Brian B. Gowen

Variation in Lipid A Structure in the Pathogenic Yersiniae

Important pathogens in the genus Yersinia include the plague bacillus Yersinia pestis and two enteropathogenic species, Yersinia pseudotuberculosis and Yersinia enterocolitica. A shift in growth temperature induced changes in the number and type of acyl groups on the lipid A of all three species. After growth at 37°C, Y. pestis lipopolysaccharide (LPS) contained the tetra‐acylated lipid IVA and smaller amounts of lipid IVA modified with C10 or C12 acyl groups, Y. pseudotuberculosis contained the same forms as part of a more heterogeneous population in which lipid IVA modified with C16:0 predominated, and Y. enterocolitica produced a unique tetra‐acylated lipid A. When grown at 21°C, however, the three yersiniae synthesized LPS containing predominantly hexa‐acylated lipid A. This more complex lipid A stimulated human monocytes to secrete tumour necrosis factor‐α, whereas the lipid A synthesized by the three species at 37°C did not. The Y. pestis phoP gene was required for aminoarabinose modification of lipid A, but not for the temperature‐dependent acylation changes. The results suggest that the production of a less immunostimulatory form of LPS upon entry into the mammalian host is a conserved pathogenesis mechanism in the genus Yersinia, and that species‐specific lipid A forms may be important for life cycle and pathogenicity differences.

T-705 (favipiravir) and related compounds: novel broad-spectrum inhibitors of RNA viral infections.

Abstract A series of pyrazinecarboxamide derivatives T-705 (favipiravir), T-1105 and T-1106 were discovered to be candidate antiviral drugs. These compounds have demonstrated good activity in treating viral infections in laboratory animals caused by various RNA viruses, including influenza virus, arenaviruses, bunyaviruses, West Nile virus (WNV), yellow fever virus (YFV), and foot-and-mouth disease virus (FMDV). Treatment has in some cases been effective when initiated up to 5–7 days after virus infection, when the animals already showed signs of illness. Studies on the mechanism of action of T-705 have shown that this compound is converted to the ribofuranosyltriphosphate derivative by host enzymes, and this metabolite selectively inhibits the influenza viral RNA-dependent RNA polymerase without cytotoxicity to mammalian cells. Interestingly, these compounds do not inhibit host DNA and RNA synthesis and inosine 5′-monophosphate dehydrogenase (IMPDH) activity. From in vivo studies using several animal models, the pyrazinecarboxamide derivatives were found to be effective in protecting animals from death, reducing viral burden, and limiting disease manifestations, even when treatment was initiated after virus inoculation. Importantly, T-705 imparts its beneficial antiviral effects without significant toxicity to the host. Prompt development of these compounds is expected to provide effective countermeasures against pandemic influenza virus and several bioweapon threats, all of which are of great global public health concern given the current paucity of highly effective broad-spectrum drugs.

In Vitro and In Vivo Activities of T-705 against Arenavirus and Bunyavirus Infections

ABSTRACT There is a need for the development of effective antivirals for the treatment of severe viral diseases caused by members of the virus families Bunyaviridae and Arenaviridae. The pyrazine derivative T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has demonstrated remarkable antiviral activity against influenza virus and, to a lesser degree, against some other RNA viruses (Y. Furuta, K. Takahashi, Y. Fukuda, M. Kuno, T. Kamiyama, K. Kozaki, N. Nomura, H. Egawa, S. Minami, Y. Watanabe, H. Narita, and K. Shiraki, Antimicrob. Agents Chemother., 46:977-981, 2002). Here, we report that T-705 is highly active against a panel of bunyaviruses (La Crosse, Punta Toro, Rift Valley fever, and sandfly fever viruses) and arenaviruses (Junin, Pichinde, and Tacaribe viruses) by cytopathic effect and virus yield reduction cell-based assays. The 50% effective concentrations for T-705 ranged from 5 to 30 μg/ml and 0.7 to 1.2 μg/ml against the bunyaviruses and arenaviruses examined, respectively. We also demonstrate that orally administered T-705 is efficacious in treating Punta Toro virus in the mouse and hamster infection models, as well as Pichinde virus infection in hamsters. When administered twice daily for 5 to 6 days, beginning 4 h pre- or 24 h post-Punta Toro virus challenge, a 30-mg/kg of body weight/day dose provided complete protection from death and limited viral burden and liver disease. A dose of 50 mg/kg/day was found to be optimal for treating Pichinde infection and limiting viral replication and disease severity. In general, T-705 was found to be more active than ribavirin in cell-based assays and in vivo, as reflected by substantially greater therapeutic indexes. Our results suggest that T-705 may be a viable alternative for the treatment of life-threatening bunyaviral and arenaviral infections.

