Angela J. Toepp

Regulatory IgDhi B Cells Suppress T Cell Function via IL-10 and PD-L1 during Progressive Visceral Leishmaniasis

During visceral leishmaniasis (VL), Th1-based inflammation is induced to control intracellular parasites. Inflammation-based pathology was shown to be dampened by IL-10 and eventual programmed death 1–mediated T cell exhaustion. Cell type(s) responsible for the initiation of T cell–produced IL-10 during VL are unknown. CD19+, CD5−, CD1d−, IgDhi regulatory B cells from healthy controls produced IL-10 in the absence of infection or stimulation, in contrast to IgDlo/neg B cells. IgDhi B cells may have a de novo versus induced regulatory program. The population of IgDhi B cells increased 3-fold as VL progressed. B cells from VL dogs were necessary and sufficient to suppress Th1 cell effector function. IgDhi B cells induced IL-10 production by T cells and IgDlo B cells. Blockage of B cell–specific PD-L1 restored Th1 responses. IgDhi regulatory B cells represent a novel regulatory B cell that may precipitate T cell exhaustion during VL.

Immunologic progression of canine leishmaniosis following vertical transmission in United States dogs

Canine leishmaniosis (CanL) is caused by Leishmania infantum, an obligate intracellular protozoan parasite, endemic in U.S. hunting dog populations. CanL has been found in dogs in 28 states and two Canadian provinces. Previous studies by our group, (Boggiatto et al., 2011), demonstrated that vertical transmission of Leishmania was the predominant means of transmission within U.S. dogs. Very little is known regarding how this alternative means of transmission, alters the long-term immunity and clinical presentation of leishmaniosis in dogs born to a positive bitch. This study follows the immunological progression of CanL in three pups after birth to an infected bitch. During the course of the study, these dogs were tested every six months over the course of six years. Both immunologic (IFN-γ, T cell proliferation, antibody production) and parasitological parameters (qPCR) of vertically-infected dogs were measured. Within the six years after birth to an L. infantum-infected, oligosymptomatic bitch, all dogs had at least one L. infantum PCR-positive test. Interestingly, despite living in the same location for their entire lives and being full siblings, these pups demonstrate three different disease progression patterns of L. infantum infection. One dog progressed to oligosymptomatic disease, maintaining a positive titer and had intermittent positive PCR results. One asymptomatic dog had positive serological titers and demonstrated a robust CD4(+) immune response to infection. The third dog had a negligible response to L. infantum antigen and was healthy. This work demonstrates the biologic variability associated with vertically-transmitted infection similar to the variety of presentations observed during vector-borne leishmaniosis.

Recovery of antigen-specific T cell responses from dogs infected with Leishmania (L.) infantum by use of vaccine associated TLR-agonist adjuvant

Visceral leishmaniasis (VL), caused by infection with the obligate intracellular protozoan parasite Leishmania infantum, is a fatal disease of dogs and humans. Protection against VL requires a T helper 1 (Th1) skewed CD4+ T response, but despite this knowledge, there are currently no approved-to-market vaccines for humans and only three veterinary-use vaccines globally. As VL progresses from asymptomatic to symptomatic, L. infantum-specific interferon gamma (IFNγ) driven-Th1 responses become dampened and a state of immune exhaustion established. T cell exhaustion and other immunoregulatory processes, starting during asymptomatic disease, are likely to hinder vaccine-induced responses if vaccine is administered to infected, but asymptomatic and seronegative, individuals. In this study we evaluated how immune exhaustion, shown previously by our group to worsen in concert with VL progression, effected the capacity of vaccine candidate antigen/toll-like receptor (TLR) agonist combinations to promote protective CD4+ T cell responses during progressive VL. In conjunction with Th1 responses, we also evaluated concomitant stimulation of immune-balanced IL-10 regulatory cytokine production by these vaccine products in progressive VL canine T cells. Vaccine antigen L111f in combination with TLR agonists significantly recovered CD4+ T cell IFNγ intracellular production in T cells from asymptomatic VL dogs. Vaccine antigen NS with TLR agonists significantly recovered CD4+ T cell production in both endemic control and VL dogs. Combinations of TLR agonists and vaccine antigens overcame L. infantum induced cellular exhaustion, allowing robust Th1 CD4+ T cell responses from symptomatic dogs that previously had dampened responses to antigen alone. Antigen-agonist adjuvants can be utilized to promote more robust vaccine responses from infected hosts in endemic areas where vaccination of asymptomatic, L. infantum-infected animals is likely.

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.

