A localizing nanocarrier formulation enables multi-target immune responses to multivalent replicating RNA with limited systemic inflammation.

RNA vaccines possess significant clinical promise in counteracting human diseases caused by infectious or cancerous threats. Self-amplifying replicon RNA (repRNA) has been thought to offer the potential for enhanced potency and dose sparing. However, repRNA is a potent trigger of innate immune responses in vivo, which can cause reduced transgene expression and dose-limiting reactogenicity, as highlighted by recent clinical trials. Here, we report that multivalent repRNA vaccination, necessitating higher doses of total RNA, could be safely achieved in mice by delivering multiple repRNAs with a localizing cationic nanocarrier formulation (LION). Intramuscular delivery of multivalent repRNA by LION resulted in localized biodistribution accompanied by significantly upregulated local innate immune responses and the induction of antigen-specific adaptive immune responses in the absence of systemic inflammatory responses. In contrast, repRNA delivered by lipid nanoparticles (LNPs) showed generalized biodistribution, a systemic inflammatory state, an increased body weight loss, and failed to induce neutralizing antibody responses in a multivalent composition. These findings suggest that in vivo delivery of repRNA by LION is a platform technology for safe and effective multivalent vaccination through mechanisms distinct from LNP-formulated repRNA vaccines.

Correlates of GLA family adjuvants' activities.

Lipopolysaccharide (LPS) is a well-defined agonist of Toll-like receptor (TLR) 4 that activates innate immune responses and influences the development of the adaptive response during infection with Gram-negative bacteria. Many years ago, Dr. Edgar Ribi separated the adjuvant activity of LPS from its toxic effects, an effort that led to the development of monophosphoryl lipid A (MPL). MPL, derived from Salmonella minnesota R595, has progressed through clinical development and is now used in various product-enabling formulations to support the generation of antigen-specific responses in several commercial and preclinical vaccines. We have generated several synthetic lipid A molecules, foremost glucopyranosyl lipid adjuvant (GLA) and second-generation lipid adjuvant (SLA), and have advanced these to clinical trial for various indications. In this review we summarize the potential and current positioning of TLR4-based adjuvant formulations in approved and emerging vaccines.

Adjuvants.

Developing new vaccines against emerging pathogens or pathogens where variability of antigenic sites presents a challenge, the inclusion of stimulators of the innate immune system is critical to mature the immune response in a way that allows high avidity recognition while preserving the ability to react to drifted serovars. The innate immune system is an ancient mechanism for recognition of nonself and the first line of defense against pathogen insult. By triggering innate receptors, adjuvants can boost responses to vaccines and enhance the quality and magnitude of the resulting immune response. This chapter: (1) describes the innate immune system, (2) provides examples of how adjuvants are formulated to optimize their effectiveness, and (3) presents examples of how adjuvants can improve outcomes of immunization.

Evolution of antigen-specific immune responses in cutaneous leishmaniasis patients.

AIMS: Despite immunization appearing to be the most appropriate strategy for long-term control of the vector-borne leishmaniases, no sustainable vaccine is currently available against any form of leishmaniasis. We therefore evaluated, in the context of vaccine antigen candidates, antigen-specific immune response at various stages of cutaneous leishmaniasis (CL). METHODS AND RESULTS: Peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers and CL patients (caused by either Leishmania major or L tropica) were incubated with crude Leishmania proteins (soluble Leishmania antigen; SLA), single recombinant proteins (TSA, LeIF, LmSTI1) or chimeric fusion proteins (LEISH-F2 and LEISH-F3). The concentrations of immune modulatory cytokines were then determined. While we did not detect appreciable antigen-specific IL-5 secretion, SLA induced secretion of interleukin (IL)-10 in cultures from early active lesion CL patients and even from healthy individuals. Conversely, interferon (IFN)-γ responses to SLA and recombinant proteins followed a similar pattern, developing only in the late active CL lesion phase. Once established, antigen-specific IFN-γ responses persisted in cured CL patients. CONCLUSION: Together, our results provide further insight into the development of immune responses during CL and further validate the selection of LEISH-F2 and LEISH-F3 as vaccine antigen candidates.

Skin tests for the detection of Mycobacterial infections: achievements, current perspectives, and implications for other diseases.

