CpG ODN D35 improves the response to abbreviated low-dose pentavalent antimonial treatment in non-human primate model of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) affects the lives of 0.7-1 million people every year causing lesions that take months to heal. These lesions can result in disfiguring scars with psychological, social and economic consequences. Antimonials are the first line of therapy for CL, however the treatment is lengthy and linked to significant toxicities; further, its efficacy is variable and resistant parasites are emerging. Shorter or lower dose antimonial treatment regimens, which would decrease the risk of adverse events and improve patient compliance, have shown reduced efficacy and further increase the risk emergence of antimonial-resistant strains. The progression of lesions in CL is partly determined by the immune response it elicits, and previous studies showed that administration of immunomodulatory type D CpG ODNs, magnifies the immune response to Leishmania and reduces lesion severity in nonhuman primates (NHP) challenged with Leishmania major or Leishmania amazonensis. Here we explored whether the addition of a single dose of immunomodulating CpG ODN D35 augments the efficacy of a short-course, low-dose pentavalent antimonial treatment regimen. Results show that macaques treated with D35 plus 5mg/kg sodium stibogluconate (SbV) for 10 days had smaller lesions and reduced time to re-epithelization after infection with Leishmania major. No toxicities were evident during the studies, even at doses of D35 10 times higher than those used in treatment. Critically, pentavalent antimonial treatment did not modify the ability of D35 to induce type I IFNs. The findings support the efficacy of D35 as adjuvant therapy for shorter, low dose pentavalent antimonial treatment.

A Review: The Current In Vivo Models for the Discovery and Utility of New Anti-leishmanial Drugs Targeting Cutaneous Leishmaniasis.

The current in vivo models for the utility and discovery of new potential anti-leishmanial drugs targeting Cutaneous Leishmaniasis (CL) differ vastly in their immunological responses to the disease and clinical presentation of symptoms. Animal models that show similarities to the human form of CL after infection with Leishmania should be more representative as to the effect of the parasite within a human. Thus, these models are used to evaluate the efficacy of new anti-leishmanial compounds before human clinical trials. Current animal models aim to investigate (i) host-parasite interactions, (ii) pathogenesis, (iii) biochemical changes/pathways, (iv) in vivo maintenance of parasites, and (v) clinical evaluation of drug candidates. This review focuses on the trends of infection observed between Leishmania parasites, the predictability of different strains, and the determination of parasite load. These factors were used to investigate the overall effectiveness of the current animal models. The main aim was to assess the efficacy and limitations of the various CL models and their potential for drug discovery and evaluation. In conclusion, we found that the following models are the most suitable for the assessment of anti-leishmanial drugs: L. major-C57BL/6 mice (or-vervet monkey, or-rhesus monkeys), L. tropica-CsS-16 mice, L. amazonensis-CBA mice, L. braziliensis-golden hamster (or-rhesus monkey). We also provide in-depth guidance for which models are not suitable for these investigations.

A comprehensive review of cutaneous leishmaniasis in kerman province, southeastern iran-narrative review article.

BACKGROUND: Cutaneous leishmaniasis (CL) remains a serious public health concern in Kerman Province, eastern Iran. This study was aimed to conduct a comprehensive review and highlights various aspects of CL in the province of Kerman. METHODS: This article mainly focuses on the studies published in the past 26 years on CL in the province. Current data for the present status were obtained through the provincial health system. RESULTS: Bam was the most infected district (63.6%), followed by Kerman (24.7%) and other districts to a less extent. Leishmania tropica is the major causative agent (95.5%) of CL in Kerman province, however, L. major accounts for 4.5% of the total cases. Bam, Kerman and southern districts of Kerman province were purely anthroponotic CL (ACL), while Rafsanjan, Baft, and Sirjan showed both ACL and zoonotic CL (ZCL). In contrast, Orzoieh district was merely endemic to ZCL type. Phlebotomus sergenti was the main vector in ACL foci while Ph. papatasi was the major vector in the ZCL district of Orzoieh. Localized CL was the most prevalent form (80%) of the disease, while leishmaniasis recidivans was the most uncommon clinical manifestation (18.7%). CONCLUSION: Due to recent rises in CL disease both in regard of increases in incidence rate and expansion of the disease to new foci, and presence of various risk factors in the province, control measures and health strategies should have high priorities to help treat the existing cases and prevent the expansion of the disease to new areas.

