Pathogenesis and transmission of swine origin A(H3N2)v influenza viruses in ferrets

Recent isolation of a novel swine-origin influenza A H3N2 variant virus [A(H3N2)v] from humans in the United States has raised concern over the pandemic potential of these viruses. Here, we analyzed the virulence, transmissibility, and receptor-binding preference of four A(H3N2)v influenza viruses isolated from humans in 2009, 2010, and 2011. High titers of infectious virus were detected in nasal turbinates and nasal wash samples of A(H3N2)v-inoculated ferrets. All four A(H3N2)v viruses possessed the capacity to spread efficiently between cohoused ferrets, and the 2010 and 2011 A(H3N2)v isolates transmitted efficiently to naïve ferrets by respiratory droplets. A dose-dependent glycan array analysis of A(H3N2)v showed a predominant binding to α2-6–sialylated glycans, similar to human-adapted influenza A viruses. We further tested the viral replication efficiency of A(H3N2)v viruses in a relevant cell line, Calu-3, derived from human bronchial epithelium. The A(H3N2)v viruses replicated in Calu-3 cells to significantly higher titers compared with five common seasonal H3N2 influenza viruses. These findings suggest that A(H3N2)v viruses have the capacity for efficient replication and transmission in mammals and underscore the need for continued public health surveillance.Seasonal epidemics and periodic pandemics are an ever-present international public health burden. During seasonal epidemics, the risk for hospitalization and death are highest among persons at either end of the age spectrum as well as for individuals with underlying medical conditions (1, 2). Although the overall impact of the 2009 influenza pandemic, caused by an H1N1 virus [A(H1N1)pdm09] was more modest than those of prior pandemics, the disproportionate disease burden among children and younger adults distinguished this pandemic from seasonal influenza (3–5). The A(H1N1)pdm09 virus emerged from swine, with a unique constellation of genes from human, avian, and swine influenza viruses not previously observed in nature. Swine represent a unique host because of their ability to be infected by influenza viruses from multiple species and serve as a reservoir for specific subtypes of influenza capable of infecting humans (6–8). Triple-reassortant swine (TRS) H1N1 viruses, which share host gene-lineage origins with A(H1N1)pdm09 viruses, have been responsible for sporadic human cases since 2005 (9, 10). The emergence of the A(H1N1)pdm09 virus, to which the majority of children and younger adults had little preexisting immunity, highlights the public health threat posed by other swine-origin influenza virus subtypes.H3N2 viruses have circulated in humans since their pandemic emergence in 1968 and are generally associated with uncomplicated disease in young healthy adults. However, epidemics caused by H3N2 viruses have been more severe than those caused by seasonal H1N1 or influenza B viruses (11, 12). In 1997–1998, human H3N2 viruses infected swine and spread widely in North American swine (6–8). In particular, TRS H3N2 viruses with a human lineage polymerase subunit polymerase basic 1 (PB1) gene, avian lineage PB2 and polymerase acidic (PA) genes, and swine lineage nucleoprotein (NP), matrix (M), and nonstructural (NS) genes, referred to as the triple-reassortant internal gene (TRIG) constellation, have been isolated widely in pigs throughout the United States (6–8, 13). From the late 1990s to 2009, these novel variants of H3N2 viruses [A(H3N2)v] were limited to transmission among swine, with only occasional detections of transmission to humans (14). However, between September and November 2010, five cases of human infection with the novel swine-origin A(H3N2)v were reported (15). Although all five recovered fully from their illness, two of the five cases were hospitalized. In 2011, 12 additional human cases were documented in the United States, with limited human-to-human transmission in some cases (15–17). The 2011 A(H3N2)v viruses are similar to other A(H3N2)v viruses isolated from previous human infections over the past 2 y but are unique in that the M gene is derived from the A(H1N1)pdm09 virus. Antigenic characterization showed that the A(H3N2)v viruses are distinct from current seasonal H3N2 viruses but exhibit a low degree of serologic cross-reactivity with human H3N2 viruses that circulated in the early 1990s (6, 8, 13), suggesting that children born after this time period may be particularly susceptible to infection.The use of the ferret model has become indispensable for understanding the virulence and transmission of influenza viruses (18–20), partly because ferrets and humans share similar lung physiology as well as because human and avian influenza viruses exhibit similar patterns of binding to sialic acids, the receptor for influenza viruses distributed throughout the respiratory tract in both species (21, 22). In this study, we used glycan microarrays to determine the receptor-binding preference of the A(H3N2)v viruses isolated from humans. The culture model of bronchial epithelial Calu-3 cells was used to assess viral replication, and the ferret model was used to assess pathogenicity and transmissibility. Notably, the 2010 and 2011 swine-origin H3N2 viruses replicated even more efficiently than human seasonal influenza viruses in human airway Calu-3 cells and exhibited efficient respiratory-droplet (RD) transmission in ferrets. These findings suggest that swine-origin H3N2 viruses have the potential to cause additional human disease.     

Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus

The emergence of human infection with a novel H7N9 influenza virus in China raises a pandemic concern. Chicken H9N2 viruses provided all six of the novel reassortant’s internal genes. However, it is not fully understood how the prevalence and evolution of these H9N2 chicken viruses facilitated the genesis of the novel H7N9 viruses. Here we show that over more than 10 y of cocirculation of multiple H9N2 genotypes, a genotype (G57) emerged that had changed antigenicity and improved adaptability in chickens. It became predominant in vaccinated farm chickens in China, caused widespread outbreaks in 2010–2013 before the H7N9 viruses emerged in humans, and finally provided all of their internal genes to the novel H7N9 viruses. The prevalence and variation of H9N2 influenza virus in farmed poultry could provide an important early warning of the emergence of novel reassortants with pandemic potential.Human infection with a novel avian-origin H7N9 influenza A virus causing severe respiratory symptoms and mortality was first reported in eastern China in March 2013 (1). To date, the novel virus has caused two outbreaks of human infection, including 375 known cases and 115 deaths as of 11 March 2014 (2). Phylogenetic analysis suggests that the virus is a triple reassortant of H7, N9, and H9N2 avian influenza viruses (3, 4). The H7 and N9 genes may have been transferred from migratory birds to domestic ducks and then to chickens in the live poultry markets (3–5), after which reassortment with enzootic H9N2 viruses formed the H7N9 viruses identified in humans (3–5).H9N2 influenza virus has low pathogenicity for avians, replicating mainly in the upper respiratory tract and causing mild or no overt signs of illness in specific pathogen-free (SPF) chickens (6). In 1994, the H9N2 subtype was first identified in chicken farms in the Guangdong province of south China (7); it has since become widespread in chickens and has caused great economic loss from reduced egg production and highly lethal coinfections (8–11). To reduce the impact of H9N2 infection in chickens, the flocks have been vaccinated since 1998 with commercial inactivated vaccines, such as A/chicken/Guangdong/SS/1994 (Ck/GD/SS/94), A/chicken/Shandong/6/1996 (Ck/SD/6/96), and A/chicken/Shanghai/F/1998 (Ck/SH/F/98) (8, 12, 13). These H9N2 vaccines initially limited the outbreaks and virus spread. However, despite multiple doses, the H9N2 vaccines became less effective, especially after 2007, and H9N2 influenza virus continues to circulate in vaccinated chicken flocks and has caused sporadic disease outbreaks (8, 10, 12–20). However, the recent prevalence and molecular evolution of the H9N2 viruses in chickens especially in the flocks receiving large-scale vaccination, and their role in the emergence of human H7N9 virus, are not fully understood. In this study, we systematically investigated the prevalence and evolution of H9N2 viruses mainly focusing on farm chickens and their role in the genesis of the novel H7N9 viruses.     

