Javeed A. Shah

Prime-Boost Vaccination with HIV-1 Gag Protein and Cytosine Phosphate Guanosine Oligodeoxynucleotide, Followed by Adenovirus, Induces Sustained and Robust Humoral and Cellular Immune Responses

A prophylactic vaccine for HIV-1 will probably require the induction and maintenance of both humoral and cellular immunity. One current strategy to achieve such long term immune responses is a prime-boost vaccination approach using a DNA priming inoculation, followed by recombinant viral boost. In this report we use a novel prime-boost approach in which the priming injections consist of recombinant HIV-1 Gag protein mixed with cytosine phosphate guanosine oligodeoxynucleotide (CpG ODN), followed by recombinant adenoviral boost expressing HIV-1 Gag. Analysis of the immune responses indicates that HIV-1 Gag protein plus CpG ODN immunization alone induces potent humoral as well as Th1 and CD8+ T cell responses. Boosting with recombinant adenovirus strikingly enhances CD8+, but not Th1, T cell responses, resulting in CD8+ T cell responses far greater in magnitude than Th1 responses. Furthermore, the Th1 and CD8+ T cell responses following prime-boost immunization were seen in both lymphoid and peripheral mucosal organs and were sustained over several months. Together, these data suggest a new immunization approach for elicitation of long term humoral and cellular immune responses.

Dendritic Cells Are Responsible for the Capacity of CpG Oligodeoxynucleotides to Act as an Adjuvant for Protective Vaccine Immunity Against Leishmania major in Mice

Vaccination with leishmanial Ag and CpG oligodeoxynucleotides (ODN) confers sustained cellular immunity and protection to infectious challenge up to 6 mo after immunization. To define the cellular mechanism by which CpG ODN mediate their adjuvant effects in vivo, the functional capacity of distinct dendritic cell (DC) subsets was assessed in the lymph nodes (LNs) of BALB/c mice, 36 h after immunization with the leishmanial antigen (LACK) and CpG ODN. After this immunization, there was a striking decrease in the frequency of the CD11c+B220+ plasmacytoid DCs with a proportionate increase in CD11c+CD8−B220− cells. CD11c+CD8+B220− cells were the most potent producers of interleukin (IL)-12 p70 and interferon (IFN)-γ, while plasmacytoid DCs were the only subset capable of secreting IFN-α. In terms of antigen presenting capacity, plasmacytoid DCs were far less efficient compared with the other DC subsets. To certify that DCs were responsible for effective vaccination, we isolated CD11c+ and CD11c− cells 36 h after immunization and used such cells to elicit protective immunity after adoptive transfer in naive, Leishmania major susceptible BALB/c mice. CD11c+ cells but not 10-fold higher numbers of CD11c− cells from such immunized mice mediated protection. Therefore, the combination of LACK antigen and CpG ODN adjuvant leads to the presence of CD11c+ DCs in the draining LN that are capable of vaccinating naive mice in the absence of further antigen or adjuvant.

Human TOLLIP regulates TLR2 and TLR4 signaling and its polymorphisms are associated with susceptibility to tuberculosis

Tuberculosis, one of the leading causes of death worldwide, stimulates inflammatory responses with beneficial and pathologic consequences. The regulation and nature of an optimal inflammatory response to Mycobacterium tuberculosis remains poorly understood in humans. Insight into mechanisms of negative regulation of the TLR-mediated innate immune response to M. tuberculosis could provide significant breakthroughs in the design of new vaccines and drugs. We hypothesized that TOLLIP and its common variants negatively regulate TLR signaling in human monocytes and are associated with susceptibility to tuberculosis. Using short hairpin RNA knockdown of TOLLIP in peripheral blood human monocytes, we found that TOLLIP suppresses TNF and IL-6 production after stimulation with TLR2 and TLR4 ligands. In contrast, secretion of the anti-inflammatory cytokine IL-10 was induced by TOLLIP. We also discovered two common polymorphisms that are associated with either decreased levels of mRNA expression (rs3750920) or increased IL-6 production (rs5743899) in a sample of 56 healthy volunteers. Furthermore, in a case-population study in Vietnam with 760 cord blood samples and 671 TB case patients, we found that SNPs rs3750920 and rs5743899 were associated with susceptibility to tuberculosis (p = 7.03 × 10−16 and 6.97 × 10−7, respectively). These data demonstrate that TOLLIP has an anti-inflammatory effect on TLR signaling in humans and that TOLLIP deficiency is associated with an increased risk of tuberculosis. To our knowledge, these data also show the first associations of TOLLIP polymorphisms with any infectious disease. These data also implicate an unexpected mechanism of negative regulation of TLR signaling in human tuberculosis pathogenesis.