Modular multiantigen T cell epitope-enriched DNA vaccine against human leishmaniasis.

The leishmaniases are protozoal diseases that severely affect large populations in tropical and subtropical regions. There are only limited treatment options and preventative measures. Vaccines will be important for prevention, control and elimination of leishmaniasis, and could reduce the transmission and burden of disease in endemic populations. We report the development of a DNA vaccine against leishmaniasis that induced T cell-based immunity and is a candidate for clinical trials. The vaccine antigens were selected as conserved in various Leishmania species, different endemic regions, and over time. They were tested with T cells from individuals cured of leishmaniasis, and shown to be immunogenic and to induce CD4(+) and CD8(+) T cell responses in genetically diverse human populations of different endemic regions. The vaccine proved protective in a rodent model of infection. Thus, the immunogenicity of candidate vaccine antigens in human populations of endemic regions, as well as proof of principle for induction of specific immune responses and protection against Leishmania infection in mice, provides a viable strategy for T cell vaccine development.

Methodology of clinical trials aimed at assessing interventions for cutaneous leishmaniasis.

The current evidence-base for recommendations on the treatment of cutaneous leishmaniasis (CL) is generally weak. Systematic reviews have pointed to a general lack of standardization of methods for the conduct and analysis of clinical trials of CL, compounded with poor overall quality of several trials. For CL, there is a specific need for methodologies which can be applied generally, while allowing the flexibility needed to cover the diverse forms of the disease. This paper intends to provide clinical investigators with guidance for the design, conduct, analysis and report of clinical trials of treatments for CL, including the definition of measurable, reproducible and clinically-meaningful outcomes. Having unified criteria will help strengthen evidence, optimize investments, and enhance the capacity for high-quality trials. The limited resources available for CL have to be concentrated in clinical studies of excellence that meet international quality standards.

Assessment of interferon-γ levels and leishmanin skin test results in persons recovered for leishmaniasis.

Patients who recover from leishmaniasis usually show development of strong immunity and induction of interferon-γ (IFN-γ) and delayed type hypersensitivity. In a randomized trial, we analyzed the IFN-γ response by using a Quantiferon-Leishmania assay against three Leishmania peptide antigens and compared it with results of the leishmanin skin test (LST) in persons residing in areas in Iran to which zoonotic cutaneous leishmaniasis (ZCL, 181 persons), anthroponotic cutaneous leishmaniasis (ACL, 104 persons), and zoonotic visceral leishmaniasis (ZVL, 67 persons) are endemic. The percentage of persons with an IFN-γ-positive response (> 0.2 IU/mL) to three antigens and the mean concentration of IFN-γ induced by the antigens were higher for persons from areas endemic for ZVL than for persons from areas endemic for ZCL and ACL. The percentage of persons with LST-positive results (≥ 5 mm indurations) was 99%, 94%, and 70% for areas with ZCL, ACL, and ZVL, respectively. Our data indicate that the LST is significantly more sensitive than IFN-γ levels in persons who have been cured of cutaneous leishmaniasis than in persons who have been cured of ZVL.

Comparison of short-course multidrug treatment with standard therapy for visceral leishmaniasis in India: an open-label, non-inferiority, randomised controlled trial.

