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Influenza viruses undergo continual antigenic evolution allowing mutant viruses to evade host immunity acquired to previous virus strains. Antigenic phenotype is often assessed through pairwise measurement of cross-reactivity between influenza strains using the hemagglutination inhibition (HI) assay. Here, we extend previous approaches to antigenic cartography, and simultaneously characterize antigenic and genetic evolution by modeling the diffusion of antigenic phenotype over a shared virus phylogeny. Using HI data from influenza lineages A/H3N2, A/H1N1, B/Victoria and B/Yamagata, we determine patterns of antigenic drift across viral lineages, showing that A/H3N2 evolves faster and in a more punctuated fashion than other influenza lineages. We also show that year-to-year antigenic drift appears to drive incidence patterns within each influenza lineage. This work makes possible substantial future advances in investigating the dynamics of influenza and other antigenically-variable pathogens by providing a model that intimately combines molecular and antigenic evolution. Every year, seasonal influenza, commonly called flu, infects up to one in five people around the world, and causes up to half a million deaths. Even though the human immune system can detect and destroy the virus that causes influenza, people can catch flu many times throughout their lifetimes because the virus keeps evolving in an effort to avoid the immune system. This antigenic drift—so-called because the antigens displayed by the virus keep changing—also explains why influenza vaccines become less effective over time and need to be reformulated every year. It is possible to determine which antigens are displayed by a new strain of the virus by observing how blood samples that respond to known strains respond to the new strain. This information about the “antigenic phenotype” of the virus can be plotted on an antigenic map in which strains with similar antigens cluster together. Gene sequencing has shown that there are four subtypes of the flu virus that commonly infect people; but the relationship between changes in antigenic phenotype and changes in gene sequences of the influenza virus is poorly understood. Bedford et al. have now developed an approach to combine antigenic maps with genetic information about the four subtypes of the human flu virus. This revealed that the antigenic phenotype of H3N2—a subtype that is becoming increasingly common—evolved faster than the other three subtypes. Further, a correlation was observed between antigenic drift and the number of new influenza cases per year for each flu strain. This suggests that knowing which antigenic phenotypes are present at the start of flu season could help predict which strains of the virus will predominate later on. The work of Bedford et al. provides a useful framework to study influenza, and could help to pinpoint which changes in viral genes cause the changes in antigens. This information could potentially speed up the development of new flu vaccines for each flu season.

Influenza B viruses have circulated in humans for over 80 y, causing a significant disease burden. Two antigenically distinct lineages (“B/Victoria/2/87-like” and “B/Yamagata/16/88-like,” termed Victoria and Yamagata) emerged in the 1970s and have cocirculated since 2001. Since 2015 both lineages have shown unusually high levels of epidemic activity, the reasons for which are unclear. By analyzing over 12,000 influenza B virus genomes, we describe the processes enabling the long-term success and recent resurgence of epidemics due to influenza B virus. We show that following prolonged diversification, both lineages underwent selective sweeps across the genome and have subsequently taken alternate evolutionary trajectories to exhibit epidemic dominance, with no reassortment between lineages. Hemagglutinin deletion variants emerged concomitantly in multiple Victoria virus clades and persisted through epistatic mutations and interclade reassortment—a phenomenon previously only observed in the 1970s when Victoria and Yamagata lineages emerged. For Yamagata viruses, antigenic drift of neuraminidase was a major driver of epidemic activity, indicating that neuraminidase-based vaccines and cross-reactivity assays should be employed to monitor and develop robust protection against influenza B morbidity and mortality. Overall, we show that long-term diversification and infrequent selective sweeps, coupled with the reemergence of hemagglutinin deletion variants and antigenic drift of neuraminidase, are factors that contributed to successful circulation of diverse influenza B clades. Further divergence of hemagglutinin variants with poor cross-reactivity could potentially lead to circulation of 3 or more distinct influenza B viruses, further complicating influenza vaccine formulation and highlighting the urgent need for universal influenza vaccines.