A common solution to group 2 influenza virus neutralization

The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.Influenza viruses are a significant and persistent threat to human health worldwide. Annual epidemics cause 3–5 million cases of severe illness and up to 0.5 million deaths (1), and periodic influenza pandemics have the potential to kill millions (2). Inhibitors against the viral surface glycoprotein neuraminidase are widely used for the treatment of influenza infections, but their efficacy is being compromised by the emergence of drug-resistant viral strains (3). Vaccination remains the most effective strategy to prevent influenza virus infection. However, protective efficacy is suboptimal in the highest risk groups: infants, the elderly, and the immunocompromised (1). Furthermore, because immunity after vaccination is typically strain-specific and influenza viruses evolve rapidly, vaccines must be updated almost annually. The antigenic composition of the vaccine is based on a prediction of strains likely to circulate in the coming year, therefore, mismatches between vaccine strains and circulating strains occur that can render the vaccine less effective (4). Consequently, there is an urgent need for new prophylactic and therapeutic interventions that provide broad protection against influenza.Immunity against influenza viruses is largely mediated by neutralizing antibodies that target the major surface glycoprotein hemagglutinin (HA) (5, 6). Identification of antigenic sites on HA indicates that influenza antibodies are primarily directed against the immunodominant HA head region (7), which mediates endosomal uptake of the virus into host cells by binding to sialic acid receptors (8). Because of high mutation rates in the HA head region and its tolerance for antigenic changes, antibodies that target the HA head are typically only effective against strains closely related to the strain(s) by which they were elicited, although several receptor binding site-targeting antibodies with greater breadth have been structurally characterized (9–15). In contrast, antibodies that bind to the membrane-proximal HA stem region tend to exhibit much broader neutralizing activity and can target strains within entire subtypes and groups (16–25) as well as across influenza types (24). These stem-directed antibodies inhibit major structural rearrangements in HA that are required for the fusion of viral and host endosomal membranes and thus, prevent the release of viral contents into the cell (8). The stem region is less permissive for mutations than the head and relatively well-conserved across divergent influenza subtypes.Anti-stem antibodies are elicited in some, but not all, individuals during influenza infection or vaccination (20, 26) and thus, hold great promise as potential broad spectrum prophylactic or therapeutic agents and for the development of a universal influenza vaccine (27–29). The majority of the known heterosubtypic stem binding antibodies neutralize influenza A virus subtypes belonging to group 1 (17–20, 23, 25). Furthermore, two antibodies that target a similar epitope in the HA stem, like most heterosubtypic group 1 antibodies, are able to more broadly recognize both group 1 and 2 influenza A viruses (22) or influenza A and B viruses (24). Strikingly, group 2-specific broadly neutralizing Abs (bnAbs) seem to be rare, because only one has been reported to date (21). CR8020 uniquely targets a distinct epitope in the stem in close proximity to the viral membrane at the HA base and binds lower down the stem than any other influenza HA antibody (21).In the discovery process that led to the isolation of bnAb CR8020, we recovered additional group 2-specific bnAbs. Here, we describe one such bnAb, CR8043, which recognizes a similar but nonidentical footprint on the HA as CR8020 and approaches the HA from a different angle. Furthermore, these two bnAbs are derived from different germ-line genes and, consequently, use distinct sets of interactions for HA recognition. Thus, the human immune system is able to recognize this highly conserved epitope in different ways using different germ-line genes. Hence, this valuable information can be used for the design of therapeutics and vaccines targeting this site of vulnerability in group 2 influenza A viruses that include the pandemic H3N2 subtype.     

