Evaluating the fitness of PA/I38T-substituted influenza A viruses with reduced baloxavir susceptibility in a competitive mixtures ferret model.

Baloxavir is approved in several countries for the treatment of uncomplicated influenza in otherwise-healthy and high-risk patients. Treatment-emergent viruses with reduced susceptibility to baloxavir have been detected in clinical trials, but the likelihood of widespread occurrence depends on replication capacity and onward transmission. We evaluated the fitness of A/H3N2 and A/H1N1pdm09 viruses with the polymerase acidic (PA) I38T-variant conferring reduced susceptibility to baloxavir relative to wild-type (WT) viruses, using a competitive mixture ferret model, recombinant viruses and patient-derived virus isolates. The A/H3N2 PA/I38T virus showed a reduction in within-host fitness but comparable between-host fitness to the WT virus, while the A/H1N1pdm09 PA/I38T virus had broadly similar within-host fitness but substantially lower between-host fitness. Although PA/I38T viruses replicate and transmit between ferrets, our data suggest that viruses with this amino acid substitution have lower fitness relative to WT and this relative fitness cost was greater in A/H1N1pdm09 viruses than in A/H3N2 viruses.

Baloxavir treatment of ferrets infected with influenza A(H1N1)pdm09 virus reduces onward transmission.

Influenza viruses cause seasonal outbreaks and pose a continuous pandemic threat. Although vaccines are available for influenza control, their efficacy varies each season and a vaccine for a novel pandemic virus manufactured using current technology will not be available fast enough to mitigate the effect of the first pandemic wave. Antivirals can be effective against many different influenza viruses but have not thus far been used extensively for outbreak control. Baloxavir, a recently licensed antiviral drug that targets the influenza virus endonuclease, has been shown to reduce virus shedding more effectively than oseltamivir, a widely used neuraminidase inhibitor drug. Thus it is possible that treatment with baloxavir might also interrupt onward virus transmission. To test this, we utilized the ferret model, which is the most commonly used animal model to study influenza virus transmission. We established a subcutaneous baloxavir administration method in ferrets which achieved similar pharmacokinetics to the approved human oral dose. Transmission studies were then conducted in two different locations with different experimental setups to compare the onward transmission of A(H1N1)pdm09 virus from infected ferrets treated with baloxavir, oseltamivir or placebo to naïve sentinel ferrets exposed either indirectly in adjacent cages or directly by co-housing. We found that baloxavir treatment reduced infectious viral shedding in the upper respiratory tract of ferrets compared to placebo, and reduced the frequency of transmission amongst sentinels in both experimental setups, even when treatment was delayed until 2 days post-infection. In contrast, oseltamivir treatment did not substantially affect viral shedding or transmission compared to placebo. We did not detect the emergence of baloxavir-resistant variants in treated animals or in untreated sentinels. Our results support the concept that antivirals which decrease viral shedding could also reduce influenza transmission in the community.

Epidemiology of repeat influenza infection in Queensland, Australia, 2005-2017.

Natural infection with the influenza virus is believed to generate cross-protective immunity across both types and subtypes. However, less is known about the persistence of this immunity and thus the susceptibility of individuals to repeat infection. We used 13 years (2005-2017) of surveillance data from Queensland, Australia, to describe the incidence and distribution of repeat influenza infections. Consecutive infections that occurred within 14 days of prior infection were considered a mixed infection; those that occurred more than 14 days later were considered separate (repeat) infections. Kaplan-Meier plots were used to investigate the probability of reinfection over time and the Prentice, Williams and Peterson extension of the Cox proportional hazards model was used to assess the association of age and gender with reinfection. Among the 188 392 notifications received during 2005-2017, 6165 were consecutively notified for the same individual (3.3% of notifications), and 2958 were mixed infections (1.6%). Overall, the probability of reinfection was low: the cumulative incidence was <1% after one year, 4.6% after five years, and 9.6% after ten years. The majority of consecutive infections were the result of two type A infections (43%) and were most common among females (adjusted hazard ratio (aHR): 1.15, 95% confidence interval (CI) 1.09-1.21), children aged less than 5 years (relative to adults aged 18-64 years aHR: 1.58, 95% CI 1.47-1.70) and older adults aged at least 65 years (aHR: 1.35; 95% CI 1.24-1.47). Our study suggests consecutive infections are possible but rare. These findings have implications for our understanding of population immunity to influenza.

