Immune response profiles from humans experimentally exposed to Phlebotomus duboscqi bites.

Introduction: Cutaneous leishmaniasis is a neglected vector-borne parasitic disease prevalent in 92 countries with approximately one million new infections annually. Interactions between vector saliva and the human host alter the response to infection and outcome of disease. Methods: To characterize the human immunological responses developed against saliva of Phlebotomus duboscqi, a Leishmania major (L. major) vector, we repeatedly exposed the arms of 14 healthy U.S volunteers to uninfected P. duboscqi bites. Blood was collected a week after each exposure and used to assess total IgG antibodies against the proteins of P. duboscqi salivary gland homogenate (SGH) and the levels of IFN-gamma and IL-10 from peripheral blood mononuclear cells (PBMCs) stimulated with SGH or recombinant sand fly proteins. We analyzed skin punch biopsies of the human volunteer arms from the insect bite site and control skin site after multiple P. duboscqi exposures (four volunteers) using immunohistochemical staining. Results: A variety of immediate insect bite skin reactions were observed. Late skin reactions to insect bites were characterized by macular hyperpigmentation and/or erythematous papules. Hematoxylin and eosin staining showed moderate mononuclear skin infiltrate with eosinophils in those challenged recently (within 2 months), eosinophils were not seen in biopsies with recall challenge (6 month post bites). An increase in plasma antigen-specific IgG responses to SGH was observed over time. Western Blot results showed strong plasma reactivity to five P. duboscqi salivary proteins. Importantly, volunteers developed a cellular immunity characterized by the secretion of IFN-gamma upon PBMC stimulation with P. duboscqi SGH and recombinant antigens. Discussion: Our results demonstrate that humans mounted a local and systemic immune response against P. duboscqi salivary proteins. Specifically, PduM02/SP15-like and PduM73/adenosine deaminase recombinant salivary proteins triggered a Th1 type immune response that might be considered in future development of a potential Leishmania vaccine.

Analytical validation of quantitative SARS-CoV-2 subgenomic and viral load laboratory developed tests conducted on the Panther Fusion® (Hologic) with preliminary application to clinical samples.

OBJECTIVE: Validate the performance characteristics of two analyte specific, laboratory developed tests (LDTs) for the quantification of SARS-CoV-2 subgenomic RNA (sgRNA) and viral load on the Hologic Panther Fusion® using the Open Access functionality. METHODS: Custom-designed primers/probe sets targeting the SARS-CoV-2 Envelope gene (E) and subgenomic E were optimized. A 20-day performance validation following laboratory developed test requirements was conducted to assess assay precision, accuracy, analytical sensitivity/specificity, lower limit of detection and reportable range. RESULTS: Quantitative SARS-CoV-2 sgRNA (LDT-Quant sgRNA) assay, which measures intermediates of replication, and viral load (LDT-Quant VLCoV) assay demonstrated acceptable performance. Both assays were linear with an R2 and slope equal to 0.99 and 1.00, respectively. Assay precision was evaluated between 4-6 Log10 with a maximum CV of 2.6% and 2.5% for LDT-Quant sgRNA and LDT-Quant VLCoV respectively. Using negative or positive SARS-CoV-2 human nasopharyngeal swab samples, both assays were accurate (kappa coefficient of 1.00 and 0.92). Common respiratory flora and other viral pathogens were not detected and did not interfere with the detection or quantification by either assay. Based on 95% detection, the assay LLODs were 729 and 1206 Copies/mL for the sgRNA and VL load LDTs, respectively. CONCLUSION: The LDT-Quant sgRNA and LDT-Quant VLCoV demonstrated good analytical performance. These assays could be further investigated as alternative monitoring assays for viral replication; and thus, medical management in clinical settings which could inform isolation/quarantine requirements.

Potential Biomarkers for Asymptomatic Visceral Leishmaniasis among Iraq-Deployed U.S. Military Personnel.

