Experimental design and safety The central aim of this study was to assess how the route and dose of BCG vaccination influence systemic and tissue-resident T cell immunity, and protection after Mtb challenge. Rhesus macaques were vaccinated with 5 × 10 7 colony-forming units (CFUs) of BCG by intradermal (ID high), AE or IV routes, or with a combination of both AE (5 × 10 7 CFUs) and ID

Despite widespread use of the bacille Calmette-Guérin (BCG) vaccine, tuberculosis (TB) remains a leading cause of global mortality from a single infectious agent (Mycobacterium tuberculosis or Mtb). Here, over two independent Mtb challenge studies, we demonstrate that subcutaneous vaccination of rhesus macaques (RMs) with rhesus cytomegalovirus vectors encoding Mtb antigen inserts (hereafter referred to as RhCMV/TB)-which elicit and maintain highly effector-differentiated, circulating and tissue-resident Mtb-specific CD4 and CD8 memory T cell responses-can reduce the overall (pulmonary and extrapulmonary) extent of Mtb infection and disease by 68%, as compared to that in unvaccinated controls, after intrabronchial challenge with the Erdman strain of Mtb at ∼1 year after the first vaccination. Fourteen of 34 RhCMV/TB-vaccinated RMs (41%) across both studies showed no TB disease by computed tomography scans or at necropsy after challenge (as compared to 0 of 17 unvaccinated controls), and ten of these RMs were Mtb-culture-negative for all tissues, an exceptional long-term vaccine effect in the RM challenge model with the Erdman strain of Mtb. These results suggest that complete vaccine-mediated immune control of highly pathogenic Mtb is possible if immune effector responses can intercept Mtb infection at its earliest stages.

No abstract

Viral replication relies on the host to supply nucleosides. Host enzymes involved in nucleoside biosynthesis are potential targets for antiviral development. Ribavirin (a known antiviral drug) is such an inhibitor that suppresses guanine biosynthesis; depletion of the intracellular GTP pool was shown to be the major mechanism to inhibit flavivirus. Along similar lines, inhibitors of the pyrimidine biosynthesis pathway could be targeted for potential antiviral development. Here we report on a novel antiviral compound (NITD-982) that inhibits host dihydroorotate dehydrogenase (DHODH), an enzyme required for pyrimidine biosynthesis. The inhibitor was identified through screening 1.8 million compounds using a dengue virus (DENV) infection assay. The compound contains an isoxazole-pyrazole core structure, and it inhibited DENV with a 50% effective concentration (EC 50 ) of 2.4 nM and a 50% cytotoxic concentration (CC 50 ) of >5 M. NITD-982 has a broad antiviral spectrum, inhibiting both flaviviruses and nonflaviviruses with nanomolar EC 90 s. We also show that (i) the compound inhibited the enzymatic activity of recombinant DHODH, (ii) an NITD-982 analogue directly bound to the DHODH protein, (iii) supplementing the culture medium with uridine reversed the compoundmediated antiviral activity, and (iv) DENV type 2 (DENV-2) variants resistant to brequinar (a known DHODH inhibitor) were cross resistant to NITD-982. Collectively, the results demonstrate that the compound inhibits DENV through depleting the intracellular pyrimidine pool. In contrast to the in vitro potency, the compound did not show any efficacy in the DENV-AG129 mouse model. The lack of in vivo efficacy is likely due to the exogenous uptake of pyrimidine from the diet or to a high plasma protein-binding activity of the current compound.

These data indicate that TCN-032 may provide immediate immunity and therapeutic benefit in influenza A infection, with no apparent emergence of resistant virus. TCN-032 was safe with no evidence of immune exacerbation based on serum cytokine expression. Clinicaltrials.gov registry number. NCT01719874.

