Renato Porrozzi

Protection against Cutaneous Leishmaniasis Induced by Recombinant Antigens in Murine and Nonhuman Primate Models of the Human Disease

ABSTRACT Leishmaniasis affects approximately 2 million people each year throughout the world. This high incidence is due in part to the lack of an efficacious vaccine. We present evidence that the recombinant leishmanial antigens LmSTI1 and TSA, which we identified and characterized previously, induce excellent protection in both murine and nonhuman primate (rhesus monkey) models of human cutaneous leishmaniasis. The remarkable protection induced by LmSTI1 and TSA in an animal model that is evolutionarily close to humans qualifies this antigen combination as a promising candidate subunit vaccine against human leishmaniasis.

Comparative Evaluation of Enzyme-Linked Immunosorbent Assays Based on Crude and Recombinant Leishmanial Antigens for Serodiagnosis of Symptomatic and Asymptomatic Leishmania infantum Visceral Infections in Dogs

ABSTRACT The diagnosis of visceral leishmaniasis remains difficult in rural areas where the disease is endemic, and serologic methods still need assessment, as they are not very sensitive for the detection of asymptomatic infectious dogs. Here we present data on the development of enzyme-linked immunosorbent assay (ELISA)-based methods for the detection of antibodies against recombinant leishmanial antigens (namely, the recombinant K26 [rK26] and rK39 antigens from Leishmania infantum and the rA2 protein from Leishmania donovani) in comparison to ELISAs employing crude soluble antigen (CSA). The assays utilized sera from known negative controls (n = 25) and clinically asymptomatic (n = 50) and symptomatic (n = 50) dogs with confirmed L. infantum infections. Additional studies were also done using sera from animals harboring other infections (n = 14) for the evaluation of cross-reactivity. Our study indicated that rK26 and rK39 used in ELISAs provided very high sensitivities for the detection of symptomatic dogs (94% and 100%, respectively), followed by CSA (88%) and rA2 (70%). Conversely, rA2 was more sensitive for asymptomatic dogs (88%) than rK39 and rK26 (both 66%) and CSA (30%). Some cross-reactivity in sera from dogs with other infections (Leishmania braziliensis and Leptospira interrogans) was identified, but the rA2 protein provided the greatest specificity (98%). Data further indicate that all three recombinant proteins must be used in parallel to detect essentially all infected dogs. Efforts should be made to develop a cheap and reliable serologic test based on epitope selection from these diagnostic markers for the sensitive detection of L. infantum-infected dogs.

Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease

We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79% in attenuated parasite-vaccinated monkeys, versus 75% in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-gamma) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application.

Sequence analysis and PCR-RFLP profiling of the hsp70 gene as a valuable tool for identifying Leishmania species associated with human leishmaniasis in Brazil.

Since the first report of the genus Leishmania, many species have been described. In Brazil, human leishmaniasis has been associated with eight Leishmania species, of which seven are responsible for cutaneous leishmaniasis (CL). In some endemic areas, CL is associated with only one species; however, in other areas, such as the Amazon, the etiology of CL can be assigned to many species. The multitude of highly similar Leishmania species in Brazil makes it difficult to develop an appropriate method of typing them. Most Leishmania species were first described based on epidemiological and biological characteristics, and these were later corroborated by Multilocus Enzyme Electrophoresis (MLEE), the gold standard technique for identifying Leishmania species. In an attempt to overcome the limitations of MLEE, many PCR-based methods have been developed and used for parasite identification. In the present study, we analyzed the sequence of the hsp70 gene in Leishmania species associated with human leishmaniasis in Brazil. This analysis led to the identification of restriction enzymes that could be used for PCR-RFLP-based identification. The results obtained were in complete agreement with those obtained by MLEE, suggesting that PCR-RFLP analysis of hsp70 could soon replace MLEE for routine Leishmania typing.

Species diversity of Leishmania (Viannia) parasites circulating in an endemic area for cutaneous leishmaniasis located in the Atlantic rainforest region of northeastern Brazil.