Potential antigenic explanation for atypical H1N1 infections among middle-aged adults during the 2013–2014 influenza season

Influenza viruses typically cause the most severe disease in children and elderly individuals. However, H1N1 viruses disproportionately affected middle-aged adults during the 2013–2014 influenza season. Although H1N1 viruses recently acquired several mutations in the hemagglutinin (HA) glycoprotein, classic serological tests used by surveillance laboratories indicate that these mutations do not change antigenic properties of the virus. Here, we show that one of these mutations is located in a region of HA targeted by antibodies elicited in many middle-aged adults. We find that over 42% of individuals born between 1965 and 1979 possess antibodies that recognize this region of HA. Our findings offer a possible antigenic explanation of why middle-aged adults were highly susceptible to H1N1 viruses during the 2013–2014 influenza season. Our data further suggest that a drifted H1N1 strain should be included in future influenza vaccines to potentially reduce morbidity and mortality in this age group.Seasonal H1N1 (sH1N1) viruses circulated in the human population for much of the last century and, as of 2009, most humans had been exposed to sH1N1 strains. In 2009, an antigenically distinct H1N1 strain began infecting humans and caused a pandemic (1–3). Elderly individuals were less susceptible to 2009 pandemic H1N1 (pH1N1) viruses because of cross-reactive antibodies (Abs) elicited by infections with older sH1N1 strains (3–7). pH1N1 viruses have continued to circulate on a seasonal basis since 2009. Influenza viruses typically cause a higher disease burden in children and elderly individuals (8) but pH1N1 viruses caused unusually high levels of disease in middle-aged adults during the 2013–2014 influenza season (9–12). For example, a significantly higher proportion of individuals aged 30- to 59-y-old were hospitalized in Mexico with laboratory-confirmed pH1N1 cases in 2013–2014 relative to 2011–2012 (11).Most neutralizing influenza Abs are directed against the hemagglutinin (HA) glycoprotein. International surveillance laboratories rely primarily on ferret anti-influenza sera for detecting HA antigenic changes (13). For these assays, sera are isolated from ferrets recovering from primary influenza infections. Seasonal vaccine strains are typically updated when human influenza viruses acquire HA mutations that prevent the binding of primary ferret anti-influenza sera. Our laboratory and others have demonstrated that sera isolated from ferrets recovering from primary pH1N1 infections are dominated by Abs that recognize an epitope involving residues 156, 157, and 158 of the Sa HA antigenic site (14, 15). The pH1N1 component of the seasonal influenza vaccine has not been updated since 2009 because very few pH1N1 isolates possess mutations in residues 156, 157, and 158. The majority of isolates from the 2013–2014 season have been labeled as antigenically similar to the A/California/07/2009 vaccine strain (9).It is potentially problematic that major antigenic changes of influenza viruses are mainly determined using antisera isolated from ferrets recovering from primary influenza infections. Unlike experimental ferrets, humans are typically reinfected with antigenically distinct influenza strains throughout their life (16). In the 1950s, it was noted that the human immune system preferentially mounts Ab responses that cross-react to previously circulating influenza strains, as opposed to new Ab responses that exclusively target newer viral strains (17). This process, which Thomas Francis Jr. termed “original antigenic sin,” has been experimentally recapitulated in ferrets (14, 18), mice (19–21), and rabbits (22). Our group and others recently demonstrated that the specificity of pH1N1 Ab responses can be shaped by prior sH1N1 exposures (14, 23–26). We found that ferrets sequentially infected with sH1N1 and pH1N1 viruses mount Ab responses dominated against epitopes that are conserved between the viral strains (14). These studies indicate that primary ferret antisera may not be fully representative of human influenza immunity.It has been proposed that increased morbidity and mortality of middle-aged adults during the 2013–2014 influenza season is primarily a result of low vaccination rates within these populations (27). An alternative explanation is that recent pH1N1 strains have acquired a true antigenic mutation that has been mislabeled as “antigenically neutral” by assays that rely on primary ferret antisera. Here we complete a series of experiments to determine if recent pH1N1 strains possess a mutation that prevents binding of Abs in middle-aged humans who have been previously exposed to different H1N1 strains.     

