Faculty Bibliography

Zika virus infection is associated with the development of Guillain-Barré syndrome (GBS), a neurological autoimmune disorder caused by immune recognition of gangliosides and other components at nerve membranes. Using a high-throughput ELISA, we have analyzed the anti-glycolipid antibody profile, including gangliosides, of plasma samples from patients with Zika infections associated or not with GBS in Salvador, Brazil. We have observed that Zika patients that develop GBS present higher levels of anti-ganglioside antibodies when compared to Zika patients without GBS. We also observed that a broad repertoire of gangliosides was targeted by both IgM and IgG anti-self antibodies in these patients. Since Zika virus infects neurons, which contain membrane gangliosides, antigen presentation of these infected cells may trigger the observed autoimmune anti-ganglioside antibodies suggesting direct infection-induced autoantibodies as a cause leading to GBS development. Collectively, our results establish a link between anti-ganglioside antibodies and Zika-associated GBS in patients.

Secretion of specific proteins contributes to pathogenesis and immune responses in tuberculosis and other bacterial infections, yet the kinetics of protein secretion and fate of secreted proteins in vivo are poorly understood. We generated new monoclonal antibodies that recognize the Mycobacteriumtuberculosis secreted protein Ag85B and used them to establish and characterize a sensitive enzyme-linked immunosorbent assay (ELISA) to quantitate Ag85B in samples generated in vitro and in vivo We found that nutritional or culture conditions had little impact on the secretion of Ag85B and that there is considerable variation in Ag85B secretion by distinct strains in the M. tuberculosis complex: compared with the commonly used H37Rv strain (lineage 4), Mycobacteriumafricanum (lineage 6) secretes less Ag85B, and two strains from lineage 2 secrete more Ag85B. We also used the ELISA to determine that the rate of secretion of Ag85B is 10- to 100-fold lower than that of proteins secreted by Gram-negative and Gram-positive bacteria, respectively. ELISA quantitation of Ag85B in lung homogenates of M. tuberculosis H37Rv-infected mice revealed that although Ag85B accumulates in the lungs as the bacterial population expands, the amount of Ag85B per bacterium decreases nearly 10,000-fold at later stages of infection, coincident with the development of T cell responses and arrest of bacterial population growth. These results indicate that bacterial protein secretion in vivo is dynamic and regulated, and quantitation of secreted bacterial proteins can contribute to the understanding of pathogenesis and immunity in tuberculosis and other infections.IMPORTANCE Bacterial protein secretion contributes to host-pathogen interactions, yet the process and consequences of bacterial protein secretion during infection are poorly understood. We developed a sensitive ELISA to quantitate a protein (termed Ag85B) secreted by M. tuberculosis and used it to find that Ag85B secretion occurs with slower kinetics than for proteins secreted by Gram-positive and Gram-negative bacteria and that accumulation of Ag85B in the lungs is markedly regulated as a function of the bacterial population density. Our results demonstrate that quantitation of bacterial proteins during infection can reveal novel insights into host-pathogen interactions.

Infection with M. tuberculosis is associated with inconsistent and incomplete elimination of the bacteria, despite development of antigen-specific T cell responses. One mechanism employed by M. tuberculosis is to limit availability of antigen for activation of CD4 T cells. We examined the utility of systemic administration of epitope peptides to activate pre-existing T cells in mice infected with M. tuberculosis. We found that systemic peptide administration: 1) selectively activates T cells specific for the epitope peptide; 2) loads MHC class II on lung macrophages and dendritic cells; 3) activates CD4 T cells in the lung parenchyma; 4) has little antimycobacterial activity. Further studies revealed that CD4 T cells in lung lesions are distant from the infected cells, suggesting that, even if they can be activated, the positioning of CD4 T cells and their direct interactions with infected cells may be limiting determinants of immunity in TB.