Immunological and molecular advances have modernized diagnostic testing for many diseases. Although interferon gamma-release and polymerase chain reaction assays have been developed to detect Mycobacterium tuberculosis (Mtb) infection, purified protein derivative (PPD)-based tuberculin skin testing (TST) remains the most widely used method. Indeed, the TST is a simple and cost-effective tool that can be easily applied for widespread screening for Mtb infection. However, the lack of specificity has been a limitation of these tests, and, more recently, supply issues have arisen. Building upon the skin tests that historically have been used within TB and leprosy control programs, we discuss recent developments using modern technologies for improving mycobacterial skin testing as well as practical advantages inherent to the technique. Furthermore, we outline how this knowledge could be applied to develop similar tests that could benefit diagnostic strategies for other infections. KEY POINTS: • Skin testing provides a significantly cheaper alternative to most modern technologies. • Skin tests provide a lab-independent diagnostic strategy that can be widely administered. • Diseases for which T cell responses are more robust or durable than antibody responses are accessible for skin testing.

Computationally Designed Bispecific MD2/CD14 Binding Peptides Show TLR4 Agonist Activity.

Toll-like receptor 4 plays an important role in the regulation of the innate and adaptive immune response. The majority of TLR4 activators currently in clinical use are derivatives of its prototypic ligand LPS. The discovery of innovative TLR4 activators has the potential of providing new therapeutic immunomodulators and adjuvants. We used computational design methods to predict and optimize a total of 53 cyclic and linear peptides targeting myeloid differentiation 2 (MD2) and cluster of differentiation 14 (CD14), both coreceptors of human TLR4. Activity of the designed peptides was first assessed using NF-κB reporter cell lines expressing either TLR4/MD2 or TLR4/CD14 receptors, then binding to CD14 and MD2 confirmed and quantified using MicroScale Thermophoresis. Finally, we incubated select peptides in human whole blood and observed their ability to induce cytokine production, either alone or in synergy with LPS. Our data demonstrate the advantage of computational design for the discovery of new TLR4 peptide activators with little structural resemblance to known ligands and indicate an efficient strategy with which to identify TLR4 targeting peptides that could be used as easy-to-produce alternatives to LPS-derived molecules in a variety of settings.

Development of LepReact, a defined skin test for paucibacillary leprosy and low-level M. leprae infection.

The persistence of new leprosy cases in endemic areas such as India, Brazil, Bangladesh, and the Philippines has encouraged studies of chemoprophylaxis among contacts of patients. Epidemiological screening tools to enable early detection of infected individuals in endemic populations would be critical to target individuals most in need of intervention. Despite decades of attempts, however, there still are no tests available for the early detection of low-level infection with Mycobacterium leprae. In this report, we describe the development of a leprosy skin test using M. leprae-specific antigens. We selected the chimeric LID-1 fusion protein, formulated to achieve maximum performance at a minimal dose, as a skin test candidate based on its ability to elicit delayed-type hypersensitivity (DTH) reactions in M. leprae immune guinea pigs in a sensitive and specific manner, i.e., with no cross-reactivity observed with other mycobacterial species. Importantly, evaluations in armadillos indicated that intradermal inoculation of formulated LID-1 could distinguish uninfected from M. leprae-infected animals manifesting with symptoms distinctly similar to the PB presentation of patients. Together, our data provide strong proof-of-concept for developing an antigen-specific skin test to detect low-level M. leprae infection. Such a test could, when applied with appropriate use of chemo- and/or immunoprophylaxis, be instrumental in altering the evolution of clinical disease and M. leprae transmission, thus furthering the objective of zero leprosy.

Social and environmental conditions related to Mycobacterium leprae infection in children and adolescents from three leprosy endemic regions of Colombia.

BACKGROUND: Leprosy is is still considered a public health issue and in Colombia 7-10% of new cases are found in children, indicating both active transmission and social inequality. We hypothesized that circulating antibodies against Natural Octyl Disaccharide-Leprosy IDRI Diagnostic (NDO-LID) (a combination of Mycobacterium leprae antigens) could reveal the social and environmental aspects associated with higher frequencies of M. leprae infection among children and adolescents in Colombia. METHODS: An observational cross-sectional study was conducted involving sampling from 82 children and adolescents (younger than 18 years of age) who had household contact with index leprosy patients diagnosed in the last 5 years. Data were analyzed through bivariate analysis made by applying a Pearson x2 test for qualitative variables, while quantitative variables, depending on their distribution, were analyzed using either a Student's t-test or Mann-Whitney U test. Multivariate analysis was performed using a multiple regression and binomial logistic approach. RESULTS: A bivariate analysis demonstrated that antibody titers against NDO-LID were significantly greater in children and adolescents with a low socioeconomic status that had: lived in vulnerable areas of the UAChR shared region; eaten armadillo meat; exposure of over 10 years to an index case and; not received BCG immunization. Moreover, a multivariate analysis showed that residing in the UAChR region has a strong association with a greater possibility of M. leprae infection. CONCLUSIONS: M. leprae transmission persists among young Colombians, and this is associated with social and environmental conditions. An intensification of efforts to identify new leprosy cases in vulnerable and forgotten populations where M. leprae transmission continues therefore appears necessary.