BACKGROUND: Improved treatment approaches are needed for visceral leishmaniasis. We assessed the efficacy and safety of three potential short-course combination treatments compared with the standard monotherapy in India. METHODS: Standard treatment (1 mg/kg amphotericin B infusion on alternate days for 30 days, total dose 15 mg/kg) was compared with three drug combinations (single injection of 5 mg/kg liposomal amphotericin B and 7-day 50 mg oral miltefosine or single 10-day 11 mg/kg intramuscular paromomycin; or 10 days each of miltefosine and paromomycin) in an open-label, parallel-group, non-inferiority, randomised controlled trial in two hospital sites in Bihar, India. Patients aged 5-60 years with parasitologically confirmed visceral leishmaniasis were randomly assigned one of the four treatments by the trial statistician by use of a computer-generated list. Clinical assessments were done at the end of treatment (15 days on combination treatment; 31 days for standard treatment) and after 45 days and 6 months. The primary endpoint was definitive cure (defined as no sign or symptom of visceral leishmaniasis and parasitologically cured to the last follow-up). Analyses were done both by intention to treat and per protocol. This trial is registered with ClinicalTrials.gov, number NCT00696969. FINDINGS: Between June, 2008, and July, 2009, 634 patients were assigned amphotericin B (n=157), liposomal amphotericin B with miltefosine (n=160) or paromomycin (n=158), or miltefosine and paromomycin (n=159). 618 patients were in the per-protocol population. There were two relapses in each group. The numbers with definitive cure at 6 months for the intention-to-treat population were 146 (cure rate 93·0%; CI 87·5-96·3) for amphotericin B, 156 (97·5%; 93·3-99·2) for liposomal amphotericin B and miltefosine, 154 (97·5%; 93·24-99·2) for liposomal amphotericin B and paromomycin, and 157 (98·7%; 95·1-99·8) for miltefosine and paromomycin. All combinations were non-inferior to the standard treatment, in both the intention-to-treat and per-protocol populations. Patients in the combination groups had fewer adverse events than did those assigned standard treatment. INTERPRETATION: Combination treatments for visceral leishmaniasis are efficacious and safe, and decrease the duration of therapy, thereby encouraging adherence and reducing emergence of drug-resistant parasites. FUNDING: Drugs for Neglected Diseases initiative and the Indian Council of Medical Research.

Leishmaniasis vaccines: past, present and future.

No vaccine exists against any form of leishmaniasis. Because recovery from infection is usually accompanied by a strong immunity and because it is possible to protect experimental animals against live challenge, hope for the development of a vaccine for humans has been high. However, leishmaniasis is a disease of the poor and the market for a vaccine is very limited. Until a few years ago, with minimal resources, only a pragmatic approach was possible for testing the first-generation vaccines (i.e. killed whole parasites). Recently, funding has become available for developing defined second-generation vaccines, including recombinant proteins and DNA constructs. With new adjuvants also being developed there is new hope, and several new vaccines are in development against leishmaniasis.

Leishmaniasis recidivans among school children in Bam, South-east Iran, 1994-2006.

BACKGROUND: Leishmaniasis recidivans (LR) is a rare phenomenon in the world with high morbidity in children. METHODS: Overall 22 838 school children were examined during 1994-2006. Diagnosis was performed by combination of methods as clinical appearance, direct smears, cultures, polymerase chain reaction (PCR) and histology. RESULTS: Ninety-eight cases were diagnosed as LR with duration of lesions varying from 2 to 8 years and diameter of lesions 1-5 cm, yellowish-brown appearance with papules around or in the scar. Most of the lesions (95%) were on the face. No amastigote was found in direct smears. Identification of nine random isolates by PCR confirmed all species to be L. tropica. Tissue sections showed typical granulomatous reactions with various inflammatory cells but no visible amastigote was seen. CONCLUSIONS: The presence of LR as an important cause of morbidity has future implications for treatment regimens and immunoprophylaxis.

Immunological stimulation for the treatment of leishmaniasis: a modality worthy of serious consideration.

Instead of relying on drugs to reduce the parasite burden of leishmaniasis, and waiting for the effector immune response to develop in time to control the parasites, immunotherapy in conjunction with chemotherapy can rapidly induce the effector immune response. With a safe and potent drug plus an affordable therapeutic vaccine (immunostimulant), which remains to be developed, a single visit by patients with visceral or cutaneous leishmaniasis might be sufficient to induce a quick and lasting recovery. Drug toxicity and the emergence of resistance could also be dramatically reduced compared with present long-term monotherapy. Immunotherapy could be an effective addition to chemotherapy for leishmaniasis.

