The influenza A virus host shutoff factor PA-X is rapidly turned over in a strain-specific manner

The influenza A endoribonuclease PA-X regulates virulence and transmission of the virus by reducing host gene expression and thus regulating immune responses to influenza A virus. Despite this key function in viral biology, the levels of PA-X protein remain markedly low during infection, and previous results suggest that these low levels are not solely the result of regulation of the level of translation and RNA stability. How PA-X is regulated post-translationally remains unknown. We now report that the PA-X protein is rapidly turned over. PA-X from multiple viral strains are short-lived, although the half-life of PA-X ranges from ∼30 minutes to ∼3.5 hours depending on the strain. Moreover, sequences in the variable PA-X C-terminal domain are primarily responsible for regulating PA-X half-life, although the N-terminal domain also accounts for some differences among strains. Interestingly, we find that the PA-X from the 2009 pandemic H1N1 strain has a longer half-life compared to the other variants we tested. This PA-X isoform has been reported to have a higher host shutoff activity, suggesting a role for protein turnover in regulating PA-X activity. Collectively, this study reveals a novel regulatory mechanism of PA-X protein levels that may impact host shutoff activity during influenza A virus infection. IMPORTANCE The PA-X protein from influenza A virus reduces host immune responses to infection through suppressing host gene expression, including genes encoding the antiviral response. Thus, it plays a central role in influenza A virus biology. Despite its key function, PA-X was only discovered in 2012 and much remains to be learned including how PA-X activity is regulated to promote optimal levels of viral infection. In this study, we reveal that PA-X protein levels are very low likely because of rapid turnover. We show that instability is a conserved property among PA-X variants from different strains of influenza A virus, but that the half-lives of PA-X variants differ. Moreover, the longer half-life of PA-X from the 2009 pandemic H1N1 strain correlates with its reported higher activity. Therefore, PA-X stability may be a way to regulate its activity and may contribute to the differential virulence of influenza A virus strains.

Pathogenicity and transmissibility assessment of two strains of human influenza virus isolated in China in 2018

Objective Influenza season occurs every year in China, but its presentation was unusual in the period from December 2017 to early 2018. During this period, influenza activity was increasing across the country and was much greater than during the same period in previous years, with great harm to people’s health. Methods In this study, we isolated two human influenza virus strains—A/Hebei/F076/2018(H1N1) and B/Hebei/16275B/2018—from patients with severe influenza in Hebei, China, during the flu season in January 2018, and explored their genetic characteristics, pathogenicity, and transmissibility. Results A/Hebei/F076/2018(H1N1) belongs to the human-like H1N1 influenza virus lineage, whereas B/Hebei/16275B/2018 belongs to the Victoria lineage and is closely related to the World Health Organization reference strain B/Brisbane/60/2008. Pathogenicity tests revealed that A/Hebei/F076/2018(H1N1) replicated much more strongly in mice, with mice exhibiting 40% mortality, whereas B/Hebei/16275B/2018 was not lethal. Both viruses could be transmitted through direct contact and by the aerosol route between guinea pigs, but the H1N1 strain exhibited higher airborne transmissibility. Conclusions These results may contribute to the monitoring of influenza mutation and the prevention of an influenza outbreak.

Intradermal versus Intramuscular Administration of Influenza Vaccination: Rapid Review and Meta-analysis

BackgroundVaccinations are essential for prevention of influenza. We synthesized the published literature on the immunogenicity and safety of the influenza vaccine at reduced or full intradermal doses compared with full intramuscular doses.MethodsA rapid review of the literature was completed. MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials were searched for studies published from 2010 until June 5th, 2020. All studies comparing intradermal and intramuscular influenza vaccinations were included. Random-effects meta-analyses of immunogenicity and safety outcomes were conducted.ResultsA total of 30 relevant studies were included. Seroconversion rates were equivalent between the 3 mcg, 6 mcg, 7.5 mcg, and 9 mcg intradermal vaccine doses and the 15 mcg intramuscular vaccine dose for each of the H1N1, H3N2, and B strains, but significantly higher with the 15 mcg intradermal compared with the 15 mcg intramuscular dose, for the H1N1 (RR 1.10, 95% CI: 1.01-1.20) and B strains (RR 1.40, 95% CI: 1.13-1.73). Seroprotection rates for the 9 mcg and 15 mcg intradermal doses were equivalent with the 15 mcg intramuscular dose for all the three strains, except for the 15 mcg intradermal dose for the H1N1 strain which was significantly higher (RR 1.05, 95% CI: 1.01-1.09). Local adverse events were significantly higher with intradermal doses. Fever and chills were significantly higher with the 9 mcg intradermal dose, while all other systemic adverse events were equivalent for all doses.ConclusionReduced dose intradermal influenza vaccination appears to be a reasonable alternative to standard dose intramuscular vaccination because of the similarity in immunogenicity.

