) antigens was previously shown to induce Th1 immune responses and protection in BALB/c mice against Leishmania donovani and L. amazonensis (A2) or L. donovani and L. mexicana (NH) infections. In this work, we investigated the protective efficacy of A2 and NH DNA vaccines, in BALB/c mice, against L. amazonensis or L. chagasi challenge infection. Immunization with either A2 (A2-pCDNA3) or NH (NH-VR1012) DNA induced an elevated IFN-gamma production before infection; however, only A2 DNA immunized mice were protected against both Leishmania species and displayed a sustained IFN-gamma production and very low IL-4 and IL-10 levels, after challenge. Mice immunized with NH/A2 DNA produced higher levels of IFN-gamma in response to both specific recombinant proteins (rNH or rA2), but displayed higher IL-4 and IL-10 levels and increased edema and parasite loads after L. amazonensis infection, as compared to A2 DNA immunized animals. These data extend the characterization of the immune responses induced by NH and A2 antigens as potential candidates to compose a defined vaccine and indicate that a highly polarized type 1 immune response is required for improvement of protective levels of combined vaccines against both L. amazonensis and L. chagasi infections.     

Leishmania major: nature of immunity induced by immunization with a mutagenized avirulent clone of the parasite in mice

A chemically mutagenized avirulent form of Leishmania major was used to immunize BALB/c and C57B1/6 mice against challenge with virulent L. major. Immunity was elicited when the avirulent parasite was injected intravenously or intraperitoneally, but not subcutaneously. In fact, the latter route of immunization sometimes resulted in exacerbation of a subsequent infection with virulent L. major. Mice immunized with avirulent L. major developed upon challenge with virulent L. major cutaneous lesions which were significantly smaller and contained substantially fewer parasites than lesions on control nonimmune animals. Finally, the protection conferred by immunization with avirulent L. major could be adoptively transferred with T cells of the CD4+ lineage but not the CD8+ lineage.     

Leishmania amazonensis: humoral response to amastigote excreted-antigens in murine leishmaniasis

With the purpose of studying the antigenic role that factors excreted by Leishmania amastigotes might have during murine infection, immunoblots were carried out with sera from C57BL/6 and BALB/c mice infected with two strains of Leishmania (L.) amazonensis, NR and IFLA/BR. Both strains differ widely in virulence in BALB/c mice. BALB/c but not C57BL/6 sera recognized several excretion products. The excreted antigens showed a strong response towards IgG1 and IgG2a isotypes whilst they reacted only weakly against IgG2b and IgG3. A low-molecular weight antigen (about 20 kDa) excreted by both Leishmania strains was strongly recognized by IgG1 from BALB/c mice sera infected with IFLA/BR, the most virulent strain. Sera from NR infected mice were incapable of recognizing this antigen in spite of its presence in NR excreted products. The results indicate that the humoral immune response to excreted antigens of amastigotes depends on both the host genetic background and the parasite strain.     

Leukotrienes are essential for the control of Leishmania amazonensis infection and contribute to strain variation in susceptibility

Leukotrienes (LTs) are known to be produced by macrophages when challenged with Leishmania, but it is not known whether these lipid mediators play a role in host defense against this important protozoan parasite. In this study, we investigated the involvement of LTs in the in vitro and in vivo response to Leishmania amazonensis infection in susceptible (BALB/c) and resistant (C3H/HePAS) mice. Pharmacologic or genetic deficiency of LTs resulted in impaired leishmanicidal activity of peritoneal macrophages in vitro. In contrast, addition of LTB4 increased leishmanicidal activity and this effect was dependent on the BLT1 receptor. LTB4 augmented NO production in response to L. amazonensis challenge, and studies with a NO synthesis inhibitor revealed that NO was critical for the enhancement of macrophage leishmanicidal activity. Interestingly, macrophages from resistant mice produced higher levels of LTB4 upon L. amazonensis challenge than did those from susceptible mice. In vivo infection severity, as assessed by footpad swelling following s.c. promastigote inoculation, was increased when endogenous LT synthesis was abrogated either pharmacologically or genetically. Taken together, these results for the first time reveal an important role for LTB4 in the protective response to L. amazonensis, identify relevant leishmanicidal mechanisms, and suggest that genetic variation in LTB4 synthesis might influence resistance and susceptibility patterns to infection.     