Avian influenza H5N1 viral and bird migration networks in Asia

The spatial spread of the highly pathogenic avian influenza virus H5N1 and its long-term persistence in Asia have resulted in avian influenza panzootics and enormous economic losses in the poultry sector. However, an understanding of the regional long-distance transmission and seasonal patterns of the virus is still lacking. In this study, we present a phylogeographic approach to reconstruct the viral migration network. We show that within each wild fowl migratory flyway, the timing of H5N1 outbreaks and viral migrations are closely associated, but little viral transmission was observed between the flyways. The bird migration network is shown to better reflect the observed viral gene sequence data than other networks and contributes to seasonal H5N1 epidemics in local regions and its large-scale transmission along flyways. These findings have potentially far-reaching consequences, improving our understanding of how bird migration drives the periodic reemergence of H5N1 in Asia.Migratory birds play important roles in the geographic spread of various zoonotic agents (1). Among these agents, the avian influenza viruses (AIVs) have been shown to be transmitted over long distances during the seasonal migration of birds (2, 3). Wild waterfowl, in particular, are considered the natural reservoir of low-pathogenic avian influenza (LPAI) viruses and have been shown to spread LPAI viruses along migratory flyways in Asia, Africa, and the Americas (4–7). However, one of the fundamental unknowns remaining is the role played by wild birds in the regional spread of AIV (8–11).Highly pathogenic avian influenza (HPAI) H5N1 first appeared in Asia in 1996 (12), and subsequently spread to Europe, the Middle East, and Africa, causing many human casualties and major economic loss in the booming Asian poultry sector. Despite the low transmissibility of HPAI H5N1 from birds to humans and from humans to humans, the high fatality rate reported in humans after the onset of the epidemic and the potential for H5N1 to become pandemic through migratory bird flyways raised serious concerns (13). The Qinghai lineage of H5N1, in particular, expanded from Qinghai to Eurasia and into the Indian subcontinent and northern and central Africa along migratory flyways. It was also shown experimentally that some species of birds shed the virus before the onset of clinical signs or with no clinical signs (14, 15). This suggests that the large-scale transmission of H5N1 by migratory birds could potentially go undetected. Using satellite telemetry, Gaidet et al. reported that one infected white-faced whistling duck (Dendrocygna viduata) survived HPAI H5N2 infection and was able to migrate for at least 655 km, when tracked with a satellite transmitter for 47 d (16). Other studies have shown that the direction of the geographic spread of HPAI H5N1 is consistent with the major bird migration routes (17, 18). A number of studies have also suggested that long-distance migration may lead to immunosuppression in birds and migratory performance is negatively affected by viral infections (19–21). However, it should be noted that HPAI H5N1 is rarely reported in living and healthy wild birds (22–24).Recently, HPAI H5N1 clade 2.3.2, the dominant subclade in Asia, was detected in migratory birds during their migration in Mongolia, South Korea, and Japan, and was shown to be associated with wild waterfowl infections (25–27). Furthermore, an isolate of HPAI H5N1 from a common buzzard (Buteo buteo) in Bulgaria showed close genetic proximity to clade 2.3.2.1 isolates from wild birds in the Tyva Republic and Mongolia, suggesting that the HPAI H5N1 viruses of clade 2.3.2 have spread westward and pose a public health threat (28). These numerous studies have directed our attention to the roles played by migratory birds in the spread of HPAI H5N1 viruses in the last decade.In this study, we constructed networks of bird and viral gene migrations to evaluate the roles of migratory birds in the spread of HPAI H5N1 clades 2.3.2 before 2007, and 2.3.2.1 on and after 2007 (clade 2.3.2 for abbreviation) in Asia. We assembled a unique database of satellite tracking data on wild bird migration patterns, records of HPAI H5N1 outbreaks, and both the viral hemagglutinin (HA) gene and whole-genome nucleotide sequences over the period 2003–2012. The objective of this study was to analyze the association between the networks of bird migration, the networks of viral gene flow, and the timing of HPAI H5N1 outbreaks at different geographic locations.     