Links between recognition and degradation of cytoplasmic viral RNA in innate immune response.

Recognition and degradation of viral RNA are essential for antiviral innate immune responses. Cytoplasmic viral RNA is recognized by retinoic acid-inducible gene I (RIG-I)-like receptors, which trigger type I interferon (IFN) production. Secreted type I IFN activates ubiquitously expressed type I IFN receptor and induces IFN-stimulated genes (ISGs). To suppress viral replication, several nucleases degrade viral RNA. RNase L is an ISG with endonuclease activity that degrades viral RNA, producing small RNA that activates RIG-I, resulting in the amplification of type I IFN production. Moreover, recent studies have elucidated novel links between viral RNA recognition and degradation. The RNA exosome is a protein complex that includes nucleases and is essential for host and viral RNA decay. Although the small RNAs produced by the RNA exosome do not activate RIG-I, several accessory factors of the RNA exosome promote RIG-I activation. Zinc-finger antiviral protein (ZAP) is an accessory factor that recognizes viral RNA and promotes viral RNA degradation via the RNA exosome. ZAPS is an alternative splicing form of ZAP and promotes RIG-I oligomerization and ATPase activity, resulting in RIG-I activation. DDX60 is another cofactor involved in the viral RNA degradation via the RNA exosome. The DDX60 protein promotes RIG-I signaling in a cell-type specific manner. These observations imply that viral RNA degradation and recognition are linked to each other. In this review, I discuss the links between recognition and degradation of viral RNA.

Mapping the pulmonary environment of animals protected from virulent H1N1 influenza infection using the TLR-2 agonist Pam2Cys.

We have previously shown that intranasal administration of the Toll-like receptor-2 agonist, S-(2,3-bis(palmitoyloxy)propyl) cysteine (Pam2Cys), provides immediate and antigen independent protection against challenge with influenza virus. Here we characterize the cellular pulmonary environments of mice which had either been treated with Pam2Cys or placebo and then challenged with influenza virus. We show that Pam2Cys treatment results in the influx of innate immune cells into the lungs and that depletion of phagocytic cells from this influx using clodronate-loaded liposomes caused a reduction in the number of interstitial macrophages and monocytes. This resulted in abolition of the protective effect indicating the importance of this cellular subset in Pam2Cys-mediated protection.

Reducing the impact of influenza-associated secondary pneumococcal infections.

When administered prophylactically, we show that the Toll-like receptor-2 (TLR-2) agonist PEG-Pam2Cys (pegylated-S-(2,3-bis(palmitoyloxy)propyl)cysteine) not only mediates potent anti-viral activity against influenza virus but also reduces the impact of secondary infections with Streptococcus pneumoniae (the pneumococcus) by reducing (i) pulmonary viral and bacterial burdens, (ii) the levels of proinflammatory cytokines that normally accompany influenza and S. pneumoniae secondary infections and (iii) the vascular permeability of the pulmonary tract that can allow bacterial invasion of the blood in mice. We also show that an inactivated detergent-disrupted influenza virus vaccine formulated with the Pam2Cys-based adjuvant R4-Pam2Cys provides the host with both immediate and long-term protection against secondary pneumococcal infections following influenza virus infection through innate and specific immune mechanisms, respectively. Vaccinated animals generated influenza virus-specific immune responses that provided the host with long-term protection against influenza virus and its sequelae. This vaccine, which generates an immediate response, provides an additional countermeasure, which is ideal for use even in the midst of an influenza outbreak.

Intranasal administration of the TLR2 agonist Pam2Cys provides rapid protection against influenza in mice.