Objectives  To identify the aetiological agents of cutaneous leishmaniasis and to investigate the genetic polymorphism of Leishmania (Viannia) parasites circulating in an area with endemic cutaneous leishmaniasis (CL) in the Atlantic rainforest region of northeastern Brazil.

Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identification of the parasite species

In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

Epidemiological and clinical features of Leishmania (Viannia) braziliensis American cutaneous and mucocutaneous leishmaniasis in the State of Espírito Santo, Brazil

Between 1985 and 2000, epidemiological surveys of the American tegumentary leishmaniasis (ATL) were carried out in several rural and urban communities in Espírito Santo, Brazil. A total of 100 stocks of Leishmania (comprising isolates from both human and canine hosts with ATL) were identified by two methods of molecular characterization, using specific monoclonal antibodies and multilocus enzyme electrophoresis. Parasite isolates from 19 municipalities were found to belong to the same zymodeme and serodeme type as of the Leishmania (Viannia) braziliensis reference strain. In contrast, our genotyping studies have shown intra-specific variation among these parasites (comparisons of the variability of the internal transcribed spacers between the small and large subunits of the rRNA genes of the 22 stocks studied revealed at least 11 genotypes). Two main clusters of L. (V.) braziliensis genotypes were observed, representing parasites collected from different endemic regions in the state, where transmission reflects distinct eco-epidemiological features. Infection with this pathogen was associated with the characteristic disease forms, but neither the clinical outcome nor the response to treatment could be related to the genetic polymorphism of the isolates, as defined by using the proposed methodology.

The Genetic Structure of Leishmania infantum Populations in Brazil and Its Possible Association with the Transmission Cycle of Visceral Leishmaniasis

Leishmania infantum is the etiologic agent of visceral leishmaniasis (VL) in the Americas, Mediterranean basin and West and Central Asia. Although the geographic structure of L. infantum populations from the Old World have been described, few studies have addressed the population structure of this parasite in the Neotropical region. We employed 14 microsatellites to analyze the population structure of the L. infantum strains isolated from humans and dogs from most of the Brazilian states endemic for VL and from Paraguay. The results indicate a low genetic diversity, high inbreeding estimates and a depletion of heterozygotes, which together indicate a predominantly clonal breeding system, but signs of sexual events are also present. Three populations were identified from the clustering analysis, and they were well supported by F statistics inferences and partially corroborated by distance-based. POP1 (111 strains) was observed in all but one endemic area. POP2 (31 strains) is also well-dispersed, but it was the predominant population in Mato Grosso (MT). POP3 (31 strains) was less dispersed, and it was observed primarily in Mato Grosso do Sul (MS). Strains originated from an outbreak of canine VL in Southern Brazil were grouped in POP1 with those from Paraguay, which corroborates the hypothesis of dispersal from Northeastern Argentina and Paraguay. The distribution of VL in MS seems to follow the west-east construction of the Bolivia-Brazil pipeline from Corumbá municipality. This may have resulted in a strong association of POP3 and Lutzomyia cruzi, which is the main VL vector in Corumbá, and a dispersion of this population in this region that was shaped by human interference. This vector also occurs in MT and may influence the structure of POP2. This paper presents significant advances in the understanding of the population structure of L. infantum in Brazil and its association with eco-epidemiological aspects of VL.

Further Evidence of an Association between the Presence of Leishmania RNA Virus 1 and the Mucosal Manifestations in Tegumentary Leishmaniasis Patients