Efficacy of killed whole-parasite vaccines in the prevention of leishmaniasis: a meta-analysis.

Despite decades of investigation in countries on three continents, an efficacious vaccine against Leishmania infections has not been developed. Although some indication of protection was observed in some of the controlled trials conducted with "first-generation" whole, inactivated Leishmania parasite vaccines, convincing evidence of protection was lacking. After reviewing all previously published or unpublished randomized, controlled field efficacy clinical trials of prophylactic candidate vaccines, a meta-analysis of qualified trials was conducted to evaluate whether there was some evidence of protection revealed by considering the results of all trials together. The findings indicate that the whole-parasite vaccine candidates tested do not confer significant protection against human leishmaniasis.

First generation leishmaniasis vaccines: a review of field efficacy trials.

First generation candidate vaccines against leishmaniasis, prepared using inactivated whole parasites as their main ingredient, were considered as promising because of their relative ease of production and low cost. These vaccines have been the subject of many investigations over several decades and are the only leishmaniasis vaccine candidates which have undergone phase 3 clinical trial evaluation. Although the studies demonstrated the safety of the vaccines and several studies showed reasonable immunogenicity and some indication of protection, an efficacious prophylactic vaccine is yet to be identified. Despite this overall failure, these trials contributed significantly to increasing knowledge on human leishmaniasis immunology. To provide a collective view, this review discusses the methods and findings of field efficacy trials of first generation leishmaniasis vaccine clinical trials conducted in the Old and New Worlds.

Consultation meeting on the development of therapeutic vaccines for post kala azar dermal leishmaniasis.

BACKGROUND: Post kala azar dermal leishmaniasis (PKDL) is a disease that appears after treatment of visceral leishmaniasis (VL). The highest incidence of PKDL in the world is in Sudan. Many patients heal spontaneously within 6 months but those who don't are difficult to treat, often requiring months of daily injections. These patients harbour parasite in their skin and are believed to be a source of infection and possibly epidemics. Present treatment modalities of PKDL are inadequate and impractical due to cost, duration of treatment required and side effects. New approach for treatment of PKDL is required. A joint meeting of the UNICEF/UNDP/World Bank/WHO Special Programme for research and training in Tropical Disease (TDR) and the Infectious Disease Research Institute (IDRI) Seattle, USA was held to review the progress of therapeutic vaccines and plan the development of treatment modalities for PKDL. METHODS: The history of leishmaniasis vaccine development for prophylaxis and therapy was reviewed. Other than previous infection - simulated by inoculation of live Leishmania as a vaccine (leishmanization), none of the preparations of killed parasite with or without adjuvants have shown significant prophylactic efficacy. Killed L. major absorbed with alum and mixed with BCG remains to be tested as a prophylactic vaccine. RESULTS: Killed parasite preparations i.e. L. mexicana mixed with BCG and L. amazonensis (combined with low dose of antimonial), have shown efficacy in immunotherapy and immuno-chemotherapy, respectively. In addition combined full antimonial plus alum-absorbed autoclaved L. major vaccine has been shown to significantly improve therapy of refractory PKDL patients. These are all crude preparations of parasites and are difficult to define and standardize. However, there is now a new, second generation vaccine, Leish-111f + MPL-SE, composed of a recombinant protein comprising three leishmanial antigens and a defined adjuvant in clinical development. CONCLUSION AND RECOMMENDATIONS: Immuno-chemotherapy has the potential of becoming a practical and affordable treatment modality for PKDL and other forms of leishmaniasis. The encouraging results with alum-autoclaved L. major + antimonial should be pursued. However, before further trials, availability of the vaccine and its production under Good Manufacturing Product, hence quality control must be assured. Following satisfactory safety profile of Leish-111f+MPL-SE, clinical trials using this vaccine initially with antimonials should be initiated. Similarly immunotherapy of VL should be considered with the view to controlling development of PKDL. Some immunological studies are required prior to initiation of immunotherapy in VL patients.