New tricks for old dogs: countering antibiotic resistance in tuberculosis with host-directed therapeutics

Despite the availability of Mycobacterium tuberculosis (Mtb) drugs for over 50 years, tuberculosis (TB) remains at pandemic levels. New drugs are urgently needed for resistant strains, shortening duration of treatment, and targeting different stages of the disease, especially for treatment during human immunodeficiency virus co‐infection. One solution to the conundrum that antibiotics kill the bacillus yet select for resistance is to target the host rather than the pathogen. Here, we discuss recent progress in so‐called ‘host‐directed therapeutics’ (HDTs), focusing on two general mechanistic strategies: (i) HDTs that disrupt Mtb pathogenesis in macrophages and (ii) immunomodulatory HDTs that facilitate protective immune responses that kill Mtb or reduce deleterious responses that exacerbate disease. HDTs hold significant promise as adjunctive therapies in that they are less likely to engender resistance, will likely have efficacy against antibiotic‐resistant strains, and may have activity against non‐replicating Mtb. However, TB is a complex and variegated disease, and human populations exhibit significant diversity in their immune responses to it, which presents a complicated landscape for HDTs to navigate. Nevertheless, we suggest that a detailed mechanistic understanding of drug action, together with careful selection of disease stage targets and dosing strategies may overcome such limitations and allow the development of HDTs as effective adjunctive treatment options for TB.

Human ULK1 Variation and Susceptibility to Mycobacterium tuberculosis Infection

BACKGROUND Unlike tuberculosis, few studies have evaluated a host genetic basis for variability in susceptibility to latent Mycobacterium tuberculosis infection (LTBI). We performed a candidate gene association study of autophagy-related genes and LTBI. METHODS We enrolled close contacts of individuals with pulmonary tuberculosis, assessed LTBI status, and determined clinical and sociodemographic risk factors for LTBI. In participants who self-identified as Asian or black, we compared haplotype-tagging single-nucleotide polymorphisms (SNPs) in ULK1 and GABARAP between cases (n = 143) and controls (n = 106). Using CRISPR/Cas9 in U937 monocytes, we investigated the effect of ULK1 deficiency on cytokine expression, autophagy, and M. tuberculosis replication. RESULTS In Asian participants, we identified 2 ULK1 SNPs (rs12297124 and rs7300908) associated with LTBI. After adjustment for population admixture and clinical risk for LTBI, each rs12297124 minor allele conferred 80% reduction in LTBI risk (odds ratio, 0.18; 95% confidence interval, .07-.46). Compared with controls, ULK1-deficient cells exhibited decreased tumor necrosis factor secretion after stimulation with Toll-like receptor ligands and M. tuberculosis whole-cell lysate, increased M. tuberculosis replication, and decreased selective autophagy. CONCLUSIONS These results demonstrate a strong association of rs12297124, a noncoding ULK1 SNP, with LTBI and a role for ULK1 regulation of TNF secretion, nonspecific and M. tuberculosis-induced autophagy, and M. tuberculosis replication in monocytes.