IL-18 and Subcapsular Lymph Node Macrophages are Essential for Enhanced B Cell Responses with TLR4 Agonist Adjuvants.

Designing modern vaccine adjuvants depends on understanding the cellular and molecular events that connect innate and adaptive immune responses. The synthetic TLR4 agonist glycopyranosyl lipid adjuvant (GLA) formulated in a squalene-in-water emulsion (GLA-SE) augments both cellular and humoral immune responses to vaccine Ags. This adjuvant is currently included in several vaccines undergoing clinical evaluation including those for tuberculosis, leishmaniasis, and influenza. Delineation of the mechanisms of adjuvant activity will enable more informative evaluation of clinical trials. Early after injection, GLA-SE induces substantially more Ag-specific B cells, higher serum Ab titers, and greater numbers of T follicular helper (TFH) and Th1 cells than alum, the SE alone, or GLA without SE. GLA-SE augments Ag-specific B cell differentiation into germinal center and memory precursor B cells as well as preplasmablasts that rapidly secrete Abs. CD169+ SIGNR1+ subcapsular medullary macrophages are the primary cells to take up GLA-SE after immunization and are critical for the innate immune responses, including rapid IL-18 production, induced by GLA-SE. Depletion of subcapsular macrophages (SCMф) or abrogation of IL-18 signaling dramatically impairs the Ag-specific B cell and Ab responses augmented by GLA-SE. Depletion of SCMф also drastically reduces the Th1 but not the TFH response. Thus the GLA-SE adjuvant operates through interaction with IL-18-producing SCMф for the rapid induction of B cell expansion and differentiation, Ab secretion, and Th1 responses, whereas augmentation of TFH numbers by GLA-SE is independent of SCMф.

Utility of immunoglobulin isotypes against LID-1 and NDO-LID for, particularly IgG1, confirming the diagnosis of multibacillary leprosy.

BACKGROUND Leprosy remains a health problem in many countries, with difficulties in diagnosis resulting in delayed treatment and more severe disabilities. Antibodies against several Mycobacterium leprae antigens have, however, shown value as diagnostic and/or prognostic markers. OBJECTIVES The objective of this study was to evaluate serum immunoglobulin (Ig) IgM and IgG subclass reactivity against three M. leprae specific antigens: NDO-HSA, a conjugate formed by natural octyl disaccharide bound to human serum albumin; LID-1, the fusion protein product of the ml0405 and ml2331 genes; and NDO-LID, a combination of LID-1 and NDO. METHODS Sera from healthy controls, paucibacillary (PB) and multibacillary (MB) leprosy patients, and their respective household contacts, were evaluated for the presence of antigen-specific IgM, IgG, and IgG subclass antibodies by enzyme-linked immunosorbent assay (ELISA). The sensitivity and specificity of each ELISA were evaluated by receiver operating characteristic (ROC) curve analysis. FINDINGS Our data confirm that serum IgM antibodies against NDO-HSA and IgG antibodies against LID-1, as well as IgG/M antibodies against NDO-LID, are markedly increased in MB patients. For the first time, our data reveal a selective increase in IgG1 and IgG3 antibodies against LID-1 and NDO-LID in MB patients, demonstrating that these antibody isotypes are suitable for differentiation between MB and PB patients. ROC curve analysis indicates an improved capacity for diagnosing MB leprosy patients using the detection of IgG antibodies, particularly the IgG1 isotype, specific to LID-1 and NDO-LID over the performance levels attained with NDO-HSA. CONCLUSIONS Our findings indicate that serological tests based on the detection of antigen-specific IgG1 antibodies are a useful tool to differentiate MB from PB patients, and indicate the enhanced performance of the LID-1 and NDO-LID antigens in the serodiagnosis of leprosy.

Use of Recombinant Antigens for Sensitive Serodiagnosis of American Tegumentary Leishmaniasis Caused by Different Leishmania Species.