Telomere dysfunction causes alveolar stem cell failure

Telomere syndromes have their most common manifestation in lung disease that is recognized as idiopathic pulmonary fibrosis and emphysema. In both conditions, there is loss of alveolar integrity, but the underlying mechanisms are not known. We tested the capacity of alveolar epithelial and stromal cells from mice with short telomeres to support alveolar organoid colony formation and found that type 2 alveolar epithelial cells (AEC2s), the stem cell-containing population, were limiting. When telomere dysfunction was induced in adult AEC2s by conditional deletion of the shelterin component telomeric repeat-binding factor 2, cells survived but remained dormant and showed all the hallmarks of cellular senescence. Telomere dysfunction in AEC2s triggered an immune response, and this was associated with AEC2-derived up-regulation of cytokine signaling pathways that are known to provoke inflammation in the lung. Mice uniformly died after challenge with bleomycin, underscoring an essential role for telomere function in AEC2s for alveolar repair. Our data show that alveoloar progenitor senescence is sufficient to recapitulate the regenerative defects, inflammatory responses, and susceptibility to injury that are characteristic of telomere-mediated lung disease. They suggest alveolar stem cell failure is a driver of telomere-mediated lung disease and that efforts to reverse it may be clinically beneficial.Mutations in telomerase and telomere genes cause abnormal telomere shortening. Clinically, this molecular abnormality manifests in a spectrum of telomere syndromes that recapitulate features of age-associated pathology (1). In highly proliferative compartments, such as the bone marrow, telomere dysfunction causes stem cell exhaustion, and hematopoietic stem cell transplantation can reverse this pathology (1). More commonly, short telomeres predispose to adult-onset disease in the lung, a tissue that has slow cell turnover (1). Idiopathic pulmonary fibrosis and emphysema are the most prevalent clinical manifestations of human telomere syndromes and account for more than 80% of presentations (1, 2). The alveolar structures are preferentially affected in these disorders, and their pathology is marked by inflammation and mesenchymal abnormalities (3, 4). Affected patients are also exquisitely sensitive to pulmonary-toxic drugs, which are fatal even when there is no detectable baseline lung disease (1, 5).The mechanisms by which telomere defects provoke lung disease are not understood, but a number of observations have pointed to lung-intrinsic factors and epithelial dysfunction as candidate events (6–10). For example, in telomerase-null mice, DNA damage preferentially accumulates in the air-exposed epithelium after environmentally induced injury, such as with cigarette smoke (7). The additive effect of environmental injury and telomere dysfunction has been suggested to contribute to the susceptibility to emphysema seen in these mice (7). Moreover, humans that carry mutations in the surfactant protein C gene, SFTPC, which is expressed exclusively in type 2 alveolar epithelial cells (AEC2s), develop lung disease phenotypes similar to those seen in telomerase mutation carriers (10–12). Pulmonary fibrosis and emphysema patients have also been noted to have abnormally short telomeres in AEC2s (6, 7, 13). These observations, along with AEC2s’ regenerative capacity (14–16), led us to hypothesize that telomere dysfunction is sufficient to provoke AEC2 failure and that this event drives lung disease pathogenesis.One hurdle to modeling the consequences of telomere dysfunction in a cell type-specific manner is that laboratory mice have very long telomeres (17). In the absence of telomerase, telomere dysfunction can be generated only after several generations of breeding, precluding cell type-specific studies (18). To overcome this limitation, we designed two experimental systems. First we examined the role of telomere shortening in purified AEC2s in a stem cell assay ex vivo. For an in vivo system, we generated a model in which telomere dysfunction can be induced by deleting telomeric repeat-binding factor 2 (Trf2) (19, 20) exclusively in adult AEC2s. Trf2 functions to suppress the DNA damage response, and its loss leads to telomere dysfunction by uncapping, thus allowing cell type-specific studies within a single generation (19, 20). The latter surrogate model allowed us to test the consequences of acquired DNA damage and telomere dysfunction in the adult lung. We show here, in both late-generation telomerase-null mice and in a conditional mutant model, that telomere dysfunction restricted to AEC2s impairs stem cell function by inducing senescence. This program recapitulates the inflammatory responses and susceptibility to injury that are hallmarks of telomere-mediated lung disease.     