Evaluation of the Lon-deficient Salmonella strain as an oral vaccine candidate

We evaluated the efficacy of CS2022 (the Lon protease-deficient mutant strain of Salmonella enterica serovar Typhimurium) as a candidate live oral vaccine strain against subsequent oral challenge with a virulent strain administered to BALB/c and C57BL/6 mice. CS2022 persistently resided in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum of both strains of mice after a single oral inoculation with 1 x 10(8) colony-forming units. Finally, CS2022 almost disappeared from each tissue sample by week 12 in BALB/c mice, whereas CS2022 still resided in each tissue type at week 12 after inoculation of C57BL/6 mice. A significant increase in the serovar Typhimurium lipopolysaccharide-specific secretory immunoglobulin A (s-IgA), as measured for one of the mucosal immune responses, was detected in bile and intestinal samples of both strains of immunized mice at week 4 after immunization. In addition, the expression of gamma interferon mRNA in the spleens of both strains of immunized mice, especially those of C57BL/6 mice, was significantly increased at week 4 after immunization and was boosted during the following 5 days after the challenge was administered to the mice. Furthermore, peritoneal macrophages isolated from immunized mice at week 4 after immunization exhibited an increase in intracellular killing activity against both virulent and avirulent Salmonella. The present results suggested that salmonellae-specific s-IgA on the mucosal surfaces induced by immunization with CS2022 generally prevented mice from succumbing to an oral challenge with a virulent strain. Simultaneously, CS2022 promoted the protective immunity associated with macrophages in both strains of mice.     

Protection against Leishmania major infection by oligomannose-coated liposomes

Liposomes coated with neoglycolipids constructed with mannopentaose and dipalmitoylphosphatidylethanolamine (Man5-DPPE) have been shown to induce cellular immunity against antigens encapsulated in the liposomes. To assess whether these neoglycolipid-coated liposomes can elicit protective immune response against challenge infection, effects of immunization with soluble leishmanial antigens encapsulated in the liposomes were evaluated using Leishmania major infection in susceptible BALB/c mice. Intraperitoneal immunization of mice with leishmanial antigens in the Man5-DPPE-coated liposomes significantly suppressed footpad swelling in comparison to the control, non-immunized mice, while progression of the disease was observed in mice administered antigens in uncoated liposomes and those administered soluble antigens alone, as seen with control mice. Similarly, the number of parasites decreased substantially in local lymph nodes of mice immunized with the antigen in the Man5-DPPE-coated liposomes. Protection against L. major infection in the immunized mice also coincided with an elevated ratio of antigen-specific IgG2a/IgG1 antibodies, which is a profile of T helper-type 1-like immune response. Taken together, these results indicate the possibility that Man5-DPPE-coated liposome-encapsulated antigens could serve as a vaccine that triggers protection against infectious disease.     

Efficacy of the vaccination of C57BL/6 mice against infection with different species of Leishmania