Visceral leishmaniasis (VL) is a chronic infection caused by Leishmania (L.) donovani or L. infantum parasites. Despite having the infection, most individuals never develop the clinical disease and are able to control the parasite and remain asymptomatic. However, some progress to symptomatic VL, leading to death if untreated. The host immune response has a major role in determining the progression and severity of the clinical manifestations in VL; several immune biomarkers of symptomatic VL have been described with interferon-gamma release as a surrogate biomarker of host cellular immunity. However, new biomarkers to identify asymptomatic VL (AVL) are needed for the identification of people at risk for VL activation. In our study, levels of chemokine/cytokine in the supernatants of peripheral mononuclear blood cells (PBMC) from 35 AVL+ Iraq-deployed participants, stimulated in vitro with soluble Leishmania antigen for 72 h, were assessed by a bead-based assay that allows the measurement of multiple analytes. PBMC of AVL-negative military beneficiaries were used as controls. Monocyte Chemoattractant Protein-1, Monokine Induced by Gamma Interferon and Interleukin-8, were detected at high levels in AVL+ stimulated cultures from Iraq deployers compared to uninfected controls. Measurement of chemokine/cytokine levels can identify cellular immune responses in AVL+ asymptomatic individuals.

Validation of SARS-CoV-2 pooled testing for surveillance using the Panther Fusion® system: Impact of pool size, automation, and assay chemistry.

Combining diagnostic specimens into pools has been considered as a strategy to augment throughput, decrease turnaround time, and leverage resources. This study utilized a multi-parametric approach to assess optimum pool size, impact of automation, and effect of nucleic acid amplification chemistries on the detection of SARS-CoV-2 RNA in pooled samples for surveillance testing on the Hologic Panther Fusion® System. Dorfman pooled testing was conducted with previously tested SARS-CoV-2 nasopharyngeal samples using Hologic's Aptima® and Panther Fusion® SARS-CoV-2 Emergency Use Authorization assays. A manual workflow was used to generate pool sizes of 5:1 (five samples: one positive, four negative) and 10:1. An automated workflow was used to generate pool sizes of 3:1, 4:1, 5:1, 8:1 and 10:1. The impact of pool size, pooling method, and assay chemistry on sensitivity, specificity, and lower limit of detection (LLOD) was evaluated. Both the Hologic Aptima® and Panther Fusion® SARS-CoV-2 assays demonstrated >85% positive percent agreement between neat testing and pool sizes ≤5:1, satisfying FDA recommendation. Discordant results between neat and pooled testing were more frequent for positive samples with CT>35. Fusion® CT (cycle threshold) values for pooled samples increased as expected for pool sizes of 5:1 (CT increase of 1.92-2.41) and 10:1 (CT increase of 3.03-3.29). The Fusion® assay demonstrated lower LLOD than the Aptima® assay for pooled testing (956 vs 1503 cp/mL, pool size of 5:1). Lowering the cut-off threshold of the Aptima® assay from 560 kRLU (manufacturer's setting) to 350 kRLU improved the assay sensitivity to that of the Fusion® assay for pooled testing. Both Hologic's SARS-CoV-2 assays met the FDA recommended guidelines for percent positive agreement (>85%) for pool sizes ≤5:1. Automated pooling increased test throughput and enabled automated sample tracking while requiring less labor. The Fusion® SARS-CoV-2 assay, which demonstrated a lower LLOD, may be more appropriate for surveillance testing.

A SARS-CoV-2 Spike Ferritin Nanoparticle Vaccine Is Protective and Promotes a Strong Immunological Response in the Cynomolgus Macaque Coronavirus Disease 2019 (COVID-19) Model.

The COVID-19 pandemic has had a staggering impact on social, economic, and public health systems worldwide. Vaccine development and mobilization against SARS-CoV-2 (the etiologic agent of COVID-19) has been rapid. However, novel strategies are still necessary to slow the pandemic, and this includes new approaches to vaccine development and/or delivery that will improve vaccination compliance and demonstrate efficacy against emerging variants. Here, we report on the immunogenicity and efficacy of a SARS-CoV-2 vaccine comprising stabilized, pre-fusion spike protein trimers displayed on a ferritin nanoparticle (SpFN) adjuvanted with either conventional aluminum hydroxide or the Army Liposomal Formulation QS-21 (ALFQ) in a cynomolgus macaque COVID-19 model. Vaccination resulted in robust cell-mediated and humoral responses and a significant reduction in lung lesions following SARS-CoV-2 infection. The strength of the immune response suggests that dose sparing through reduced or single dosing in primates may be possible with this vaccine. Overall, the data support further evaluation of SpFN as a SARS-CoV-2 protein-based vaccine candidate with attention to fractional dosing and schedule optimization.