Mycobacterium tuberculosis causes chronic infection of mononuclear phagocytes, especially resident (alveolar) macrophages, recruited macrophages, and dendritic cells. Despite the importance of these cells in tuberculosis (TB) pathogenesis and immunity, little is known about the population dynamics of these cells at the sites of infection. We used a combination of congenic monocyte adoptive transfer, and pulse-chase labeling of DNA, to determine the kinetics and characteristics of trafficking, differentiation, and infection of mononuclear phagocytes during the chronic, adaptive immune phase of M. tuberculosis infection in mice. We found that Ly6Chi monocytes traffic rapidly to the lungs, where a subpopulation become Ly6Clo and remain in the lung vascular space, while the remainder migrate into the lung parenchyma and differentiate into Ly6Chi dendritic cells, CD11b+ dendritic cells, and recruited macrophages. As in humans with TB, M. tuberculosis-infected mice have increased numbers of blood monocytes; this is due to increased egress from the bone marrow, and not delayed egress from the blood. Pulse-chase labeling of dividing cells and flow cytometry analysis revealed a T1/2 of ~15 hrs for Ly6Chi monocytes, indicating that they differentiate rapidly upon entry to the parenchyma of infected lungs; in contrast, cells that differentiate from Ly6Chi monocytes turn over more slowly, but diminish in frequency in less than one week. New cells (identified by pulse-chase labeling) acquire bacteria within 1-3 days of appearance in the lungs, indicating that bacteria regularly encounter new cellular niches, even during the chronic stage of infection. Our findings that mononuclear phagocyte populations at the site of M. tuberculosis infection are highly dynamic provide support for specific approaches for host-directed therapies directed at monocytes, including trained immunity, as potential interventions in TB, by replacing cells with limited antimycobacterial capabilities with newly-recruited cells better able to restrict and kill M. tuberculosis.

Tuberculosis (TB) is a large global health problem, in part because of the long period of coevolution of the pathogen, Mycobacterium tuberculosis, and its human host. A major factor that sustains the global epidemic of TB is the lack of a sufficiently effective vaccine. While basic mechanisms of immunity that protect against TB have been identified, attempts to improve immunity to TB by vaccination have been disappointing. This Review discusses the mechanisms used by M. tuberculosis to evade innate and adaptive immunity and that likely limit the efficacy of vaccines developed to date. Despite multiple mechanisms of immune evasion, recent trials have indicated that effective TB vaccines remain an attainable goal. This Review discusses how knowledge from other systems can inform improvements on current vaccine approaches.

Antigen-specific CD4 and CD8 T cells are important components of the immune response toMycobacterium tuberculosis, yet little information is currently known regarding how the breadth, specificity, phenotype, and function ofM. tuberculosis-specific T cells correlate withM. tuberculosisinfection outcome in humans. To facilitate evaluation of humanM. tuberculosis-specific T cell responses targeting multiple different Ags, we sought to develop a high throughput and reproducible T cell response spectrum assay requiring low blood sample volumes. We describe here the optimization and standardization of a microtiter plate-based, diluted whole blood stimulation assay utilizing overlapping peptide pools corresponding to a functionally diverse panel of 60M. tuberculosisAgs. Using IFN-γ production as a readout of Ag specificity, the assay can be conducted using 50 μl of blood per test condition and can be expanded to accommodate additional Ags. We evaluated the intra- and interassay variability, and implemented testing of the assay in diverse cohorts ofM. tuberculosis-unexposed healthy adults, foreign-born adults with latentM. tuberculosisinfection residing in the United States, and tuberculosis household contacts with latentM. tuberculosisinfection in a tuberculosis-endemic setting in Kenya. TheM. tuberculosis-specific T cell response spectrum assay further enhances the immunological toolkit available for evaluatingM. tuberculosis-specific T cell responses across different states ofM. tuberculosisinfection, and can be readily implemented in resource-limited settings. Moreover, application of the assay to longitudinal cohorts will facilitate evaluation of treatment- or vaccine-induced changes in the breadth and specificity of Ag-specific T cell responses, as well as identification ofM. tuberculosis-specific T cell responses associated withM. tuberculosisinfection outcomes.

Two recent studies (Cambier et al., 2017; Madigan et al., 2017) reveal in vivo functions for specific phenolic glycolipids (PGLs) in the mycobacteria that cause tuberculosis or leprosy. M. tuberculosis (and M. marinum) PGL promotes bacterial spread to growth-permissive macrophages, while M. leprae PGL-1 induces macrophages to cause nerve demyelination characteristic of human leprosy.