Regional differences in vaccine uptake and serological responses to vaccine and circulating strains of H1N1 viruses among patients with confirmed influenza

BackgroundSeasonal epidemics of influenza are often characterized through national or international surveillance efforts to determine vaccine efficacy and vaccine strain selection, but they do not provide detailed information about local variations in factors that can influence influenza cases and disease severity.MethodsSurveillance for influenza like illness was performed in Emergency Medicine Departments in Taipei, Taiwan and Baltimore, Maryland during the winter of 2015-16. Detailed demographic and clinical data were obtained. Nasal swabs or washes were collected for influenza virus diagnosis, sequencing and isolation. Serum was collected to determine neutralizing antibody levels.ResultsH1N1 viruses dominated both sites, but more influenza cases occurred in Taipei compared to Baltimore. H1 HA clade diversity was greater in Taipei. Vaccination rates were lower in Taipei than Baltimore, but vaccination was associated with an increase in serum neutralizing antibodies to recent H1N1 strains in Taipei, but not Baltimore. There was a higher level of preexisting immunity to circulating H1N1 strains in Baltimore.ConclusionsRegional differences in preexisting immunity and H1N1 strain circulation may have contributed to the vastly different 2015-16 influenza seasons in Taipei and Baltimore and suggest immune responses to vaccination can be affected by the degree of preexisting immunity in the population.

159. efficacy of CD377, a Novel Antiviral Fc-conjugate, Against Seasonal Influenza in Lethal Mouse Infection Models

Background Cidara’s AVCs (antiviral Fc-conjugates) are novel conjugates of potent, antiviral agents with the Fc domain of human IgG1. CD377 is an AVC development candidate for prevention and treatment of influenza that has broad anti-neuraminidase activity in both enzymatic and cell-based assays and the potential to engage the immune system, as well as a long half-life. Methods Efficacy studies were conducted in BALB/c mice lethally challenged intranasally at 3x the LD95 with influenza A (H1N1, H3N2) and influenza B (both lineages). CD377 was administered as a single dose subcutaneously (SC) 2 hours after viral challenge. Body weights (BW) and health scores were monitored daily, with 20% BW loss recorded as a mortality. Results In mice challenged with a lethal dose of an H1N1 strain (A/California/12/2012), a single 0.3 mg/kg dose of CD377 administered 2 hours post-challenge was fully protective (P=0.0015 relative to vehicle) (Fig 1A). In a similar study against a mouse-adapted H3N2 subtype (A/Hong Kong/1/1968), a single dose of CD377 at 0.1 mg/kg was fully protective (P=0.0025) (Fig 1B). In both studies, only a transient loss of BW was observed before mice began recovering weight. The activity of CD377 was also evaluated against both lineages of influenza B (Fig 1C, 1D). Against influenza B/Colorado/06/2017 (Victoria), a single CD377 dose of 0.3 mg/kg was fully protective (P=0.0035) while the Fc-only control dosed at 1 mg/kg was not, as expected. Against the Yamagata lineage (B/Florida/4/2006), CD377 demonstrated full protection at a dose of only 0.03 mg/kg (P=0.0023). Conclusion A single dose of CD377 (0.3 mg/kg or less) was protective against lethal challenge with several seasonal influenza A/B subtypes. The exceptional PK profile of CD377 combined with potent broad-spectrum activity highlight its potential for use as a long-term preventative against seasonal influenza. Seasonal influenza efficacy Disclosures James Levin, PhD, Cidara Therapeutics (Shareholder) Karin Amundson, BSc, Cidara Therapeutics (Shareholder) Allen Borchardt, PhD, Cidara Therapeutics (Employee) Thanh Lam, PhD, Cidara Therapeutics (Shareholder) Tom Brady, PhD, Cidara Therapeutics (Shareholder) Alain Noncovich, PhD, Cidara Therapeutics (Shareholder) Les Tari, PhD, Cidara Therapeutics (Shareholder)

The influenza A virus host shutoff factor PA-X is rapidly turned over in a strain-specific manner