Virological and Serological Assessment of US Army Trainees Isolated for Coronavirus Disease 2019.

BACKGROUND: Laboratory screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key mitigation measure to avoid the spread of infection among recruits starting basic combat training in a congregate setting. Because viral nucleic acid can be detected persistently after recovery, we evaluated other laboratory markers to distinguish recruits who could proceed with training from those who were infected. METHODS: Recruits isolated for coronavirus disease 2019 (COVID-19) were serially tested for SARS-CoV-2 subgenomic ribonucleic acid (sgRNA), and viral load (VL) by reverse-transcriptase polymerase chain reaction (RT-PCR), and for anti- SARS-CoV-2. Cluster and quadratic discriminant analyses of results were performed. RESULTS: Among 229 recruits isolated for COVID-19, those with a RT-PCR cycle threshold >30.49 (sensitivity 95%, specificity 96%) or having sgRNA log10 RNA copies/mL <3.09 (sensitivity and specificity 96%) at entry into isolation were likely SARS-CoV-2 uninfected. Viral load >4.58 log10 RNA copies/mL or anti-SARS-CoV-2 signal-to-cutoff ratio <1.38 (VL: sensitivity and specificity 93%; anti-SARS-CoV-2: sensitivity 83%, specificity 79%) had comparatively lower sensitivity and specificity when used alone for discrimination of infected from uninfected. CONCLUSIONS: Orthogonal laboratory assays used in combination with RT-PCR may have utility in determining SARS-CoV-2 infection status for decisions regarding isolation.

A SARS-CoV-2 spike ferritin nanoparticle vaccine protects hamsters against Alpha and Beta virus variant challenge.

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a SARS-CoV-2 spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the Alpha (B.1.1.7), and Beta (B.1.351) VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 µg) or low (0.2 µg) dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose vaccinations. Dose-dependent SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques.

Emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean serum neutralizing antibody titers of 14,000 to 21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within 4 d in seven of eight animals receiving 50 µg of RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only approximately twofold relative to WA1/2020. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-CoV-like Sarbecovirus vaccine development.

A SARS-CoV-2 spike ferritin nanoparticle vaccine protects against heterologous challenge with B.1.1.7 and B.1.351 virus variants in Syrian golden hamsters.

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the B.1.1.7 and B.1.351 VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 µg) or low (0.2 µg) immunogen dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose two vaccinations. SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

The human immune response to saliva of Phlebotomus alexandri, the vector of visceral leishmaniasis in Iraq, and its relationship to sand fly exposure and infection.

BACKGROUND: Sand fly saliva exposure plays an important role in immunity against leishmaniasis where it has mostly been associated with protection. Phlebotomus (Ph.) alexandri transmits Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), in Iraq. Our group recently demonstrated that 20% of Operation Iraqi Freedom (OIF) deployers had asymptomatic VL (AVL) indicative of prior infection by the parasite L. infantum. Little is known about Ph. alexandri saliva, and the human immune response to it has never been investigated. Here, we characterize the humoral and cellular immune response to vector saliva in OIF deployers naturally exposed to bites of Ph. alexandri and characterize their immunological profiles in association to AVL. METHODOLOGY/PRINCIPAL FINDINGS: The humoral response to Ph. alexandri salivary gland homogenate (SGH) showed that 64% of 200 OIF deployers developed an antibody response. To assess the cellular immune response to saliva, we selected a subcohort of subjects based on their post-travel (median 4 months; range 1-22 months) antibody response (SGH Antibody [Ab] positive or negative) as well as their AVL status; ten never-traveled controls were also included. Banked peripheral blood mononuclear cells (PBMC), collected ~10 years after end of deployment, were stimulated with SGH for 96 hours. The levels of IFN- γ, IL-6, IL-10, IL-13 and IL-17 were determined by ELISA. Our findings indicate that OIF deployers mounted a cellular response to SGH where the anti-SGH+ asymptomatic subjects developed the highest cytokine levels. Further, stimulation with SGH produced a mixture of pro-inflammatory and anti-inflammatory cytokines. Contrary to our hypothesis, we observed no correlation between the cellular immune response to Ph. alexandri SGH and prevention from asymptomatic infection with L. infantum. CONCLUSIONS/SIGNIFICANCE: As we found, although all infected deployers demonstrated persistent disease control years after deployment, this did not correlate with anti-saliva systemic cellular response. More exposure to this vector may facilitate transmission of the L. infantum parasite. Since exposure to saliva of Ph. alexandri may alter the human immune response to bites of this vector, this parameter should be taken into consideration when considering the VL risk.