Consultative meeting to develop a strategy for treatment of cutaneous leishmaniasis. Institute Pasteur, Paris. 13-15 June, 2006.

BACKGROUND: A meeting was organized by Drugs for Neglected Diseases initiative (DNDi) and the Institute Pasteur (IP), Paris, to review the treatment for all forms of cutaneous leishmaniasis (CL) and to propose a strategy for the development of new efficacious and affordable treatments. METHOD: The global burden of CL was discussed with respect to financial impact; relation to poverty; the stigma of CL lesions and scars (particularly in young women); lack of effective, affordable, easily implemented tools and political will and resources to implement available control tools; and lack of input from pharmaceutical and biotechnology companies to develop new drugs and vaccines. RESULTS: According to the experts from different endemic countries present, the financial and social burdens of CL are high, but we have limited quantitative data. The analysis of published trials indicates that the quality of most trials is poor and requires both improvement and standardization. The available drugs are inadequate. Criteria by which different CL types could be prioritized as target disease were set. These criteria included: severity of the disease; lack of response to available drugs; overall incidence and prevalence of the disease; sequelae of the disease, (including recidivans and mucosal leishmaniasis); the impact of treatment of individuals on control of transmission and lack of other major parties involved in drug development. Based on these, the anthroponotic CL and its sequel "recidivans" caused by L. tropica and CL caused by L. braziliensis and its sequel, mucosal leishmaniasis were considered to be the target diseases. The mechanism for controlling Leishmania infection to reach a stable self healing process is a balanced immune response. Immune stimulation during chemotherapy can enhance cure. There is no adequately effective vaccine, but some encouraging results have been obtained with whole killed Leishmania parasites or imiquimod (an immuno-modulator) plus antimonials. Further studies are needed. One safety/immunogenicity clinical trial is currently ongoing with a Second Generation Vaccine (SGV). CONCLUSIONS AND RECOMMENDATIONS: There is an urgent need for new treatments for all CL types. CL should be considered as a neglected disease and organizations, such as DNDi, should include it in their list of target diseases. It was agreed that immuno-chemotherapy (with "therapeutic" vaccines or immunomodulators) has a strong potential to make an impact as a new therapy of CL with the view of shortening/reducing duration and dose of drug treatment and preventing resistance. There is also a need for safe, affordable and efficacious new chemotherapeutics. The quality of clinical trials needs to be enhanced and standardized. Short and long-term objectives and activities were defined as a part of meeting recommendations.

Surrogate markers of immunity to Leishmania major in leishmanin skin test negative individuals from an endemic area re-visited.

BACKGROUND: In the screening of vaccine candidates it is important to select candidates that evoke immune responses associated with protection. Valid surrogate markers against human leishmaniasis are still lacking. METHODS: A controlled injection of live Leishmania known as leishmanization, (LZ), was used to evaluate vaccine (alum-precipitated autoclaved Leishmania major with BCG) efficacy and more accurately define surrogate markers of immunity to leishmaniasis in humans. Cellular immune responses to this artificial infection were monitored in the volunteers prior to and 9 months post infection. Comparisons were made between those who developed a lesion after infection and those who did not. RESULTS: In the volunteers monitored there was no significant difference in LST, IFNgamma production, or source of IFNgamma between those who developed a lesion and those who did not after LZ, with the exception that ulcer development was associated with an enhanced number of IFNgamma secreting CD4(+) CD45RA(-) (memory) T cells. DISCUSSION: Ulcer development following LZ was lower than anticipated by a pilot study (47% versus 78%) using the same stabilate several years earlier. While this may be an effect of low viability/virulence of the LZ inocula, alternative explanations are also possible. The IFNgamma responses in the study subjects were significantly lower compared to volunteers with previous history of cutaneous leishmaniasis. The findings raise the possibility that the selection of LST-negative volunteers in an endemic area may bias the study towards potentially non/low L. major-reactive volunteers.

Immunotherapy for drug-refractory mucosal leishmaniasis.