Antigens were isolated from lysates of promastigotes of Leishmania infantum by electro-elution from polyacrylamide gels. Antigens with respective molecular weights for F2 = 94-67 kd; F5 = 30-20 kd and F6 below 20 kd, were injected intravenously in C 57 BL/6 mice. The immune sera were studied by indirect immunofluorescence; an in vivo test showed their inhibitory effect on the life cycle of several Leishmania species from the Old and the New world. Furthermore, mice immunized with F2, F5 or F6 were protected against an infection by Leishmania major. These results demonstrate that vaccination is efficient in mice differing genetically for either susceptibility (BALB/c) or partial resistance (C 57 BL/6) to Leishmania infections. Recently, we reported that a single monoclonal antibody raised against Leishmania infantum can prevent the development of Leishmania major and Leishmania mexicana amazonensis. Indeed, BALB/c mice injected subcutaneously with promastigotes pre-treated with this monoclonal antibody, did not present any cutaneous lesions over a period of 3 months. Using a mouse in vivo model--intraperitoneal injection of TG 180 mouse sarcoma cells along with monoclonal antibody pre-treated promastigotes--such an antibody-mediated inhibition was also observed against Old and New World Leishmania species. Protective monoclonal antibodies recognized by immunoblotting technique three antigenic fractions (40, 70 and 113 kd) common to several Leishmania species. Antigenic preparations from Leishmania infantum extracts were isolated by electro-elution from polyacrylamide gels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leishmania major: immune response in BALB/c mice immunized with stress-inducible protein 1 encapsulated in liposomes

Protection against leishmaniasis is depending upon generation of a Th1 type of immune response. Field trials of first generation Leishmania vaccine showed a limited efficacy even with multiple doses mainly due to lack of an appropriate adjuvant. In this study, susceptible BALB/c mice were immunized with rLmSTI1 encapsulated in liposomes to explore the extent of protection induced by Leishmania antigen encapsulated in the liposomes against challenge with Leishmania major. The results showed that s.c. immunization of BALB/c mice with liposomal rLmSTI1 induced a significant protection against challenge and a significant lower parasite burden in spleen up to 14 weeks after challenge. The protected animals showed a significantly smaller footpad thickness after challenge, and a higher level of anti-SLA IgG antibodies before and after challenge with a predominant IgG2a titer. The data supports the possibility of using liposomal Leishmania antigens as a vaccine.     

TGF-beta-associated enhanced susceptibility to leishmaniasis following intramuscular vaccination of mice with Leishmania amazonensis antigens

Leishmania amazonensis and Leishmania braziliensis are the main causal agents of anergic diffuse cutaneous leishmaniasis and hyperergic mucosal leishmaniasis in man, respectively. In this work we demonstrate that intramuscular vaccination of BALB/c mice with whole antigens of L. amazonensis (LaAg) but not L. braziliensis (LbAg) results in increased susceptibility to cutaneous leishmaniasis. LaAg vaccination resulted in an increased capacity of the draining lymph nodes to produce IL-10 and TGF-beta during antigen recall responses. In vitro cultivation with LaAg but not LbAg induced increased apoptosis of CD8+ T cells. Following infection with L. amazonensis, LaAg-vaccinated mice produced significantly more TGF-beta and a higher serum IgG1/IgG2a antibody ratio compared with LbAg-vaccinated and non-vaccinated animals. The association of TGF-beta with enhanced susceptibility to infection was confirmed in mice co-vaccinated with LaAg and neutralizing anti-TGF-beta antibodies. Upon parasite challenge, these animals developed much smaller lesion sizes and parasite burdens, comparable with non-vaccinated controls. The disease-promoting effect of LaAg vaccination is not a general event, as in contrast to BALB/c, the disease outcome in C57Bl/6 mice was unaltered. Together, these findings indicate that species-specific components of L. amazonensis activate overt TGF-beta production that predisposes more susceptible individuals to aggravated disease following vaccination.     

T cells from Leishmania major-susceptible BALB/c mice have a defect in efficiently up-regulating CXCR3 upon activation