Kinetics of Disease Progression and Host Response in a Rat Model of Bubonic Plague

Plague, caused by the gram-negative bacterium Yersinia pestis, primarily affects rodents but is also an important zoonotic disease of humans. Bubonic plague in humans follows transmission by infected fleas and is characterized by an acute, necrotizing lymphadenitis in the regional lymph nodes that drain the intradermal flea bite site. Septicemia rapidly follows with spread to spleen, liver, and other organs. We developed a model of bubonic plague using the inbred Brown Norway strain of Rattus norvegicus to characterize the progression and kinetics of infection and the host immune response after intradermal inoculation of Y. pestis. The clinical signs and pathology in the rat closely resembled descriptions of human bubonic plague. The bacteriology; histopathology; host cellular response in infected lymph nodes, blood, and spleen; and serum cytokine levels were analyzed at various times after infection to determine the kinetics and route of disease progression and to evaluate hypothesized Y. pestis pathogenic mechanisms. Understanding disease progression in this rat infection model should facilitate further investigations into the molecular pathogenesis of bubonic plague and the immune response to Y. pestis at different stages of the disease.

TLR3 deletion limits mortality and disease severity due to phlebovirus infection

TLR3 was the first member of the TLR family of pattern recognition receptors found to detect a conserved viral molecular pattern, dsRNA, yet supporting evidence for a major role in host defense against viral pathogens is limited. Punta Toro virus (PTV) has been shown to produce severe infection in mice, modeling disease caused by the related highly pathogenic Rift Valley fever phlebovirus in humans and domesticated ungulates. Using TLR3-deficient mice, we investigated the involvement of TLR3 in host defense against PTV infection. Compared with wild-type, TLR3−/− mice demonstrate increased resistance to lethal infection and have reduced liver disease associated with hepatotropic PTV infection. Infectious challenge produced comparable peak liver and serum viral loads; however, TLR3−/− mice were able to clear systemic virus at a slightly faster rate. Cytokine profiling suggests that TLR3 plays an important role in PTV pathogenesis through the overproduction of inflammatory mediators, which may be central to the observed differences in survival and disease severity. Compared with TLR3-deficient mice, IL-6, MCP-1, IFN-γ, and RANTES were all present at higher levels in wild-type animals. Most dramatic was the exaggerated levels of IL-6 found systemically and in liver tissue of infected wild-type mice; however, IL-6-deficient animals were found to be more susceptible to lethal PTV infection. Taken together, we conclude that the TLR3-mediated response to PTV infection is detrimental to disease outcome and propose that IL-6, although critical to establishing antiviral defense, contributes to pathogenesis when released in excess, necessitating its controlled production as is seen with TLR3−/− mice.

Group A Streptococcus transcriptome dynamics during growth in human blood reveals bacterial adaptive and survival strategies

The molecular basis for bacterial responses to host signals during natural infections is poorly understood. The gram-positive bacterial pathogen group A Streptococcus (GAS) causes human mucosal, skin, and life-threatening systemic infections. During the transition from a throat or skin infection to an invasive infection, GAS must adapt to changing environments and host factors. To better understand how GAS adapts, we used transcript profiling and functional analysis to investigate the transcriptome of a wild-type serotype M1 GAS strain in human blood. Global changes in GAS gene expression occur rapidly in response to human blood exposure. Increased transcription was observed for many genes that likely enhance bacterial survival, including those encoding superantigens and host-evasion proteins regulated by a multiple gene activator called Mga. GAS also coordinately expressed genes involved in proteolysis, transport, and catabolism of oligopeptides to obtain amino acids in this protein-rich host environment. Comparison of the transcriptome of the wild-type strain to that of an isogenic deletion mutant (DeltacovR) mutated in the two-component regulatory system designated CovR-CovS reinforced the hypothesis that CovR-CovS has an important role linking key biosynthetic, catabolic, and virulence functions during transcriptome restructuring. Taken together, the data provide crucial insights into strategies used by pathogenic bacteria for thwarting host defenses and surviving in human blood.

TLR3 Is Essential for the Induction of Protective Immunity against Punta Toro Virus Infection by the Double-Stranded RNA (dsRNA), Poly(I:C12U), but not Poly(I:C): Differential Recognition of Synthetic dsRNA Molecules

In the wake of RNA virus infections, dsRNA intermediates are often generated. These viral pathogen-associated molecular patterns can be sensed by a growing number of host cell cytosolic proteins and TLR3, which contribute to the induction of antiviral defenses. Recent evidence indicates that melanoma differentiation-associated gene-5 is the prominent host component mediating IFN production after exposure to the dsRNA analog, poly(I:C). We have previously reported that Punta Toro virus (PTV) infection in mice is exquisitely sensitive to treatment with poly(I:C12U), a dsRNA analog that has a superior safety profile while maintaining the beneficial activity of the parental poly(I:C) in the induction of innate immune responses. The precise host factor(s) mediating protective immunity following its administration remain to be elucidated. To assess the role of TLR3 in this process, mice lacking the receptor were used to investigate the induction of protective immunity, type I IFNs, and IL-6 following treatment. Unlike wild-type mice, those lacking TLR3 were not protected against PTV infection following poly(I:C12U) therapy and failed to produce IFN-α, IFN-β, and IL-6. In contrast, poly(I:C) treatment significantly protected TLR3−/− mice from lethal challenge despite some deficiencies in cytokine induction. There was no indication that the lack of protection was due to the fact that TLR3-deficient mice had a reduced capacity to fight infection because they were not found to be more susceptible to PTV. We conclude that TLR3 is essential to the induction of antiviral activity elicited by poly(I:C12U), which does not appear to be recognized by the cytosolic sensor of poly(I:C), melanoma differentiation-associated gene-5.