The search for novel therapeutic interventions for viral disease is a challenging pursuit, hallmarked by the paucity of antiviral agents currently prescribed. Targeting of viral proteins has the inextricable challenge of rise of resistance. Safe and effective vaccines are not possible for many viral pathogens. New approaches are required to address the unmet medical need in this area. We undertook a cell-based high-throughput screen to identify leads for development of drugs to treat respiratory syncytial virus (RSV), a serious pediatric pathogen. We identified compounds that are potent (nanomolar) inhibitors of RSV in vitro in HEp-2 cells and in primary human bronchial epithelial cells and were shown to act postentry. Interestingly, two scaffolds exhibited broad-spectrum activity among multiple RNA viruses. Using the chemical matter as a probe, we identified the targets and identified a common cellular pathway: the de novo pyrimidine biosynthesis pathway. Both targets were validated in vitro and showed no significant cell cytotoxicity except for activity against proliferative B- and T-type lymphoid cells. Corollary to this finding was to understand the consequences of inhibition of the target to the host. An in vivo assessment for antiviral efficacy failed to demonstrate reduced viral load, but revealed microscopic changes and a trend toward reduced pyrimidine pools and findings in histopathology. We present here a discovery program that includes screen, target identification, validation, and druggability that can be broadly applied to identify and interrogate other host factors for antiviral effect starting from chemical matter of unknown target/mechanism of action.

We evaluated the ability of human coronaviruses to infect primary cultures of human neural cells. Double immunofluorescence with antibodies to virus and cell markers showed infection of fetal astrocytes and of adult microglia and astrocytes by strain OC43. RNA amplification revealed infection of fetal astrocytes, adult microglia, and a mixed culture of adult oligodendrocytes and astrocytes by strain 229E. Infectious virus was released only from fetal astrocytes, with higher titers for OC43. Human coronaviruses have the capacity to infect some cells of the central nervous system, although infection of adult cells appears abortive.

Truncated human coronavirus HCoV-229E spike glycoproteins containing amino acids 407 to 547 bound to purified, soluble virus receptor, human aminopeptidase N (hAPN). Soluble hAPN neutralized the infectivity of HCoV-229E virions at 37°C, but not 4°C. Binding of hAPN may therefore trigger conformational changes in the viral spike protein at 37°C that facilitate virus entry.Human coronaviruses HCoV-229E in serogroup I and HCoV-OC43 in serogroup II are, after rhinoviruses, the second most important cause of the common cold (7, 13). The first step in HCoV-229E infection is binding of the trimeric 200-kDa viral spike glycoprotein (S) to human aminopeptidase N (hAPN, CD13) on human cells (26). The three-dimensional structures of the spike glycoproteins of the plus-strand RNAcontaining coronaviruses are not yet known. However, the structure of S is predicted to be generally similar to that of type 1 viral fusion proteins of large negative-strand RNA viruses (12,22,25). The approximately 547-amino-acid (aa)-long Nterminal S1 domain of HCoV-229E spike protein has receptorbinding activity (2), and the membrane-anchored S2 domain contains several heptad repeats (3). APNs of the normal host species are also receptors for porcine, feline, and canine coronaviruses in group I (5, 6, 15, 23). The metalloprotease APN is a 150-kDa, class II glycoprotein that is expressed as a dimer on apical membranes of polarized epithelial cells, at synaptic junctions, and on antigen-presenting cells (11,17,19,21).Soluble receptors for many viruses have been used to identify critical receptor-binding sites on the viral attachment proteins, explore the specificity of virus-receptor interactions, and characterize conformational changes in the viral attachment protein that are induced by receptor binding (10,14,18,20,28). Temperature-dependent triggering of such conformational changes in a virus attachment protein by receptors on host cell membranes leads to penetration of the viral genome into the cytoplasm of the host cell. Binding of receptor to the N-terminal domain of a type 1 viral fusion protein at 37°C can trigger a conformational change in the membrane-anchored domain, leading to fusion of the viral envelope with host cell membranes (9,10,14,27). This paper reports the expression in insect cells and characterization of a soluble recombinant hAPN protein (shAPN) and interactions of shAPN with HCoV-229E virions, the S1 domain of the viral spike glycoprotein, and truncated S1 proteins. Characterization of shAPN.A soluble form of hAPN lacking the stalk, transmembrane, and intracellular domains (anchor/ stalk-minus APN), was previously expressed in mammalian cells and shown to be enzymatically active (24). For these studies, we expressed in insect cells the 95-to 98-kDa soluble, anchor/stalk-minus protein (shAPN). The open reading frame included the BiP cleavable signal sequence (MKLCILLAVVA FVGLSGL), a linker (RS), and aa 66 to 967 of hAPN with a C-terminal V5 epitope and His tag. We purified the protein on Ni-nitrilotriacetic acid (NTA) resin...