Asymptomatic Visceral Leishmania infantum Infection in US Soldiers Deployed to Iraq.

BACKGROUND: Visceral leishmaniasis (VL), due to Leishmania infantum, is a persistent intracellular parasitic infection transmitted by the bite of infected sand flies. Symptomatic VL has been reported in U.S. soldiers with Iraq deployment. Untreated symptomatic VL can be fatal; asymptomatic VL (AVL) may establish a lifelong risk of reactivation. We report prevalence and AVL risk factors in Operation Iraqi Freedom (OIF) deployers during 2002-11. METHODS: Healthy soldiers exposed to VL endemic areas in Iraq and 50 controls who never traveled to endemic regions were recruited through military healthcare facilities (2015-17). Responses to a risk factor survey and blood samples were obtained. Leishmania research diagnostics utilized included enzyme-linked immunosorbent assay (ELISA), rk39 test strips, quantitative polymerase chain reaction (PCR), and interferon gamma release (IGRA) assays. Statistical analyses included Fisher exact test, Pearson χ2 test, Mann-Whitney U test, and logistic regression. RESULTS: 200 deployed subjects were enrolled, mostly males (84.0%), of white ethnicity (79.0%), and median age 41 (range 24-61) years. 64% were seropositive for Phlebotomus alexandri saliva antibodies. Prevalence of AVL (any positive test result) was 39/200 (19.5%, 95% confidence interval 14.4%-25.8%). Two (1.0%) PCR, 10 (5%) ELISA, and 28 (14%) IGRA samples were positive. Travel to Ninewa governorate increased risk for AVL (P = .01). CONCLUSION: AVL was identified in 19.5% of OIF deployers; travel to northwest Iraq correlated with infection. Further studies are needed to inform risk for reactivation VL in US veterans and to target additional blood safety and surveillance measures.

Transcutaneous immunization using SLA or rLACK skews the immune response towards a Th1 profile but fails to protect BALB/c mice against a Leishmania major challenge.

Leishmaniasis is an expanding health threat worldwide complicated by the absence of an effective vaccine. We investigated transcutaneous immunization (TCI) as a needle-free immunization route which exploits the abundance of antigen presenting cells in the skin to induce both mucosal and systemic immunity. Leishmania (L.) major soluble antigens (SLA) or recombinant Leishmania homolog of receptors for activated C-kinase (rLACK) antigens were delivered transcutaneously together with cholera toxin (CT), to BALB/c mice. Mice were immunized at weeks 1, 4, and 7 with PBS, CT, SLA/CT or rLACK/CT. Two weeks after the final boost, antigen-specific IgG titers, IFN-γ ELISpot, and cytokine levels were assessed in half of the mice and the remainder were challenged with an intradermal (ear) injection of 5 × 104L. major metacyclic parasites. Mice were monitored weekly and sacrificed after 7 weeks to assess the parasite burden and to study the ear lesion immunohistopathology. Our results show that TCI with SLA or rLACK yielded high levels of anti-SLA, anti-rLACK and anti-CT IgG antibodies. A Th1-type of immune response was demonstrated with a high frequency of IFN-γ secreting cells, high levels of IFN-γ production, and lower levels of IL-10 resulting in a high IFN-γ/IL-10 ratio in mice immunized with SLA/CT or rLACK/CT. After parasite challenge, rLACK immunization was not associated with protection. In addition, SLA/CT immunized mice had larger ear lesions and an increased parasite load in the ear. Immunohistochemistry of ear biopsies stained for nitric oxide synthase revealed that staining intensity was diminished in the SLA/CT group compared to the control group. This finding suggested that less parasite killing occurred at the site of the infection. In conclusion, despite a strong Th1 type profile induced by TCI, exacerbation of infection occurred after challenge with L. major. This also correlated with low induction of nitric oxide.