The protective role played by the innate immune system during early stages of infection suggests that compounds which stimulate innate responses could be used as antimicrobial or antiviral agents. In this study, we demonstrate that the Toll-like receptor-2 agonist Pam2Cys, when administered intranasally, triggers a cascade of inflammatory and innate immune signals, acting as an immunostimulant by attracting neutrophils and macrophages and inducing secretion of IL-2, IL-6, IL-10, IFN-γ, MCP-1 and TNF-α. These changes provide increased resistance against influenza A virus challenge and also reduce the potential for transmission of infection. Pam2Cys treatment also reduced weight loss and lethality associated with virulent influenza virus infection in a Toll-like receptor-2-dependent manner. Treatment did not affect the animals' ability to generate an adaptive immune response, measured by the induction of functional influenza A virus-specific CD8(+) T cells following exposure to virus. Because this compound demonstrates efficacy against distinct strains of influenza, it could be a candidate for development as an agent against influenza and possibly other respiratory pathogens.

Infection with different human influenza A subtypes affects the period of susceptibility to secondary bacterial infections in ferrets.

It is well-established that influenza virus infections predispose individuals to secondary bacterial infections (SBIs), which may result in a range of clinical outcomes from relatively mild (e.g. sinusitis and otitis media) to severe (e.g. pneumonia and septicaemia). The most common bacterial pathogen associated with SBI following influenza virus infections is Streptococcus pneumoniae(SPN). Of circulating human seasonal influenza viruses, influenza A viruses (IAV) of both the A(H1N1)pdm09 and A(H3N2) subtypes are associated with severe disease but have differing hospitalisation and complication rates. To study the interplay of these two IAV subtypes with SBI, we used a ferret model of influenza infection followed by secondary challenge with a clinical strain of SPN to determine the severity and the period of susceptibility for SBI. Ferrets challenged with SPN 5 days after infection with A(H3N2) or A(H1N1)pdm09 viruses developed severe disease that required euthanasia. When the time between viral infection and bacterial challenge was extended, A/H1N1pdm09-infected animals remained susceptible to SBI- for up to 10 days after the viral infection. For A(H3N2)- but not A(H1N1)pdm09-infected ferrets, susceptibility to SBI-associated disease could be extended out to 16 days postviral infection. While caution should be taken when extrapolating animal models to human infections, the differences between A(H3N2) and A(H1N1)pdm09 strains in duration of susceptibility to SBI observed in the ferret model, may provide some insight regarding the higher rates of SBI-associated disease associated with some strains of A(H3N2) viruses in humans.

Assessing the fitness of a dual-antiviral drug resistant human influenza virus in the ferret model.

Influenza antivirals are important tools in our fight against annual influenza epidemics and future influenza pandemics. Combinations of antivirals may reduce the likelihood of drug resistance and improve clinical outcomes. Previously, two hospitalised immunocompromised influenza patients, who received a combination of a neuraminidase inhibitor and baloxavir marboxil, shed influenza viruses resistant to both drugs. Here-in, the replicative fitness of one of these A(H1N1)pdm09 virus isolates with dual resistance mutations (NA-H275Y and PA-I38T) was similar to wild type virus (WT) in vitro, but reduced in the upper respiratory tracts of challenged ferrets. The dual-mutant virus transmitted well between ferrets in an airborne transmission model, but was outcompeted by the WT when the two viruses were co-administered. These results indicate the dual-mutant virus had a moderate loss of viral fitness compared to the WT virus, suggesting that while person-to-person transmission of the dual-resistant virus may be possible, widespread community transmission is unlikely.

Innate Immune Responses to Influenza Virus Infections in the Upper Respiratory Tract.

The innate immune system is the host's first line of immune defence against any invading pathogen. To establish an infection in a human host the influenza virus must replicate in epithelial cells of the upper respiratory tract. However, there are several innate immune mechanisms in place to stop the virus from reaching epithelial cells. In addition to limiting viral replication and dissemination, the innate immune system also activates the adaptive immune system leading to viral clearance, enabling the respiratory system to return to normal homeostasis. However, an overzealous innate immune system or adaptive immune response can be associated with immunopathology and aid secondary bacterial infections of the lower respiratory tract leading to pneumonia. In this review, we discuss the mechanisms utilised by the innate immune system to limit influenza virus replication and the damage caused by influenza viruses on the respiratory tissues and how these very same protective immune responses can cause immunopathology.

Prophylaxis of ferrets with nitazoxanide and oseltamivir combinations is more effective at reducing the impact of influenza a virus infection compared to oseltamivir monotherapy.