American tegumentary leishmaniasis (ATL) (also known as cutaneous leishmaniasis [CL]) is caused by various species of protozoa of the genus Leishmania The diagnosis is achieved on a clinical, epidemiological, and pathological basis, supported by positive parasitological exams and demonstration of leishmanin delayed-type hypersensitivity. Serological assays are not routinely used in the diagnosis because many are considered to have low sensitivity and the particular Leishmania species causing the disease can lead to variable performance. In the present study, we generated recombinant versions of two highly conserved Leishmania proteins, Leishmania (Viannia) braziliensis-derived Lb8E and Lb6H, and evaluated both in enzyme-linked immunosorbent assays (ELISA). Recombinant Lb6H (rLb6H) had better performance and reacted with 100.0% of the ATL and 89.4% of the VL samples. These reactions with rLb6H were highly specific (98.5%) when compared against those for samples from healthy control individuals. We then assessed rLb6H against sera from ATL patients infected with different species of Leishmania prevalent in Brazil [Leishmania (Leishmania) amazonensis, L (Viannia) braziliensis, and L (V) guyanensis] and samples from patients with other infectious diseases. In analyses of 500 sera, ELISA using rLb6H detected all 219 ATL samples (sensitivity of 100.0%) with an overall specificity of 93.9% (considering healthy individuals and other infectious diseases patients). Only a minority of samples from Chagas disease patients possessed antibodies against rLb6H, and all of these responses were low (with a highest reactivity index of 2.2). Taken together, our data support further evaluation of rLb6H and the potential for its routine use in the serological diagnosis of ATL.

Semi-quantitative measurement of asymptomatic L. infantum infection and symptomatic visceral leishmaniasis in dogs using Dual-Path Platform® CVL.

Infection with Leishmania causes diseases with variable presentation. The most severe form is visceral leishmaniasis (VL), caused by either L. donovani or L. infantum. Despite efforts to eliminate VL, to date, molecular detection in resource-poor settings have lacked the accuracy and rapidity that would enable widespread field use and the need for accurate, sensitive assays to detect asymptomatic Leishmania infection has become apparent. The domestic dog serves as the primary reservoir host of L. infantum. Study of this reservoir population provides an opportunity to evaluate the sensitivity and specificity of diagnostics for well-defined, symptomatic, canine visceral leishmaniasis (CVL) and asymptomatic L. infantum infection. Blood samples from an L. infantum-endemic population of US hunting dogs were evaluated with Dual-Path Platform (DPP®) CVL compared to those obtained via direct detection methods (culture- and Leishmania-specific quantitative polymerase chain reaction, qPCR) and immunofluorescence anti-Leishmania antibody test (IFAT). Statistically significant correlations were found between DPP® CVL development time and clinical status, culture status, circulating DNA levels, and IFAT titer. DPP® CVL results correlated with both clinical severity of disease and serological evidence of asymptomatic L. infantum infection. By precisely documenting the minimum time required for the development of a clear positive result in DPP® CVL, this test could be used in a rapid, semi-quantitative manner for the evaluation of asymptomatic and symptomatic CVL. Our results also indicate that a similar test could be used to improve our understanding of human VL.

Squalene emulsion potentiates the adjuvant activity of the TLR4 agonist, GLA, via inflammatory caspases, IL-18, and IFN-γ.

The synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) is a potent Th1-response-inducing adjuvant when formulated in a squalene oil-in-water emulsion (SE). While the innate signals triggered by TLR4 engagement are well studied, the contribution of SE remains unclear. To better understand the effect of SE on the adjuvant properties of GLA-SE, we compared the innate and adaptive immune responses elicited by immunization with different formulations: GLA without oil, SE alone or the combination, GLA-SE, in mice. Within the innate response to adjuvants, only GLA-SE displayed features of inflammasome activation, evidenced by early IL-18 secretion and IFN-γ production in memory CD8(+) T cells and neutrophils. Such early IFN-γ production was ablated in caspase-1/11(-/-) mice and in IL-18R1(-/-) mice. Furthermore, caspase-1/11 and IL-18 were also required for full Th1 CD4(+) T-cell induction via GLA-SE. Thus, we demonstrate that IL-18 and caspase-1/11 are components of the response to immunization with the TLR4 agonist/squalene oil-in-water based adjuvant, GLA-SE, providing implications for other adjuvants that combine oils with TLR agonists.

Accurate Serodetection of Asymptomatic Leishmania donovani Infection by Use of Defined Antigens.