Influenza affects approximately 1 billion individuals each year resulting in between 290,000 and 650,000 deaths. Young children and immunocompromised individuals are at a particularly high risk of severe illness attributable to influenza and these are also the groups of individuals in which reduced susceptibility to neuraminidase inhibitors is most frequently seen. High levels of resistance emerged with previous adamantane therapy for influenza A and despite no longer being used to treat influenza and therefore lack of selection pressure, high levels of adamantane resistance continue to persist in currently circulating influenza A strains. Resistance to neuraminidase inhibitors has remained at low levels to date and the majority of resistance is seen in influenza A H1N1 pdm09 infected immunocompromised individuals receiving oseltamivir but is also seen less frequently with influenza A H3N2 and B. Rarely, resistance is also seen in the immunocompetent. There is evidence to suggest that these resistant strains (particularly H1N1 pdm09) are able to maintain their replicative fitness and transmissibility, although there is no clear evidence that being infected with a resistant strain is associated with a worse clinical outcome. Should neuraminidase inhibitor resistance become more problematic in the future, there are a small number of alternative novel agents within the anti-influenza armoury with different mechanisms of action to neuraminidase inhibitors and therefore potentially effective against neuraminidase inhibitor resistant strains. Limited data from use of novel agents such as baloxavir marboxil and favipiravir, does however show that resistance variants can also emerge in the presence of these drugs.

Vaccine effectiveness estimates for 2015–2016 seasonal influenza vaccine are reported from Canada. Findings suggest that agent-host and immuno-epidemiologic factors beyond antigenic match—including viral genomic variation, birth (immunological) cohort effects, repeat vaccination, and potential within-season waning immunity—may influence vaccine performance. Abstract Background Vaccine effectiveness (VE) estimates for 2015–2016 seasonal influenza vaccine are reported from Canada’s Sentinel Practitioner Surveillance Network (SPSN). This season was characterized by a delayed 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09) epidemic and concurrent influenza B(Victoria) virus activity. Potential influences on VE beyond antigenic match are explored, including viral genomic variation, birth cohort effects, prior vaccination, and epidemic period. Methods VE was estimated by a test-negative design comparing the adjusted odds ratio for influenza test positivity among vaccinated compared to unvaccinated participants. Vaccine-virus relatedness was assessed by gene sequencing and hemagglutination inhibition assay. Results Analyses included 596 influenza A(H1N1)pdm09 and 305 B(Victoria) cases and 926 test-negative controls. A(H1N1)pdm09 viruses were considered antigenically related to vaccine (unchanged since 2009), despite phylogenetic clustering within emerging clade 6B.1. The adjusted VE against A(H1N1)pdm09 was 43% (95% confidence interval [CI], 25%–57%). Compared to other age groups, VE against A(H1N1)pdm09 was lower for adults born during 1957–1976 (25%; 95% CI, −16%–51%). The VE against A(H1N1)pdm09 was also lower for participants consecutively vaccinated during both the current and prior seasons (41%; 95% CI, 18%–57%) than for those vaccinated during the current season only (75%; 95% CI, 45%–88%), and the VE among participants presenting in March–April 2016 (19%; 95% CI, −15%–44%) was lower than that among those presenting during January–February 2016 (62%; 95% CI, 44%–74%). The adjusted VE for B(Victoria) viruses was 54% (95% CI, 32%–68%), despite lineage-level mismatch to B(Yamagata) vaccine. The further variation in VE as observed for A(H1N1)pdm09 was not observed for B(Victoria). Conclusions Influenza VE findings may require consideration of other agent-host and immuno-epidemiologic influences on vaccine performance beyond antigenic match, including viral genomic variation, repeat vaccination, birth (immunological) cohort effects, and potential within-season waning of vaccine protection.