ABSTRACTThe influenza A endoribonuclease PA-X regulates virulence and transmission of the virus by reducing host gene expression and thus regulating immune responses to influenza A virus. Despite this key function in viral biology, the levels of PA-X protein remain markedly low during infection, and previous results suggest that these low levels are not solely the result of regulation of the level of translation and RNA stability. How PA-X is regulated post-translationally remains unknown. We now report that the PA-X protein is rapidly turned over. PA-X from multiple viral strains are short-lived, although the half-life of PA-X ranges from ∼30 minutes to ∼3.5 hours depending on the strain. Moreover, sequences in the variable PA-X C-terminal domain are primarily responsible for regulating PA-X half-life, although the N-terminal domain also accounts for some differences among strains. Interestingly, we find that the PA-X from the 2009 pandemic H1N1 strain has a longer half-life compared to the other variants we tested. This PA-X isoform has been reported to have a higher host shutoff activity, suggesting a role for protein turnover in regulating PA-X activity. Collectively, this study reveals a novel regulatory mechanism of PA-X protein levels that may impact host shutoff activity during influenza A virus infection.IMPORTANCEThe PA-X protein from influenza A virus reduces host immune responses to infection through suppressing host gene expression, including genes encoding the antiviral response. Thus, it plays a central role in influenza A virus biology. Despite its key function, PA-X was only discovered in 2012 and much remains to be learned including how PA-X activity is regulated to promote optimal levels of viral infection. In this study, we reveal that PA-X protein levels are very low likely because of rapid turnover. We show that instability is a conserved property among PA-X variants from different strains of influenza A virus, but that the half-lives of PA-X variants differ. Moreover, the longer half-life of PA-X from the 2009 pandemic H1N1 strain correlates with its reported higher activity. Therefore, PA-X stability may be a way to regulate its activity and may contribute to the differential virulence of influenza A virus strains.

Comparability of Titers of Antibodies against Seasonal Influenza Virus Strains as Determined by Hemagglutination Inhibition and Microneutralization Assays

ABSTRACT We compared titers of antibodies against A/H1N1, A/H3N2, and B influenza virus strains collected pre- and postvaccination using hemagglutination inhibition (HI) and microneutralization (MN) assays and data from two vaccine trials: study 1, performed with a cell-grown trivalent influenza vaccine (TIVc) using cell-grown target virus in both assays, and study 2, performed with an egg-grown adjuvanted quadrivalent influenza vaccine (aQIVe) using egg-grown target virus. The relationships between HI- and MN-derived log-transformed titers were examined using different statistical techniques. Deming regression analyses showed point estimates for slopes generally close to 1 across studies and strains. The slope of regression was closest to 1 for A/H3N2 strain when either cell- or egg-grown viral target virus was used. Bland-Altman plots indicated a very small percentage of results outside 2 and 3 standard deviations. The magnitudes and directions of differences between titers in the two assays varied by study and strain. Mean differences favored the MN assay for A/H1N1 and B strains in study 1, whereas the titers determined by HI were higher than those determined by MN against the A/H3N2 strain. In study 2, mean differences favored the MN assay for A/H3N2 and B strains. Overall, the directions and magnitudes of the mean differences were similar between the two vaccines. The concordance correlation coefficient values ranged from 0.74 (A/H1N1 strain, study 1) to 0.97 (A/H3N2 strain, study 1). The comparative analysis demonstrates an overall strong positive correlation between the HI and MN assays. These data support the use of the MN assay to quantify the immune response of influenza vaccines in clinical studies, particularly for the A/H3N2 strain.

Examining immune arms in mice immunized with site-specific influenza virus mutants

Site-specific mutants as candidates for live influenza vaccines were resulted from directly introducing into the genome of the pathogenic influenza virus A/WSN/33 (H1N1) strain ts mutations derived from the genes encoding the polymerase complex proteins from some cold-adapted strains serving as attenuation donor. Here we present the data of a comparative study examining immune system arms in mice immunized intranasally with influenza virus mutants and classical cold-adapted reassortant obtained by crossing cold-adapted strain Donor A/Krasnodar/101/35/59 (H2N2) with strain A/WSN/33 (H1N1) bearing surface antigens (hemagglutinin and neuraminidase) similar to mutants. Immunophenotyping mononuclear leukocytes from immunized mice indicated at moderate suppressive effect after using site-specific mutant and the HA reassortant viruses on some immune cell subsets. All viruses in immunized mice resulted in activation of certain lymphocyte subsets including MHC II-positive cells, CD45+/CD19+ B lymphocytes and natural killer cells (CD16/32+/CD3–). Timescale and magnitude of activation markedly differed for each cell subsets. Mice immunized with mutants M26 and U2 peaked with count of CD16/32+/CD3– expressing cells on day 2 after the second immunization compared with control (p < 0.05) that may suggest about an important role for NK cells in activating immune response. In contrast, no significant changes were observed during the study in percentage of CD4+/CD25+/Fox P3 regulatory T cells, CD4+ T helpers and CD8+ cytotoxic cells, except for a sharply decreased count of activated CD4+/CD25+ cells (4-fold) on day 7 after immunization with mutant virus M26. Moreover, mutants U2 and M26 more moderately increased percentage of TLR2- and TLR4-positive cells. The viruses studied ambiguously affected count of TLR9-expressing cells in immunized animals. All viruses increased phagocytic activity in monocytes, but not neutrophils. Despite the moderate activation of innate and adaptive immunity arms, site-specific mutants more profoundly affected humoral reactions inducing increased antibody titers, so that immunogenicity of mutant viruses was higher than that of the cold-adapted reassortant. Thus, the findings hold a promise of using site-specific mutants as live influenza vaccines.