BACKGROUND: Pentavalent antimony (Sb(v)) is the mainstay therapy for mucosal leishmaniasis (ML), but it is toxic, and relapses are common. Immunotherapy using a mixture of killed parasites, with or without bacille Calmette-Guerin, is an alternative but is used sporadically because of inconsistent results. METHODS: We developed a defined immunotherapeutic antigen preparation for use in an observational, open-label trial to treat 6 patients with ML with a history of Sb(v) therapy failure. All patients were treated with the antigens thiol-specific antioxidant, Leishmania major stress inducible protein 1, Leishmania elongation initiation factor, and Leishmania heat shock protein 83, plus granulocyte-macrophage colony-stimulating factor. Patients underwent clinical and pathological evaluations before the initiation of immunotherapy and at 3, 6, 9, 12, 18, 24, and 60 months after. RESULTS: One month after the third injection, 1 patient showed complete clinical remission (CC) and remained disease free for the duration of the study. At the 9-month follow-up examination, 5 patients showed CC, and all patients were asymptomatic at a subsequent 5-year follow-up examination. CONCLUSIONS: These data support the concept that vaccine therapy with a defined antigen combination, used with standard chemotherapy, is a safe and effective approach to treat drug-refractory ML.

Leishmaniasis vaccine candidates for development: a global overview.

A vaccine against different forms of leishmaniasis should be feasible considering the wealth of information on genetics and biology of the parasite, clinical and experimental immunology of leishmaniasis, and the availability of vaccines that can protect experimental animals against challenge with different Leishmania species. However, there is no vaccine against any form of leishmaniasis for general human use. One major factor is the lack of a conceived market for human leishmaniasis vaccines. Hence pharmaceutical industries involved in vaccine development are not interested in investing millions of dollars and a decade that is required for developing a new vaccine. Besides, leishmaniasis is a local/regional problem and not a global one. According to the estimates of the World Health Organization, 90 per cent of visceral leishmaniasis occurs in five countries (Bangladesh, Brazil, India, Nepal and Sudan). Those in need are amongst the poorest people in these countries. It should therefore be the objectives of these countries to develop a vaccine. Fortunately, both Brazil and India have designated the control of visceral leishmaniasis as a top priority for their respective Ministries of Health. The purpose of this review is to present only the vaccines in use and those in development for use in dogs or humans. This is not an exhaustive review of vaccine discovery or the principles of clinical immunology underlying vaccine development.

Failure of a killed Leishmania amazonensis vaccine against American cutaneous leishmaniasis in Colombia.

We report the results of a double-blind, randomized, placebo-controlled clinical trial of a killed whole-cell Leishmania amazonensis candidate vaccine against American cutaneous leishmaniasis (CL) in Colombia. The trial subjects were 2597 healthy volunteers with negative leishmanin skin test (LST) selected from rural Colombian soldiers who were going to patrol endemic areas. They were randomized to receive either three doses of vaccine (n=1295) or placebo (n=1302) given at 20-day intervals. An active and passive case detection system was established to follow-up volunteers for 1 year after vaccination. Safety and efficacy of the vaccine were determined by comparing local and systemic adverse reactions after each dose and the incidence of parasitologically confirmed CL. In the vaccine and placebo groups 101 (7.7%) and 88 (6.8%) volunteers developed CL respectively. The vaccine was shown to be safe but offered no protection against CL caused by L. panamensis in the proposed vaccination schedule.

Leishmanization: use of an old method for evaluation of candidate vaccines against leishmaniasis.

To establish the safety and reproducibility of live challenge with Leishmania major, a single frozen stabilate was used in two open, single-arm leishmanization (LZ) trials. A total of 42 inoculations in 28 male adult volunteers were followed until complete healing. Lesions induced by LZ are as diverse as natural infection, but much milder. Total protection was seen in 100% (11/11) of recovered volunteers. LZ used as live challenge to test vaccine candidates reduces the required sample size, is cost effective for surrogate markers studies and will induce protection in the participants who are not protected by candidate vaccines.