Insight into the molecular basis of pathogen abundance: Group A Streptococcus inhibitor of complement inhibits bacterial adherence and internalization into human cells

Streptococcal inhibitor of complement (Sic) is a secreted protein made predominantly by serotype M1 Group A Streptococcus (GAS), which contributes to persistence in the mammalian upper respiratory tract and epidemics of human disease. Unexpectedly, an isogenic sic-negative mutant adhered to human epithelial cells significantly better than the wild-type parental strain. Purified Sic inhibited the adherence of a sic negative serotype M1 mutant and of non-Sic-producing GAS strains to human epithelial cells. Sic was rapidly internalized by human epithelial cells, inducing cell flattening and loss of microvilli. Ezrin and moesin, human proteins that functionally link the cytoskeleton to the plasma membrane, were identified as Sic-binding proteins by affinity chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. Sic colocalized with ezrin inside epithelial cells and bound to the F-actin-binding site region located in the carboxyl terminus of ezrin and moesin. Synthetic peptides corresponding to two regions of Sic had GAS adherence-inhibitory activity equivalent to mature Sic and inhibited binding of Sic to ezrin. In addition, the sic mutant was phagocytosed and killed by human polymorphonuclear leukocytes significantly better than the wild-type strain, and Sic colocalized with ezrin in discrete regions of polymorphonuclear leukocytes. The data suggest that binding of Sic to ezrin alters cellular processes critical for efficient GAS contact, internalization, and killing. Sic enhances bacterial survival by enabling the pathogen to avoid the intracellular environment. This process contributes to the abundance of M1 GAS in human infections and their ability to cause epidemics.

Treatment of Late Stage Disease in a Model of Arenaviral Hemorrhagic Fever: T-705 Efficacy and Reduced Toxicity Suggests an Alternative to Ribavirin

A growing number of arenaviruses are known to cause viral hemorrhagic fever (HF), a severe and life-threatening syndrome characterized by fever, malaise, and increased vascular permeability. Ribavirin, the only licensed antiviral indicated for the treatment of certain arenaviral HFs, has had mixed success and significant toxicity. Since severe arenaviral infections initially do not present with distinguishing symptoms and are difficult to clinically diagnose at early stages, it is of utmost importance to identify antiviral therapies effective at later stages of infection. We have previously reported that T-705, a substituted pyrazine derivative currently under development as an anti-influenza drug, is highly active in hamsters infected with Pichinde virus when the drug is administered orally early during the course of infection. Here we demonstrate that T-705 offers significant protection against this lethal arenaviral infection in hamsters when treatment is begun after the animals are ill and the day before the animals begin to succumb to disease. Importantly, this coincides with the time when peak viral loads are present in most organs and considerable tissue damage is evident. We also show that T-705 is as effective as, and less toxic than, ribavirin, as infected T-705-treated hamsters on average maintain their weight better and recover more rapidly than animals treated with ribavirin. Further, there was no added benefit to combination therapy with T-705 and ribavirin. Finally, pharmacokinetic data indicate that plasma T-705 levels following oral administration are markedly reduced during the latter stages of disease, and may contribute to the reduced efficacy seen when treatment is withheld until day 7 of infection. Our findings support further pre-clinical development of T-705 for the treatment of severe arenaviral infections.

T-705 (Favipiravir) Inhibition of Arenavirus Replication in Cell Culture

ABSTRACT A number of New World arenaviruses (Junín [JUNV], Machupo [MACV], and Guanarito [GTOV] viruses) can cause human disease ranging from mild febrile illness to a severe and often fatal hemorrhagic fever syndrome. These highly pathogenic viruses and the Old World Lassa fever virus pose a significant threat to public health and national security. The only licensed antiviral agent with activity against these viruses, ribavirin, has had mixed success in treating severe arenaviral disease and is associated with significant toxicities. A novel pyrazine derivative currently in clinical trials for the treatment of influenza virus infections, T-705 (favipiravir), has demonstrated broad-spectrum activity against a number of RNA viruses, including arenaviruses. T-705 has also been shown to be effective against Pichinde arenavirus infection in a hamster model. Here, we demonstrate the robust antiviral activity of T-705 against authentic highly pathogenic arenaviruses in cell culture. We show that T-705 disrupts an early or intermediate stage in viral replication, distinct from absorption or release, and that its antiviral activity in cell culture is reversed by the addition of purine bases and nucleosides, but not with pyrimidines. Specific inhibition of viral replication/transcription by T-705 was demonstrated using a lymphocytic choriomeningitis arenavirus replicon system. Our findings indicate that T-705 acts to inhibit arenavirus replication/transcription and may directly target the viral RNA-dependent RNA polymerase.