The Immunology of a Healing Response in Cutaneous Leishmaniasis Treated with Localized Heat or Systemic Antimonial Therapy.

BACKGROUND: The effectiveness of systemic antimonial (sodium stibogluconate, Pentostam, SSG) treatment versus local heat therapy (Thermomed) for cutaneous leishmaniasis was studied previously and showed similar healing rates. We hypothesized that different curative immune responses might develop with systemic and local treatment modalities. METHODS: We studied the peripheral blood immune cells in a cohort of 54 cutaneous Leishmania major subjects treated with SSG or TM. Multiparameter flow cytometry, lymphoproliferative assays and cytokine production were analyzed in order to investigate the differences in the immune responses of subjects before, on and after treatment. RESULTS: Healing cutaneous leishmaniasis lead to a significant decline in circulating T cells and NKT-like cells, accompanied by an expansion in NK cells, regardless of treatment modality. Functional changes involved decreased antigen specific CD4+ T cell proliferation (hyporesponsiveness) seen with CD8+ T cell depletion. Moreover, the healing (or healed) state was characterized by fewer circulating regulatory T cells, reduced IFN-γ production and an overall contraction in polyfunctional CD4+ T cells. CONCLUSION: Healing from cutaneous Leishmaniasis is a dynamic process that alters circulating lymphocyte populations and subsets of T, NK and NKT-like cells. Immunology of healing, through local or systemic treatments, culminated in similar changes in frequency, quality, and antigen specific responsiveness with immunomodulation possibly via a CD8+ T cell dependent mechanism. Understanding the evolving immunologic changes during healing of human leishmaniasis informs protective immune mechanisms.

Deletion of ubiquitin fold modifier protein Ufm1 processing peptidase Ufsp in L. donovani abolishes Ufm1 processing and alters pathogenesis.

Previously, we showed Leishmania donovani Ufm1 has a Gly residue conserved at the C-terminal region with a unique 17 amino acid residue extension that must be processed prior to conjugation to target proteins. In this report, we describe for the first time the isolation and characterization of the Leishmania Ufm1-specific protease Ufsp. Biochemical analysis of L. donovani Ufsp showed that this protein possesses the Ufm1 processing activity using sensitive FRET based activity probes. The Ufm1 cleavage activity was absent in a mutant Ufsp in which the active site cysteine is altered to a serine. To examine the effects of abolition of Ufm1 processing activity, we generated a L. donovani null mutant of Ufsp (LdUfsp(-/-)). Ufm1 processing activity was abolished in LdUfsp(-/-) mutant, and the processing defect was reversed by re-expression of wild type but not the cys>ser mutant in the LdUfsp(-/-) parasites. Further LdUfsp(-/-) mutants showed reduced survival as amastigotes in infected human macrophages but not as promastigotes. This growth defect in the amastigotes was reversed by re-expression of wild type but not the cys>ser mutant in the Ufsp(-/-) indicating the essential nature of this protease for Leishmania pathogenesis. Further, mouse infection experiments showed deletion of Ufsp results in reduced virulence of the parasites. Additionally, Ufsp activity was inhibited by an anti-leishmanial drug Amphotericin B. These studies provide an opportunity to test LdUfsp(-/-) parasites as drug and vaccine targets.

Leishmania donovani argininosuccinate synthase is an active enzyme associated with parasite pathogenesis.

BACKGROUND: Gene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver. CONCLUSION/SIGNIFICANCE: Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target.

Immunity to visceral leishmaniasis using genetically defined live-attenuated parasites.