Genetic background influences the outcome of Leishmania major infection. C57BL/6 mice mount a Th1 response and resolve infection. In contrast, BALB/c mice mount a Th2 response and develop chronic lesions. This susceptible phenotype is seen even though BALB/c mice generate IFN-gamma-producing T cells at proportions similar to C57BL/6 mice in their lymph nodes (LN) early after infection. We had previously shown that chemokine receptor CXCR3 mediates immunity against L. major by recruiting IFN-gamma-producing T cells to the lesions of C57BL/6 mice. Therefore, we hypothesized that IFN-gamma-secreting T cells in BALB/c mice are unable to confer protection because they may be defective in up-regulating CXCR3. To test this hypothesis, we analyzed kinetics of CXCR3-expressing T cells in the LN and lesions of BALB/c and C57BL/6 mice during L. major infection. Additionally, we compared the ability of T cells from BALB/c and C57BL/6 mice to up-regulate CXCR3 upon activation. We found that resolution of L. major infection in C57BL/6 mice was associated with an increase in the proportion of CXCR3(+) T cells in regional LN and lesions, whereas disease progression in BALB/c mice was associated with a decrease in these populations. Anti-CD3/CD28-activated T cells from naive BALB/c but not C57BL/6 mice were defective in up-regulating CXCR3. Impaired induction of CXCR3 on BALB/c T cells was not due to lack of IFN-gamma and was mediated partially by IL-10 but not IL-4 or IL-13. We propose that defective CXCR3 up-regulation on T cells in BALB/c mice may contribute to L. major susceptibility.     

Lack of signaling by IL-4 or by IL-4/IL-13 has more attenuating effects on Leishmania amazonensis dorsal skin--than on footpad-infected mice

Lesion development in tegumentary leishmaniasis is markedly influenced by the inoculation site and the type and number of injected infective forms. This and the yet unclear contribution of Th2 cytokines as susceptibility factors to Leishmania amazonensis infection prompted us to investigate the roles of IL-4, IL-13 and IL-10 on C57BL/6 and BALB/c mice infected in the footpad (paw) or rump with low-dose L. amazonensis purified-metacyclics. Wild-type (WT) mice of either strain developed, in the rump, a single large ulcerated lesion whereas paw lesions never ulcerated and were much smaller in C57BL/6 than in BALB/c mice. However, rump-inoculated IL-4-deficient (IL-4(-/-)) C57BL/6 mice did not develop any visible lesions although parasites remained in the dermis and lymph nodes, even after systemic IL-10-receptor blocking. By comparison, all IL-4(-/-) BALB/c mice developed rump ulcers. Strikingly, only 30% of rump-infected IL-4Rα(-/-) BALB/c mice developed lesions. IL-4(-/-) mice had higher IFN-γ and lower IL-10 and IL-13 levels than WT mice. Paw-infected IL-4Rα(-/-) BALB/c mice developed minimal paw lesions. While other factors contributing to L. amazonensis susceptibility cannot be discounted, our results indicate that absent signalling by IL-4 or by IL-4/IL-13 have more intense attenuating effects on rump than on paw lesions but do not eradicate parasitism.     

Strain-dependent migration of lymphocytes to the vaginal mucosa after peripheral immunization

We have previously demonstrated a genetic predisposition among mice regarding their ability to be protected against vaginal candidiasis after peripheral immunization. Both BALB/c and (BALB/cx C57BL/6) F1 mice are protected against vaginal candidiasis after subcutaneous immunization with Candida albicans extract and C57BL/6 mice are not protected by this immunization. In the present study, the ability of F1-derived immune cells to transfer protection to naive parental strains was observed in BALB/c recipient mice, but not apparent in B6 recipient mice. This result is highly suggestive that the microenvironment of the B6 mouse is responsible for the susceptible phenotype. Genetic studies using (BALB/cx C57BL/6)F1x C57BL/6 backcross mice demonstrated that two genes appeared to regulate the protective effect of peripheral immunization to vaginal challenge. Microsatellite mapping indicated that candidate loci involved in controlling the immune response to vaginal candidiasis after peripheral immunization included the intercellular adhesion molecule-1 (ICAM-1), the Icam-1 related sequence 1, and the Fc epsilon RII (P<0.01). Thus, the ability of cells to bind to vaginal endothelial cells may play an important role in protection against vaginal candidiasis mediated by peripheral immunization.     