Infection with Leishmania donovaniis typically asymptomatic, but a significant number of individuals may progress to visceral leishmaniasis (VL), a deadly disease that threatens 200 million people in areas where it is endemic. While diagnosis of acute VL has been simplified by the use of cost-effective confirmatory serological tests, similar standardized tools are not widely available for detecting asymptomatic infection, which can be 4 to 20 times more prevalent than active disease. A simple and accurate serological test that is capable of detecting asymptomaticL. donovaniinfection will be useful for surveillance programs targeting VL control and elimination. To address this unmet need, we evaluated recombinant antigens for their ability to detect serum antibodies in 104 asymptomaticL. donovani-infected individuals (qualified as positive forL. donovani-specific antibodies by direct agglutination test [DAT]) from the Mymensingh district of Bangladesh where VL is hyperendemic. The novel proteins rKR95 and rTR18 possessed the greatest potential and detected 69% of DAT-positive individuals, with rKR95 being more robust in reactivity. Agreement in results for individuals with high DAT responses, who are more likely to progress to VL disease, was 74%. When considered along with rK39, a gold standard antigen that is used to confirm clinical diagnosis of VL but that is now becoming widely used for surveillance, rKR95 and rTR18 conferred a sensitivity of 84% based on a theoretical combined estimate. Our data indicate that incorporating rKR95 and rTR18 with rK39 in serological tests amenable to rapid or high-throughput screening may enable simple and accurate detection of asymptomatic infection. Such tests will be important tools to measureL. donovaniinfection rates, a primary goal in surveillance and a critical measurement with which to assess elimination programs.

Comparative evaluation of antibody detection tests to facilitate the diagnosis of multibacillary leprosy.

Despite control efforts, leprosy persists as a significant health concern in many regions. Diagnosis is achieved by a combination of clinical, histopathological, and bacteriological examinations, each of which presents a barrier to expeditious diagnosis, particularly by non-experts. Immunological investigations in research laboratories have clearly indicated that antibody detection tests could aid the diagnosis of leprosy. In this study, we detected circulating antibodies with two rapid diagnostic tests (RDT) involving immunochromatographic lateral flow platforms and one rapid ELISA system. Leprosy patients were identified with a high degree of sensitivity in each assay (over 80% in all; over 90% among cases with bacterial indices >1+), although critical differences were observed in specificity. While the specificity of CTK OnSite Leprosy Ab Rapid Test and InBios Leprosy Detect™ fast ELISA were high (96.4 and 93.7% in the general population, respectively), there was a marked reduction in OrangeLife NDO-LID® RDT (only 25.0%). As anticipated, seropositivity rates were marginally higher in contacts of leprosy patients than in endemic controls. Although we observed a slight drop in test band intensity when blood, rather than serum, was used to develop OnSite Leprosy Ab Rapid Tests, the sensitivity and specificity of these tests was unaffected. When we contrasted test performance with clinical and bacteriological information, we found that RDT and ELISA results positively correlated with the bacteriological index. These data indicate that these assays could be a ready replacement of invasive, insensitive, and time consuming skin slit smear procedures that additionally require expert microscopic examinations. We propose that, due to their speed and point of care applicability, the RDT could be used as an initial entry point to the diagnostic protocols, with confirmation of results attained in a highly quantitative manner following serum transfer to a reference laboratory.

Screening and identification of immunoactive peptide mimotopes for the enhanced serodiagnosis of tuberculosis.

Although serological detection is a practical strategy for early detection and diagnosis of tuberculosis (TB), inconsistent and imprecise estimates of sensitivity and specificity block its development and application for clinic. New or alternative serological antigens with improved accuracy are urgently needed. A phage-displayed random peptide library was employed to screen for immunoactive peptides using specific immunoglobulin G (IgG) of TB patients as target molecules. With two screening strategies, 20 single phages displaying different sequences were obtained and no sequence homology was found among these phages. From the results of phage-ELISA, H12, TB6, TB15, and TB18 phages showed higher affinity to IgGs from TB patients(S/N ≥2.1) and were identified as the positive clones. Significant differences in the detection values of sera from 47 TB patients and 37 healthy individuals were found for these four phage clones. According to the reactivity of 284 human sera to synthetic H12, TB6, TB15, and TB18 peptides as determined by ELISA, TB15 showed significantly higher areas under the curve (AUC) and sensitivity than other peptides, providing a lead molecule for the development of new serology diagnostic strategies for TB.

Need for, and acceptability of, rapid diagnostic tests that can facilitate the diagnosis of leprosy.