Synthesis and antiviral properties derivatives of 1-(para-tolyl)-4-aryl-5,6,7,8-tetrahydro-2,2a,8a-triazacyclopenta[cd]azulene-3-carbothioic acid arylamides

Nowadays, H1N1 – strain of so called «swine» or «mexican» subspecie A virus, which is still not studied, and which spreads through aerosol and contact is known to many world countries. Significant number of lethal cases during influenza pandemic season in Ukraine was observed. It should be noted, that patients went to the hospitals in very severe cases, which is why registered daily lethality is in 31,6% of cases. Everything listed above had made it necessary to conduct preventive measures on global and national levels. One of such measures is creation of new direct action medicinal agents. The goal of this work was the synthesis of compounds with potential antiviral properties in variety of 5,6,7,8-tetrahydro-2,2a,8a-triazacyclopenta[cd]azulen-3-carbotionic acid derivatives. We have chosen derivatives of 5,6,7,8-tetrahydro-2,2a,8a-triazacyclopenta[cd]azulen-3-carbotionic acid (6 a–e and 7 a–h), which were synthesized by boiling of 1-(para-tolyl)-4-aryl-5,6,7,8-tetrahydro-2,2a,4a-triazacyclopenta[cd]azulenes (5a or 5b) with corresponding arylisothiocyanates in dry benzene. Antiviral activity of phenylamid 1-(para-tolyl)-4-phenyl-5,6,7,8-tetrahydro-2,2a,8a-triazacyclopenta[cd]azulen-3-carbotionic acid against Flu A H1N1 California/07/2009 was researched in Southern Research Institute – SRI, Birmingham, Alabama). Variety of new derivatives of 5,6,7,8-tetrahydro-2,2a,8a-triazacyclopenta[cd]azulen-3-carbotionic acid. Composition and structure of all synthesized compounds are proven by data of element analysis and 1Н NMR spectroscopy. In conducted researches of antiviral activity of phenylamide 1-(para-tolyl)-4-phenyl-5,6,7,8-tetrahydro-2,2a,8a-triazacyclopenta[cd]azulen-3-carbotionic acid against Flu A H1N1 California/07/2009, it was found out, that synthesized compound shows pronounced antiviral activity, compared to reference medicinal agents Ribavirin and Amizon. Therefore, conducted researches confirm perspective of development of a new domestic medicinal agent with antiviral activity, based on derivatives of 5,6,7,8,-tetrahydro-2,2a,8a-triazacyclopenta[cd]azulen-3-carbotionic acid.

Inclusion of site-specific mutations in the PB1-gene of a virulent A/WSN/33 (H1N1) strain of influenza A virus changes its phenotypic characteristics

Aim. Study of changes in the phenotypic characteristics of the virulent A/WSN/33 (H1N1) strain of influenza A virus under the influence of the inclusion of site-specific mutations in the PB1-gene of this strain.Materials and methods. Using a two-step polymerase reaction in the PB1 gene of A/ WSN/33 (H1N1) strain were included ts mutations taken from the genome of attenuated CA donors-strains: A/Ann Arbor/6/60 (H2N2), A/Leningrad 134/17/57 (H2N2) and A/ Krasnodar/101/35/59. Ts-phenotype, att-phenotype, immunogenicity, as well as weight loss in mice infected with these mutants were studied in the obtained site-specific mutants.Results. It was shown that the inclusion of ts mutations from the genome of CA donorsstrains of attenuation in the PB1 gene of the virulent A/WSN/33 (H1N1) strain leads to a change in the phenotypic characteristics of this strain to different degrees.Discussion. Analysis of the genome of CA strains- donors of attenuation of influenza virus indicates the crucial importance of the presence of functional defects in the PB1– protein for the formation of the attenuation phenotype of the virus.Conclusion. The technology of site-specific mutagenesis canbe successfully used to modify the PB1 gene of a virulent influenza A virus strain in order to construct a new generation of live influenza vaccines.