Protective immune response against cutaneous leishmaniasis by prime/booster immunization regimens with vaccinia virus recombinants expressing Leishmania infantum p36/LACK and IL-12 in combination with purified p36

In susceptible mice Leishmania infection triggers a CD4(+) Th2 response that has been correlated with evasion of the host immune system. To develop approaches that might trigger a Th1 response leading to protection against Leishmania we generated vaccinia virus recombinants (VVr) expressing the relevant p36/LACK protein of Leishmania infantum (VVp36) or co-expressing p36/LACK and interleukin-12 (VVp36IL12). Susceptible BALB/c mice were immunized with the VVr in various prime/booster protocols that included purified p36/LACK protein, followed 3 weeks later by a challenge with live L. major promastigotes. The course of the infection was monitored by measuring lesion development, parasite load and immunological parameters (IFN-gamma and IL-10 secretion by in vitro-stimulated lymphocytes, and specific IgG isotypes), before and after challenge. We found protocols of prime/booster immunization (VVp36/VVp36; VVp36IL12/p36; p36/VVp36IL12) that elicited different levels of protection in infected animals. The protocol of priming with purified p36 followed by a booster with VVp36IL12 induced 52% reduction in lesion size and a two-log unit reduction in parasite load. This partial protection correlated with activation of a specific Th1 type of immune response. These protocols could be of interest in the prophylaxis against Leishmania spp. and other parasitic diseases.     

A role for Lyt-2+ T cells in resistance to cutaneous leishmaniasis in immunized mice

The role of Lyt-2+ T cells in immunologic resistance to cutaneous leishmaniasis was analyzed by comparing infection patterns in resistant C57BL/6 mice and susceptible BALB/c mice induced to heal their infections after sub-lethal irradiation or i.v. immunization, with similar mice treated in vivo with anti-Lyt-2 antibodies. Administration of anti-Lyt-2 mAb resulted in a dramatic reduction in the number of lymphoid cells expressing the Lyt-2+ phenotype. Such treatment led to enhanced disease in both resistant C57BL/6 and irradiated BALB/c mice, as assessed by lesion size, but did not affect the capacity of these mice to ultimately resolve their infections. In contrast, anti-Lyt-2 treatment totally blocked the induction of resistance in i.v. immunized mice. These results suggest, that Lyt-2+ T cells may play a role in immunity to a Leishmania major infection and that their relative importance to resistance may depend on how resistance is induced.     

Low and high-dose intradermal infection with Leishmania major and Leishmania amazonensis in C57BL/6 mice

A model of skin infection with Leishmania amazonensis with low doses of parasites is compared to infection with high doses of L. amazonensis and low and high doses of Leishmania major. C57BL/6 mice were infected with 103 or 10(6) parasites in the ear and the outcome of infection was assessed. The appearance of lesions in mice infected with 103 parasites was delayed compared to mice infected with 10(6) Leishmania and parasites were detectable at the infection site before lesions became apparent. Mice infected with L. amazonensis displayed persistent lesions, whereas infection with L. major spontaneously healed in all groups, although lymphocytes persisted at the site of infection after healing. Macrophages persisted only in L. amazonensis-infected mice. High-dose L. amazonensis-infected mice produced lower levels of IFN-γ and TNF than mice infected with L. major. No correlation between the persistence of parasites and IL-10 levels and the production of nitric oxide or urea by macrophages was found. We conclude that infection with low doses of L. amazonensis in the dermis changes the course of infection by delaying the appearance of lesions. However, low-dose infection does not change the outcomes of susceptibility and cytokine production described for subcutaneous infection with high numbers of parasites.     