Leishmaniasis is a protozoan parasitic disease endemic to the tropical and subtropical regions of the world, with three major clinical forms, self-healing cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL). Drug treatments are expensive and often result in the development of drug resistance. No vaccine is available against leishmaniasis. Subunit Leishmania vaccine immunization in animal models has shown some efficacy but little or none in humans. However, individuals who recover from natural infection are protected from reinfection and develop life-long protection, suggesting that infection may be a prerequisite for immunological memory. Thus, genetically altered live-attenuated parasites with controlled infectivity could achieve such memory. In this paper, we discuss development and characteristics of genetically altered, live-attenuated Leishmania donovani parasites and their possible use as vaccine candidates against VL. In addition, we discuss the challenges and other considerations in the use of live-attenuated parasites.

Identification and characterization of genes involved in leishmania pathogenesis: the potential for drug target selection.

Identifying and characterizing Leishmania donovani genes and the proteins they encode for their role in pathogenesis can reveal the value of this approach for finding new drug targets. Effective drug targets are likely to be proteins differentially expressed or required in the amastigote life cycle stage found in the patient. Several examples and their potential for chemotherapeutic disruption are presented. A pathway nearly ubiquitous in living cells targeted by anticancer drugs, the ubiquitin system, is examined. New findings in ubiquitin and ubiquitin-like modifiers in Leishmania show how disruption of those pathways could point to additional drug targets. The programmed cell death pathway, now recognized among protozoan parasites, is reviewed for some of its components and evidence that suggests they could be targeted for antiparasitic drug therapy. Finally, the endoplasmic reticulum quality control system is involved in secretion of many virulence factors. How disruptions in this pathway reduce virulence as evidence for potential drug targets is presented.

Cellular and humoral responses to Leishmania major virulence factors in healed cutaneous leishmaniasis and Mediterranean visceral leishmaniasis patients.

Cellular and humoral immune responses of healed cutaneous leishmaniasis and Mediterranean visceral leishmaniasis patients were evaluated against results for Leishmania major virulence proteins L. major protein disulfide isomerase (LmPDI) and mitogen-activated protein kinase kinase (MAPKK). Only MAPKK induces significant peripheral blood mononuclear cell proliferation with gamma interferon production as well as antibody responses. Thus, MAPKK may be of interest in Leishmania vaccination and serodiagnosis.

Identification and characterization of a new Leishmania major specific 3'nucleotidase/nuclease protein.

We report the characterization of a new Leishmania major gene, lmaj3'nt/nu, encoding a 382 amino acids protein, Lmaj3'NT/NU, that belongs to the 3'nucleotidase/nuclease family. Interestingly, sequence and phylogenetic analysis show that this protein is Leishmania major specific and thus constitutes a new 3'nucleotidase/nuclease subgroup. Lmaj3'NT/NU displays nuclease enzymatic activity and Western blot analysis shows that it is exclusively expressed in promastigotes. Immunofluorescence microscopy using a specific anti-Lmaj3'NT/NU shows that the protein has a plasma membrane localization. Surprisingly, contrary to the previously described Leishmania mexicana 3'NT/NU, lmaj3'nt/nu is not up-regulated when parasites are cultured under purine starvation conditions. Together, these findings suggest Lmaj3'NT/NU may constitute a new important compound of the L. major purine scavenging pathway and could be involved in sandfly parasite survival and colonization.

Identification of a new developmentally regulated Leishmania major large RAB GTPase.

Here, we describe for the first time a Leishmania specific gene encoding a large 610 amino-acid RAB GTPase (LmLRAB). LmLRAB displays high homologies with the RAB GTPase protein family between amino acids 34 and 284. It contains characteristic signatures of RAB proteins: 4 GTP binding domains, 5 RAB specific domains, 3 RAB subfamily-specific domains, and a prenylation site. lmlrab is a single copy gene, transcribed as a 3.5 kb mRNA, highly conserved in Leishmania species, and encodes a protein doublet of approximately 75 kDa. Immunofluorescence microscopy using LmLRAB-specific antibodies demonstrated that LmLRAB is confined in a structure adjacent to the kinetoplast probably corresponding to an early endosomal/golgi apparatus localization. Interestingly, using quantitative real-time RT-PCR, we showed that the lmlrab gene is up-regulated twice in amastigotes relative to promastigotes. These findings suggest that LmLRAB may play a potential role in Leishmania pathogenicity.