Tegumentary Leishmaniasis (TL) is endemic in Latin America, and Brazil contributes approximately 20 thousand cases per year. The pathogenesis of TL, however, is still not fully understood. Clinical manifestations vary from cutaneous leishmaniasis (CL) to more severe outcomes, such as disseminated leishmaniasis (DL), mucosal leishmaniasis (ML) and diffuse cutaneous leishmaniasis (DCL). Many factors have been associated with the severity of the disease and the development of lesions. Recent studies have reported that the presence of Leishmania RNA virus 1 infecting Leishmania (Leishmania RNA virus 1, LRV1) is an important factor associated with the severity of ML in experimental animal models. In the present study, 156 patients who attended Rondonias Hospital of Tropical Medicine with both leishmaniasis clinical diagnoses (109 CL; 38 ML; 5 CL+ML; 3 DL and 1 DCL) and molecular diagnoses were investigated. The clinical diagnosis were confirmed by PCR by targeting hsp70 and kDNA DNA sequences and the species causing the infection were determined by HSP70 PCR-RFPL. The presence of LVR1 was tested by RT-PCR. Five Leishmania species were detected: 121 (77.6%) samples were positive for Leishmania (Viannia) braziliensis, 18 (11.5%) were positive for Leishmania (V.) guyanensis, 3 (1.8%) for Leishmania (V.) lainsoni, 2 (1.3%) for Leishmania (Leishmania) amazonensis and 2 (1.3%) for Leishmania (V.) shawi. Six (3.9%) samples were positive for Leishmania sp. but the species could not be determined, and 4 (2.6%) samples were suggestive of mixed infection by L. (V.) braziliensis and L. (V.) guyanensis. The virus was detected in L. braziliensis (N = 54), L. guyanensis (N = 5), L. amazonensis (N = 2), L. lainsoni (N = 1) and inconclusive samples (N = 6). Patients presenting with CL+ML, DL and DCL were excluded from further analysis. Association between the presence of the virus and the disease outcome were tested among the remaining 147 patients (CL = 109 and ML = 38). Of them, 71.1% (n = 27) mucosal lesions were positive for LRV1, and 28.9% (n = 11) were negative. In cutaneous lesions, 36.7% (n = 40) were positive and 63.3% (n = 69) were negative for LRV1. The ratio P(ML|LRV1+)/P(ML|LRV1-) was 2.93 (CI95% 1.57…5.46; p<0.001), thus corroborating the hypothesis of the association between LRV1 and the occurrence of mucosal leishmaniasis, as previously described in animal models; it also indicates that LRV1 is not the only factor contributing to the disease outcome.

Clinical and Parasitological Protection in a Leishmania infantum-Macaque Model Vaccinated with Adenovirus and the Recombinant A2 Antigen

Background Visceral leishmaniasis (VL) is a severe vector-born disease of humans and dogs caused by Leishmania donovani complex parasites. Approximately 0.2 to 0.4 million new human VL cases occur annually worldwide. In the new world, these alarming numbers are primarily due to the impracticality of current control methods based on vector reduction and dog euthanasia. Thus, a prophylactic vaccine appears to be essential for VL control. The current efforts to develop an efficacious vaccine include the use of animal models that are as close to human VL. We have previously reported a L. infantum-macaque infection model that is reliable to determine which vaccine candidates are most worthy for further development. Among the few amastigote antigens tested so far, one of specific interest is the recombinant A2 (rA2) protein that protects against experimental L. infantum infections in mice and dogs. Methodology/Principal Findings Primates were vaccinated using three rA2-based prime-boost immunization regimes: three doses of rA2 plus recombinant human interleukin-12 (rhIL-12) adsorbed in alum (rA2/rhIL-12/alum); two doses of non-replicative adenovirus recombinant vector encoding A2 (Ad5-A2) followed by two boosts with rA2/rhIL-12/alum (Ad5-A2+rA2/rhIL12/alum); and plasmid DNA encoding A2 gene (DNA-A2) boosted with two doses of Ad5-A2 (DNA-A2+Ad5-A2). Primates received a subsequent infectious challenge with L. infantum. Vaccines, apart from being safe, were immunogenic as animals responded with increased pre-challenge production of anti-A2-specific IgG antibodies, though with some variability in the response, depending on the vaccine formulation/protocol. The relative parasite load in the liver was significantly lower in immunized macaques as compared to controls. Protection correlated with hepatic granuloma resolution, and reduction of clinical symptoms, particularly when primates were vaccinated with the Ad5-A2+rA2/rhIL12/alum protocol. Conclusions/Significance The remarkable clinical protection induced by A2 in an animal model that is evolutionary close to humans qualifies this antigen as a suitable vaccine candidate against human VL.