Combination therapy is an alternative approach to reduce viral shedding and improve clinical outcomes following influenza virus infections. In this study we used oseltamivir (OST), a neuraminidase inhibitor and nitazoxanide (NTZ), a host directed drug, and found in vitro that the combination of these two antivirals have a synergistic relationship. Using the ferret model of (A/Perth/265/2009, (H1N1)pdm09), virus infections, we found that the combination of NTZ and OST was more effective than either NTZ or OST independently in preventing infection and reducing duration of viral shedding. However, these benefits were only seen if treatment was administered prophylactically, as opposed to therapeutically. We also found that if prophylactically treated ferrets that had detectable virus in the upper respiratory tract, no virus was detected in the lower respiratory tract. This benefit was not observed with NTZ or OST alone. The combination of NTZ and OST enhances the antiviral effect of OST, which is the standard of care in most settings.

Antivirals targeting the polymerase complex of influenza viruses.

Current influenza antivirals have limitations with regard to their effectiveness and the potential emergence of resistance. Encouragingly, several new compounds which inhibit the polymerase of influenza viruses have recently been shown to have enhanced pre-clinical and clinical effectiveness compared to the neuraminidase inhibitors, the mainstay of influenza antiviral therapy over the last two decades. In this review we focus on four compounds which inhibit polymerase function, baloxavir marboxil, favipiravir, pimodivir and AL-794 and discuss their clinical and virological effectiveness, their propensity to select for resistance and their potential for future combination therapy with the most commonly used neuraminidase inhibitor, oseltamivir.

Inactivated whole virus particle vaccine with potent immunogenicity and limited IL-6 induction is ideal for influenza.

In contrast to current ether- or detergent-disrupted "split" vaccines (SVs) for influenza, inactivated whole influenza virus particle vaccines (WPVs) retain the original virus structure and components and as such may confer similar immunity to natural infection. In a collaboration between academia and industry, the potential of WPV as a new seasonal influenza vaccine was investigated. Each of the four seasonal influenza vaccine manufacturers in Japan prepared WPVs and SVs from the same batches of purified influenza virus. Both mice and monkeys vaccinated with the WPVs exhibited superior immune responses to those vaccinated with the corresponding SVs. Vaccination with A/California/07/2009 (H1N1) WPV enabled mice to survive a lethal challenge dose of homologous virus whereas those vaccinated with SV succumbed to infection within 6 days. Furthermore, mice vaccinated with WPV induced substantial numbers of multifunctional CD8+ T cells, important for control of antigenically drifted influenza virus strains. In addition, cytokines and chemokines were detected at early time points in the sera of mice vaccinated with WPV but not in those animals vaccinated with SV. These results indicate that WPVs induce enhanced innate and adaptive immune responses compared to equivalent doses of SVs. Notably, WPV at one fifth of the dose of SV was able to induce potent immunity with limited production of IL-6, one of the pyrogenic cytokines. We thus propose that WPVs with balanced immunogenicity and safety may set a new global standard for seasonal influenza vaccines.

Animal models used to assess influenza antivirals.

INTRODUCTION: Influenza continues to be a major public health concern. Antivirals play an important role in limiting the burden of disease and preventing infection and/or transmission. The developments of such agents are heavily dependent on pre-clinical evaluation where animal models are used to answer questions that cannot be easily addressed in human clinical trials. There are numerous animal models available to study the potential benefits of influenza antivirals but each animal model has its own pros and cons. Areas covered: In this review, the authors describe the advantages and disadvantages of using mice, ferrets, guinea pigs, cotton rats, golden hamsters and non-human primates to evaluate influenza therapeutics. Expert opinion: Animals used for evaluating influenza therapeutics differ in their susceptibility to influenza virus infection, their ability to display clinical signs of illness following viral infection and in their practical requirements such as housing. Therefore, defining the scientific question being asked and the data output required will assist in selecting the most appropriate animal model.

Extracellular Vesicles Including Exosomes Regulate Innate Immune Responses to Hepatitis B Virus Infection.