Background. We estimate vaccine effectiveness (VE) against both influenza A/subtypes and B/lineages in Canada for the 2011-2012 trivalent inactivated influenza vaccine (TIV) with components entirely unchanged from the 2010-2011 TIV and in the context of phenotypic and genotypic characterization of circulating viruses. Methods. In a test-negative case-control study VE was estimated as [1-adjustedOddsRatio] × 100 for RT-PCRconfirmed influenza in vaccinated vs nonvaccinated participants. Viruses were characterized by hemagglutination inhibition (HI) and sequencing of antigenic sites of the hemagglutinin (HA) gene. Results. There were 1507 participants. VE against A(H1N1)pdm09 was 80% (95% confidence interval [CI], 52%-92%): circulating viruses were HI-characterized as vaccine-matched and bore just 2 aminoacid (AA) differences from vaccine. VE against A/H3N2 was 51% (95% CI, 10%-73%): circulating viruses were HI-characterized as vaccine-related but bore ≥11AA differences from vaccine. VE against influenza was 51% (95% CI, 26%-67%) in total: 71% (95% CI, 40%-86%) for lineage-matched B/Victoria and 27% (95% CI, -21% to 56%) for lineagemismatched B/Yamagata. For both influenza A and B types, VE was similar among recipients of either 2010-2011 or 2011-2012 TIV alone, higher when vaccinated both seasons. Conclusions. Phenotypic and genotypic characterization of circulating and vaccine viruses enhances understanding of TIV performance, shown in 2011-2012 to be substantial against well-conserved A(HINI) pdm09 and lineagematched influenza B, suboptimal against genetic-variants of A/H3N2, and further reduced against lineage-mismatched influenza B. With unchanged vaccine components, protection may extend beyond a single season.

Influenza viruses are the cause of yearly epidemics and occasional pandemics that represent a significant challenge to public health. Current control strategies are imperfect and there is an unmet need for new antiviral therapies. Here, we report the identification of small molecule compounds able to effectively and specifically inhibit growth of influenza A and B viruses in cultured cells through targeting an assembly interface of the viral RNA-dependent RNA polymerase. Using an existing crystal structure of the primary protein–protein interface between the PB1 and PA subunits of the influenza A virus polymerase, we conducted an in silico screen to identify potential small molecule inhibitors. Selected compounds were then screened for their ability to inhibit the interaction between PB1 and PA in vitro using an ELISA-based assay and in cells, to inhibit nuclear import of a binary PB1–PA complex as well as transcription by the full viral ribonucleoprotein complex. Two compounds emerged as effective inhibitors with IC50 values in the low micromolar range and negligible cytotoxicity. Of these, one compound also acted as a potent replication inhibitor of a variety of influenza A virus strains in Madin-Darby canine kidney (MDCK) cells, including H3N2 and H1N1 seasonal and 2009 pandemic strains. Importantly, this included an oseltamivir-resistant isolate. Furthermore, potent inhibition of influenza B viruses but not other RNA or DNA viruses was seen. Overall, these compounds provide a foundation for the development of a new generation of therapeutic agents exhibiting high specificity to influenza A and B viruses.

Background. We estimated the effectiveness of inactivated influenza vaccines for the prevention of laboratory-confirmed, medically attended influenza during 3 seasons with variable antigenic match between vaccine and patient strains. Methods. Patients were enrolled during or after a clinical encounter for acute respiratory illness. Influenza infection was confirmed by culture or reverse-transcriptase polymerase chain reaction. Case-control analyses were performed that used data from patients who were ill without influenza (hereafter, “test-negative control subjects”) and data from asymptomatic control subjects from the population (hereafter, “traditional control subjects”). Vaccine effectiveness (VE) was estimated as [100 × (1 − adjusted odds ratio)]. Influenza isolates were antigenically characterized. Results. Influenza was detected in 167 (20%) of 818 patients in 2004–2005, in 51 (14%) of 356 in 2005–2006, and in 102 (11%) of 932 in 2006–2007. Analyses that used data from test-negative control subjects showed that VE was 10% (95% confidence interval [CI], −36% to 40%) in 2004–2005, 21% (95% CI,−52% to 59%) in 2005–2006, and 52% (95% CI, 22% to 70%) in 2006–2007. Using data from traditional control subjects, VE for those seasons was estimated to be 5% (95% CI, −52% to 40%), 11% (95% CI, −96% to 59%), and 37% (95% CI, −10% to 64%), respectively; confidence intervals included 0. The percentage of viruses that were antigenically matched to vaccine strains was 5% (3 of 62) in 2004–2005, 5% (2 of 42) in 2005–2006, and 91% (85 of 93) in 2006–2007. Conclusions. Influenza VE varied substantially across 3 seasons and was highest when antigenic match was optimal. VE estimates that used data from test-negative control subjects were consistently higher than those that used data from traditional control subjects.