New cases of leprosy indicate that M. leprae transmission is continuing. In the first half of 2015 we sequentially diagnosed new leprosy patients attending Cebu Skin Clinic, a referral and leprosy treatment centre in Cebu City, Philippines. The great majority of patients were characterised as multibacillary (145 of 147; 98·6%), most of whom had bacterial indices 3þ (86; 58·5%) or more than 20 skin lesions (94; 63·9%). Importantly, 65·3% estimated that slightly over a year had elapsed between their initial symptoms and diagnosis, while 26·5% reported a lapse of greater than 3 years. Many patients (73·8%) and their household contacts (79·2%) reported that their lives had already been adversely affected. This indicates that improvements to expedite diagnosis are required, and rapid diagnostic tests (RDT) appear suited for this. Questionnaires were conducted among various groups to ascertain the acceptability and perception of RDT. All groups responded favourably, with 95·9% patients, 93·2% contacts and 81·4% of the general population responding that RDT would be beneficial for leprosy. The vast majority of patients (88·6%) indicated that they would 'definitely' submit to testing, followed by 69·4% of contacts and 72·2% of the general population. The majority of patients thought their household members should also be tested, while a subset indicated that RDT use should be extended to their respective communities. We propose that, due to their ease of use, point of care applicability and acceptability within target populations, RDT could be used as an entry point to inform the diagnostic process for leprosy.

Tuberculosis and leprosy infections in the Marshallese population of Arkansas, USA.

The cross-immunity between tuberculosis and leprosy is unknown. The aim of this pilot study was to evaluate the occurrence of Mycobacterium tuberculosis and M. leprae infection in Marshallese adult volunteers in Springdale, Arkansas, U.S.A., a population that experiences high rates of leprosy and tuberculosis. We used immunodiagnostic testing for tuberculosis and leprosy infection and found significant prevalence of latent tuberculosis infection (19.0%), and asymptomatic Mycobacterium leprae infection (22.2%). We found a negative association between presence of antibodies to Mycobacterium leprae and a positive interferon-γ release assay for Mycobacterium tuberculosis infection, prevalence odds ratio = 0.1 (95% CI = 0.0, 0.9). Although these findings require confirmation on a larger scale, they are supportive of the existence of cross-immunity.

Interleukin 17A acts synergistically with interferon γ to promote protection against Leishmania infantum infection.

Interleukin 17 (IL-17) is an inflammatory cytokine that plays a protective role against intracellular parasites. The role of IL-17 during Leishmania infection remains controversial and poorly defined. We evaluated whether IL-17 participates in the host immune response to Leishmania infantum. IL-17A is present in sera from patients with visceral leishmaniasis and decreases after successful treatment. In C57BL/6 infected mice, higher production of IL-17A coincided with the peak of parasitism. Il17ra(-/-) mice were more susceptible to infection and also exhibited reduced inflammatory infiltration and interferon γ (IFN-γ)-expressing CD4+ T-cell frequencies than wild-type mice. The frequencies of FoxP3+ CD4+ T cells and interleukin 10 (IL-10)-expressing CD4+ T cells were increased in Il17ra(-/-) mice. We also demonstrated that IL-17A acts synergistically with IFN-γ to potentiate NO production and leishmanicidal activity in infected macrophages. Therefore, our results indicate that L. infantum induces IL-17A production, which promotes the control of parasite replication by strengthening T-helper type 1 responses and NO production and prevents regulatory T-cell and IL-10-expressing T-cell expansion.

Zoonotic Leprosy in the Southeastern United States.

Nine-banded armadillos (Dasypus novemcinctus) are naturally infected with Mycobacterium leprae and have been implicated in zoonotic transmission of leprosy. Early studies found this disease mainly in Texas and Louisiana, but armadillos in the southeastern United States appeared to be free of infection. We screened 645 armadillos from 8 locations in the southeastern United States not known to harbor enzootic leprosy for M. leprae DNA and antibodies. We found M. leprae-infected armadillos at each location, and 106 (16.4%) animals had serologic/PCR evidence of infection. Using single-nucleotide polymorphism variable number tandem repeat genotyping/genome sequencing, we detected M. leprae genotype 3I-2-v1 among 35 armadillos. Seven armadillos harbored a newly identified genotype (3I-2-v15). In comparison, 52 human patients from the same region were infected with 31 M. leprae types. However, 42.3% (22/52) of patients were infected with 1 of the 2 M. leprae genotype strains associated with armadillos. The geographic range and complexity of zoonotic leprosy is expanding.