Safety Analysis of Leishmania Vaccine Used in a Randomized Canine Vaccine/Immunotherapy Trial

In Leishmania infantum-endemic countries, controlling infection within dogs, the domestic reservoir, is critical to public health. There is a need for safe vaccines that prevent canine progression with disease and transmission to others. Protective vaccination against Leishmania requires mounting a strong, inflammatory, Type 1 response. Three commercially available canine vaccines on the global veterinary market use saponin or inflammatory antigen components (Letifend) as a strong pro-inflammatory adjuvant. There is very little information detailing safety of saponin as an adjuvant in field trials. Safety analyses for the use of vaccine as an immunotherapeutic in asymptomatically infected animals are completely lacking. Leishmania infantum, the causative agent of canine leishmaniasis, is enzootic within U.S. hunting hounds. We assessed the safety of LeishTec® after use in dogs from two different clinical states: 1) without clinical signs and tested negative on polymerase chain reaction and serology or 2) without clinical signs and positive for at least one Leishmania diagnostic test. Vaccine safety was assessed after all three vaccinations to quantify the number and severity of adverse events. Vaccinated animals had an adverse event rate of 3.09%, whereas placebo animals had 0.68%. Receiving vaccine was correlated with the occurrence of mild, site-specific, reactions. Occurrence of severe adverse events was not associated with having received vaccine. Infected, asymptomatic animals did not have a higher rate of adverse events. Use of vaccination is, therefore, likely to be safe in infected, asymptomatic animals.

Randomized, controlled, double-blinded field trial to assess Leishmania vaccine effectiveness as immunotherapy for canine leishmaniosis

Better tools are necessary to eliminate visceral leishmaniasis (VL). Modeling studies for regional Leishmania elimination indicate that an effective vaccine is a critical tool. Dogs are the reservoir host of L. infantum in Brazil and the Mediterranean basin, and therefore are an important target for public health interventions as well as a relevant disease model for human VL. No vaccine has been efficacious as an immunotherapy to prevent progression of already diagnostically positive individuals to symptomatic leishmaniasis. We performed a double-blinded, block-randomized, placebo-controlled, vaccine immunotherapy trial testing the efficacy of a recombinant Leishmania A2 protein, saponin-adjuvanted, vaccine, LeishTec®, in owned hunting dogs infected with L. infantum. The primary outcome was reduction of clinical progression, with reduction of mortality as a secondary outcome. Vaccination as an immunotherapy reduced the risk of progression to clinically overt leishmaniasis by 25% in asymptomatic dogs (RR: 1.33 95% C.I. 1.009-1.786 p-value: 0.0450). Receiving vaccine vs. placebo reduced all-cause mortality in younger asymptomatic dogs by 70% (RR: 3.19 95% C.I.: 1.185-8.502 p-value = 0.0245). Vaccination of infected-healthy animals with an anti-Leishmania vaccine significantly reduced clinical progression and decreased all-cause mortality. Use of vaccination in infected-healthy dogs can be a tool for Leishmania control.

Leishmania-Derived Trimannose Modulates the Inflammatory Response To Significantly Reduce Leishmania major-Induced Lesions

ABSTRACT Leishmania lipophosphoglycan (LPG) is a key virulence factor, initiating inflammation resulting in cutaneous lesions. LPG is capped by various oligosaccharides. How these glycans are recognized and how they alter the course of Leishmania infection are poorly understood. Previous studies synthesized α-1,2-trimannose cap sugars on latex beads and demonstrated that C57BL/6 mice coinoculated with Leishmania major and trimannose-coated beads produced significantly higher levels of interleukin-12p40 (IL-12p40) and other proinflammatory, type 1 cytokines than mice inoculated with L. major alone within the first 48 h of infection. However, as L. major infection typically progress over weeks to months, the role of trimannose in altering disease progression over the course of infection was unknown. Wild-type mice were inoculated with either trimannose-coated or carrier (uncoated) beads, infected with L. major alone, coinoculated with carrier beads and L. major, or coinoculated with trimannose-coated beads and L. major. Trimannose treatment of L. major-infected mice decreased the parasite load and significantly decreased the lesion size at 14 days postinfection (p.i.) compared to results for nontreated, infected mice. Infected, trimannose-treated mice had decreased IL-12p40 and IL-10 secretion and increased interferon gamma secretion at 14 days p.i. Mannose receptor knockout (MR−/−) mice lack the ability to detect trimannose. When MR−/− mice were infected with L. major and treated with trimannose beads, they did not have decreased lesion size. Leishmania-derived trimannose represents a novel immunomodulator that provides early type 1-skewed cytokine production to control the parasite load and alter the course of cutaneous leishmaniasis.

Temporal Analysis of a Case Series of Leprosy Patients From a Low Endemic Area in Brazil and the Magnitude of Disability

Leprosy is an infectious disease caused by Mycobacterium leprae infection. The incubation period after the primary infection can be as long as decades. This bacterium has a tropism for macrophages and Schwann cells and as result neuropathies are frequent. The state of Rio Grande do Norte in northeastern Brazil has areas of low and high endemicity for leprosy. The aim of this study was to analyze a leprosy time series from Rio Grande do Norte, from 2005 to 2014, based on data provided by the Notifiable Diseases Information System from the State Secretariat of Health. This was a quantitative, descriptive and analytical study. We considered variables such as sex, age, clinical form, operational classification and disability grade at diagnosis. There were 3,426 cases of leprosy reported of which 1,781 were females (52%). Of those patients, 29% presented disability at diagnosis. The high level of disability in patients along with the lack of information regarding disability levels indicates the need for more effective measures, with early diagnosis to decrease morbidity as leprosy still remains as a serious public health problem.