Influenza viruses cause substantial morbidity and mortality every year despite seasonal vaccination. mRNA-based vaccines have the potential to elicit more protective immune responses, but for maximal breadth and durability, it is desirable to deliver both the viral hemagglutinin and neuraminidase glycoproteins. Delivering multiple antigens individually, however, complicates manufacturing and increases cost, thus it would be beneficial to express both proteins from a single mRNA. Here, we develop an mRNA genetic configuration that allows the simultaneous expression of unmodified, full-length NA and HA proteins from a single open reading frame. We apply this approach to glycoproteins from contemporary influenza A and B viruses and, after vaccination, observe high levels of functional antibodies and protection from disease in female mouse and male ferret challenge models. This approach may further efforts to utilize mRNA technology to improve seasonal vaccine efficacy by efficiently delivering multiple viral antigens simultaneously and in their native state. Here, the authors report the development of a genetic platform for mRNA-LNP vaccines that encodes the two major influenza virus glycoprotein genes into a single mRNA molecule. They show that this approach is generalizable to diverse influenza virus strains and is immunogenic and protective in mouse and ferret models of influenza disease.

Highly effective and broad-spectrum influenza vaccines are urgently required to prevent influenza outbreaks. Hemagglutinin (HA), M2 ectodomain (M2e), and nucleoprotein (NP) are crucial target antigens for the development of universal influenza vaccines. To generate a novel multivalent influenza vaccine, the HA genes of influenza B Yamagata (BY) and Victoria (BV) strains, and the NP gene of H1N1 were cloned into the E1 region of the chimpanzee adenoviral vector, AdC68, and M2e epitopes of H1N1 and H3N2 were fused to the loop region of the AdC68 fiber, resulting in the recombinant adenoviral vector vaccine, AdC-Flu-Tet. The immunoprotective effects of AdC-Flu-Tet were evaluated in the mouse models. The results showed that AdC-Flu-Tet successfully induced robust humoral and cellular immune responses and conferred full protection against H1N1, H3N2, BY, and BV infections. In conclusion, AdC-Flu-Tet is a promising candidate as a novel influenza vaccine with high protective efficacy.

Influenza viruses cause annual seasonal epidemics and occasional pandemics of human respiratory disease. Influenza virus infections represent a serious public health and economic problem, which are most effectively prevented through vaccination. However, influenza viruses undergo continual antigenic variation, which requires either the annual reformulation of seasonal influenza vaccines or the rapid generation of vaccines against potential pandemic virus strains. The segmented nature of influenza virus allows for the reassortment between two or more viruses within a co-infected cell, and this characteristic has also been harnessed in the laboratory to generate reassortant viruses for their use as either inactivated or live-attenuated influenza vaccines. With the implementation of plasmid-based reverse genetics techniques, it is now possible to engineer recombinant influenza viruses entirely from full-length complementary DNA copies of the viral genome by transfection of susceptible cells. These reverse genetics systems have provided investigators with novel and powerful approaches to answer important questions about the biology of influenza viruses, including the function of viral proteins, their interaction with cellular host factors and the mechanisms of influenza virus transmission and pathogenesis. In addition, reverse genetics techniques have allowed the generation of recombinant influenza viruses, providing a powerful technology to develop both inactivated and live-attenuated influenza vaccines. In this review, we will summarize the current knowledge of state-of-the-art, plasmid-based, influenza reverse genetics approaches and their implementation to provide rapid, convenient, safe and more effective influenza inactivated or live-attenuated vaccines.