Genetic Variation in Toll-Interacting Protein Is Associated With Leprosy Susceptibility and Cutaneous Expression of Interleukin 1 Receptor Antagonist

Leprosy is a chronic disease characterized by skin and peripheral nerve pathology and immune responses that fail to control Mycobacterium leprae. Toll-interacting protein (TOLLIP) regulates Toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) signaling against mycobacteria. We analyzed messenger RNA (mRNA) expression of candidate immune genes in skin biopsy specimens from 85 individuals with leprosy. TOLLIP mRNA was highly and specifically correlated with IL-1R antagonist (IL-1Ra). In a case-control gene-association study with 477 cases and 1021 controls in Nepal, TOLLIP single-nucleotide polymorphism rs3793964 TT genotype was associated with increased susceptibility to leprosy (recessive, P = 1.4 × 10(-3)) and with increased skin expression of TOLLIP and IL-1Ra. Stimulation of TOLLIP-deficient monocytes with M. leprae produced significantly less IL-1Ra (P < .001), compared with control. These data suggest that M. leprae upregulates IL-1Ra by a TOLLIP-dependent mechanism. Inhibition of TOLLIP may decrease an individuals susceptibility to leprosy and offer a novel therapeutic target for IL-1-dependent diseases.

A Functional Toll-Interacting Protein Variant Is Associated with Bacillus Calmette-Guérin–Specific Immune Responses and Tuberculosis

Rationale: The molecular mechanisms that regulate tuberculosis susceptibility and bacillus Calmette‐Guérin (BCG)‐induced immunity are mostly unknown. However, induction of the adaptive immune response is a critical step in host control of Mycobacterium tuberculosis. Toll‐interacting protein (TOLLIP) is a ubiquitin‐binding protein that regulates innate immune responses, including Toll‐like receptor signaling, which initiate adaptive immunity. TOLLIP variation is associated with susceptibility to tuberculosis, but the mechanism by which it regulates tuberculosis immunity is poorly understood. Objectives: To identify functional TOLLIP variants and evaluate the role of TOLLIP variation on innate and adaptive immune responses to mycobacteria and susceptibility to tuberculosis. Methods: We used human cellular immunology approaches to characterize the role of a functional TOLLIP variant on monocyte mRNA expression and M. tuberculosis‐induced monocyte immune functions. We also examined the association of TOLLIP variation with BCG‐induced T‐cell responses and susceptibility to latent tuberculosis infection. Measurements and Main Results: We identified a functional TOLLIP promoter region single‐nucleotide polymorphism, rs5743854, which was associated with decreased TOLLIP mRNA expression in infant monocytes. After M. tuberculosis infection, TOLLIP‐deficient monocytes demonstrated increased IL‐6, increased nitrite, and decreased bacterial replication. The TOLLIP‐deficiency G/G genotype was associated with decreased BCG‐specific IL‐2+ CD4+ T‐cell frequency and proliferation. This genotype was also associated with increased susceptibility to latent tuberculosis infection. Conclusions: TOLLIP deficiency is associated with decreased BCG‐specific T‐cell responses and increased susceptibility to tuberculosis. We hypothesize that the heightened antibacterial monocyte responses after vaccination of TOLLIP‐deficient infants are responsible for decreased BCG‐specific T‐cell responses. Activating TOLLIP may provide a novel adjuvant strategy for BCG vaccination.

The common HAQ STING variant impairs cGAS-dependent antibacterial responses and is associated with susceptibility to Legionnaires’ disease in humans

The cyclic GMP-AMP synthase (cGAS)-STING pathway is central for innate immune sensing of various bacterial, viral and protozoal infections. Recent studies identified the common HAQ and R232H alleles of TMEM173/STING, but the functional consequences of these variants for primary infections are unknown. Here we demonstrate that cGAS- and STING-deficient murine macrophages as well as human cells of individuals carrying HAQ TMEM173/STING were severely impaired in producing type I IFNs and pro-inflammatory cytokines in response to Legionella pneumophila, bacterial DNA or cyclic dinucleotides (CDNs). In contrast, R232H attenuated cytokine production only following stimulation with bacterial CDN, but not in response to L. pneumophila or DNA. In a mouse model of Legionnaires’ disease, cGAS- and STING-deficient animals exhibited higher bacterial loads as compared to wild-type mice. Moreover, the haplotype frequency of HAQ TMEM173/STING, but not of R232H TMEM173/STING, was increased in two independent cohorts of human Legionnaires’ disease patients as compared to healthy controls. Our study reveals that the cGAS-STING cascade contributes to antibacterial defense against L. pneumophila in mice and men, and provides important insight into how the common HAQ TMEM173/STING variant affects antimicrobial immune responses and susceptibility to infection. Trial registration ClinicalTrials.gov DRKS00005274, German Clinical Trials Register