The innate immune system is essential for controlling viral infection. Hepatitis B virus (HBV) persistently infects human hepatocytes and causes hepatocellular carcinoma. However, the innate immune response to HBV infection in vivo remains unclear. Using a tree shrew animal model, we showed that HBV infection induced hepatic interferon (IFN)-γ expression during early infection. Our in vitro study demonstrated that hepatic NK cells produced IFN-γ in response to HBV only in the presence of hepatic F4/80(+) cells. Moreover, extracellular vesicles (EVs) released from HBV-infected hepatocytes contained viral nucleic acids and induced NKG2D ligand expression in macrophages by stimulating MyD88, TICAM-1, and MAVS-dependent pathways. In addition, depletion of exosomes from EVs markedly reduced NKG2D ligand expression, suggesting the importance of exosomes for NK cell activation. In contrast, infection of hepatocytes with HBV increased immunoregulatory microRNA levels in EVs and exosomes, which were transferred to macrophages, thereby suppressing IL-12p35 mRNA expression in macrophages to counteract the host innate immune response. IFN-γ increased the hepatic expression of DDX60 and augmented the DDX60-dependent degradation of cytoplasmic HBV RNA. Our results elucidated the crucial role of exosomes in antiviral innate immune response against HBV. ACCESSION NUMBER: Accession number of RNA-seq data is DRA004164 (DRA in DDBJ).

Inactivated Influenza Vaccine That Provides Rapid, Innate-Immune-System-Mediated Protection and Subsequent Long-Term Adaptive Immunity.

UNLABELLED: The continual threat to global health posed by influenza has led to increased efforts to improve the effectiveness of influenza vaccines for use in epidemics and pandemics. We show in this study that formulation of a low dose of inactivated detergent-split influenza vaccine with a Toll-like receptor 2 (TLR2) agonist-based lipopeptide adjuvant (R4Pam2Cys) provides (i) immediate, antigen-independent immunity mediated by the innate immune system and (ii) significant enhancement of antigen-dependent immunity which exhibits an increased breadth of effector function. Intranasal administration of mice with vaccine formulated with R4Pam2Cys but not vaccine alone provides protection against both homologous and serologically distinct (heterologous) viral strains within a day of administration. Vaccination in the presence of R4Pam2Cys subsequently also induces high levels of systemic IgM, IgG1, and IgG2b antibodies and pulmonary IgA antibodies that inhibit hemagglutination (HA) and neuraminidase (NA) activities of homologous but not heterologous virus. Improved primary virus nucleoprotein (NP)-specific CD8(+) T cell responses are also induced by the use of R4Pam2Cys and are associated with robust recall responses to provide heterologous protection. These protective effects are demonstrated in wild-type and antibody-deficient animals but not in those depleted of CD8(+) T cells. Using a contact-dependent virus transmission model, we also found that heterologous virus transmission from vaccinated mice to naive mice is significantly reduced. These results demonstrate the potential of adding a TLR2 agonist to an existing seasonal influenza vaccine to improve its utility by inducing immediate short-term nonspecific antiviral protection and also antigen-specific responses to provide homologous and heterologous immunity. IMPORTANCE: The innate and adaptive immune systems differ in mechanisms, specificities, and times at which they take effect. The innate immune system responds within hours of exposure to infectious agents, while adaptive immunity takes several days to become effective. Here we show, by using a simple lipopeptide-based TLR2 agonist, that an influenza detergent-split vaccine can be made to simultaneously stimulate and amplify both systems to provide immediate antiviral protection while giving the adaptive immune system time to implement long-term immunity. Both types of immunity induced by this approach protect against vaccine-matched as well as unrelated virus strains and potentially even against strains yet to be encountered. Conferring dual functionality to influenza vaccines is beneficial for improving community protection, particularly during periods between the onset of an outbreak and the time when a vaccine becomes available or in scenarios in which mass vaccination with a strain to which the population is immunologically naive is imperative.

TLR Agonists as Modulators of the Innate Immune Response and Their Potential as Agents Against Infectious Disease.

Immunotherapies that can either activate or suppress innate immune responses are being investigated as treatments against infectious diseases and the pathology they can cause. The objective of these therapies is to elicit protective immune responses thereby limiting the harm inflicted by the pathogen. The Toll-like receptor (TLR) signaling pathway plays critical roles in numerous host immune defenses and has been identified as an immunotherapeutic target against the consequences of infectious challenge. This review focuses on some of the recent advances being made in the development of TLR-ligands as potential prophylactic and/or therapeutic agents.