Intradermal inoculations of low doses of Leishmania major and Leishmania amazonensis metacyclic promastigotes induce different immunoparasitic processes and status of protection in BALB/c mice

In order to simulate the natural long term parasitisms which may occur in mammals infected with Leishmania, cutaneous leishmaniases due to Leishmania major or Leishmania amazonensis were induced using a model based on the inoculation of 10-1000 metacyclic promastigotes into the ear dermis of BALB/c mice. The final outcome of these parasitisms was dependent upon the number of inoculated parasites. Only some of the mice inoculated with ten parasites displayed cutaneous lesions, whereas most mice infected with 100 metacyclics and all mice infected with 1000 metacyclics developed progressive lesions. We found, using the latter experimental conditions, that the onset of the pathology was associated with: (a) parasite multiplication in the inoculation site and the draining lymph node correlating with an increase of the lymph node cell number, especially in L. major-infected mice; and (b) the detection of lymph node cells, at least in part CD4(+) T lymphocytes, able to produce high levels of interferon-gamma, interleukin (IL)-4, IL-10 and IL-13. Thereafter, mice infected by L. major harboured few parasites in the ear and had a 100-fold reduction in lymph node parasite load between 23 and 40 weeks post-inoculation. In contrast, the parasite loads of L. amazonensis-infected mice remained stable in the ear and increased in nodes during the same period of time. Only L. major-infected mice that exhibited cutaneous lesions in the primary site were resistant to the re-inoculation of 1000 metacyclic promastigotes, whereas all L. amazonensis-primary infected mice remained susceptible to a second homologous challenge. These results are the first to document that a status of resistance to re-infection, referred to concomitant immunity, is coupled to the development of primary progressive lesions in L. major-infected BALB/c mice. Such a protective status is absent in L. amazonensis-infected BALB/c mice.     

Resolution of an infection with Leishmania braziliensis confers complete protection to a subsequent challenge with Leishmania major in BALB/c mice

Both Leishmania major and L. braziliensis induce cutaneous leishmaniasis in BALB/c mice. Whereas BALB/c mice die of infection with L. major, they cure an infection with L. braziliensis. We report here that after curing an infection with L. braziliensis, BALB/c mice are resistant to challenge with L. major. When challenged with L. major, L. braziliensis pre-treated BALB/c mice mounted a delayed-type hypersensitivity response to L. major and produced high amounts of interferon-gamma (IFN-gamma) but low amounts of interleukin-4. The IFN-gamma produced by the L. braziliensis pre-infected mice was involved in the protection seen against L. major challenge since treating the mice with a neutralizing anti-IFN-gamma abrogated the protection. This suggests that cross-reactive antigen epitopes exist between L. braziliensis and L. major and that pre-infection with L. braziliensis primes BALB/c mice to epitopes on L. major that can elicit a protective Th1 response to the parasite.     

Mimicry of apoptotic cells by exposing phosphatidylserine participates in the establishment of amastigotes of Leishmania (L) amazonensis in mammalian hosts

Signaling through exposed phosphatidylserine (PS) is fundamental for the TGFbeta1-dependent, noninflammatory phagocytosis of apoptotic cells. This same mechanism operates in the internalization of amastigotes of Leishmania (L) amazonensis (L(L)a) in a process quoted as apoptotic mimicry. Now we show that the host modulates PS exposure by the amastigotes and, as a consequence, BALB/c mice-derived amastigotes expose significantly more PS than those derived from C57BL/6 mice. Due to this difference in the density of surface PS molecules, the former are significantly more infective than the latter, both in vivo, in F1 (BALB/c x C57BL/6) mice, and in vitro, in thioglycollate-derived macrophages from this same mouse strain. PS exposure increases with progression of the lesion and reaches its maximum value in amastigotes obtained at the time point when the lesion in C57BL/6 mice begins to decrease in size and the lesions in BALB/c mice are still growing in size. Synthesis of active TGFbeta1, induction of IL-10 message, and inhibition of NO synthesis correlate with the amount of surface PS displayed by viable (propidium iodide-negative) infective amastigote. Furthermore, we also show that, similar to what happens with apoptotic cells, amastigotes of L(L)a are internalized by macropinocytosis. This mechanism of internalization is consistent with the large phagolysosomes characteristic of L(L)a infection. The intensity of macrophage macropinocytic activity is dependent on the amount of surface PS displayed by the infecting amastigote.