Species-Specific Risk Factors, Treatment Decisions and Clinical Outcomes for Laboratory Isolates of Less Common Nontuberculous Mycobacteria in Washington State.

Rationale: Nontuberculous mycobacteria (NTM) are a diverse group of environmental organisms that infrequently cause human disease. Understanding of the epidemiologic and clinical characteristics associated with NTM disease is needed to refine diagnostic and treatment strategies, particularly among the less commonly isolated species. Objectives: To improve knowledge of geographic variance of NTM species, to correlate detailed clinical information with isolation of specific NTM, and to examine the decision to treat and outcomes for specific NTM. Methods: Mycobacterial cultures submitted to the University of Washington mycobacterial laboratory from 1998 to 2011 were examined. We report isolation frequency and demographic information from all samples with clinical variables. We also examined treatment decisions and outcomes in a subset of patients with Mycobacterium abscessus complex, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium porcinum, and Mycobacterium xenopi. Results: Cultures of NTM were available from 3,470 patients, 937 of whom had clinical data available. When we compared patients born within or outside Washington State, we found that the mycobacterial species frequency varied. Among 168 patients with one of the studied environmental mycobacteria, 72% had major comorbid conditions. Bronchiectasis was common among patients with pulmonary isolation of any NTM, including those with nonpathogenic M. gordonae. Although mortality was high (37%), few deaths were directly attributable to mycobacterial infection. Among 56 patients who met American Thoracic Society criteria for NTM lung disease, 22 were treated, and 19 of those had M. abscessus complex and M. kansasii. The treatment regimens used tended to follow published guidelines. Conclusions: Isolation of NTM varied by geographic region of origin and location within Washington State. Several clinical risk factors were specific to individual species. Comorbid conditions were common in patients with and without mycobacterial disease. Among patients with one of the studied organisms, there was a high mortality rate more frequently related to comorbid conditions than to mycobacterial disease.

Washington state satellite HIV clinic program: a model for delivering highly effective decentralized care in under-resourced communities*

ABSTRACT To improve access to high-quality HIV care in underserved regions of Western Washington (WA) State, we collaborated with the WA State Department of Health (DOH) and community partners to launch four satellite HIV clinics. Here, we describe this innovative clinical care model, present an estimate of costs, and evaluate patient care outcomes, including virologic suppression rates. To accomplish this, we assessed virologic suppression rates 12 months before and 12 months after the satellite clinics opened, comparing people living with HIV (PLWH) who enrolled in the satellite clinics versus all PLWH in the same regions who did not. We also determined virologic suppression rates in 2015 comparing satellite clinic versus non-satellite clinic patients and compared care quality indicators between the satellite clinics and the parent academic clinic. Results demonstrate that the change in virologic suppression rate 12 months before to 12 months after the satellite clinics opened was higher for patients who enrolled in the satellite clinics compared to all those in the same region who did not (18% versus 6%, p < 0.001). Virologic suppression in 2015 was significantly higher for satellite clinic than non-satellite clinic patients at three of four sites. Care quality indicators were met at a high level at the satellite clinics, comparable to the parent academic clinic. Overall, through community partnerships and WA DOH support, the satellite clinic program increased access to best practice HIV care and improved virologic suppression rates in difficult-to-reach areas. This model could be expanded to other regions with inadequate access to HIV practitioners, though financial support is necessary.