Vaccination of monoglycosylated hemagglutinin induces cross-strain protection against influenza virus infections

The 2009 H1N1 pandemic and recent human cases of H5N1, H7N9, and H6N1 in Asia highlight the need for a universal influenza vaccine that can provide cross-strain or even cross-subtype protection. Here, we show that recombinant monoglycosylated hemagglutinin (HA_mg) with an intact protein structure from either seasonal or pandemic H1N1 can be used as a vaccine for cross-strain protection against various H1N1 viruses in circulation from 1933 to 2009 in mice and ferrets. In the HA_mg vaccine, highly conserved sequences that were originally covered by glycans in the fully glycosylated HA (HA_fg) are exposed and thus, are better engulfed by dendritic cells (DCs), stimulated better DC maturation, and induced more CD8+ memory T cells and IgG-secreting plasma cells. Single B-cell RT-PCR followed by sequence analysis revealed that the HA_mg vaccine activated more diverse B-cell repertoires than the HA_fg vaccine and produced antibodies with cross-strain binding ability. In summary, the HA_mg vaccine elicits cross-strain immune responses that may mitigate the current need for yearly reformulation of strain-specific inactivated vaccines. This strategy may also map a new direction for universal vaccine design.HA glycoprotein on the surface of influenza virus is a major target for infectivity-neutralizing antibodies. However, the antigenic drift and shift of this protein mean that influenza vaccines must be reformulated annually to include HA proteins of the viral strains predicted for the upcoming flu season (1). This time-consuming annual reconfiguration process has led to efforts to develop new strategies and identify conserved epitopes recognized by broadly neutralizing antibodies as the basis for designing universal vaccines to elicit antibodies with a broad protection against various strains of influenza infection (2–6). Previous studies have shown that the stem region of HA is more conserved and able to induce cross-reactive and broadly neutralizing antibodies (7–9) to prevent the critical fusion of viral and endosomal membranes in the influenza lifecycle (10–14). Other broadly neutralizing antibodies have been found to bind regions near the receptor binding site of the globular domain, although these antibodies are fewer in number (15, 16).Posttranslational glycosylation of HA plays an important role in the lifecycle of the influenza virus and also contributes to the structural integrity of HA and the poor immune response of the infected hosts. Previously, we trimmed down the size of glycans on avian influenza H5N1 HA with enzymes and showed that H5N1 HA with a single N-linked GlcNAc at each glycosylation site [monoglycosylated HA (HA_mg)] produces a superior vaccine with more enhanced antibody response and neutralization activity against the homologous influenza virus than the fully glycosylated HA (HA_fg) (17). Here, to test whether the removal of glycans from HA contributes to better immune responses and possibly protects against heterologous strains of influenza viruses, we compared and evaluated the efficacy of HA glycoproteins with various lengths of glycans as potential vaccine candidates.     

Heterosubtypic antibody recognition of the influenza virus hemagglutinin receptor binding site enhanced by avidity

Continual and rapid mutation of seasonal influenza viruses by antigenic drift necessitates the almost annual reformulation of flu vaccines, which may offer little protection if the match to the dominant circulating strain is poor. S139/1 is a cross-reactive antibody that neutralizes multiple HA strains and subtypes, including those from H1N1 and H3N2 viruses that currently infect humans. The crystal structure of the S139/1 Fab in complex with the HA from the A/Victoria/3/1975 (H3N2) virus reveals that the antibody targets highly conserved residues in the receptor binding site and contacts antigenic sites A, B, and D. Binding and plaque reduction assays show that the monovalent Fab alone can protect against H3 strains, but the enhanced avidity from binding of bivalent IgG increases the breadth of neutralization to additional strains from the H1, H2, H13, and H16 subtypes. Thus, antibodies making relatively low affinity Fab interactions with the receptor binding site can have significant antiviral activity when enhanced by avidity through bivalent interactions of the IgG, thereby extending the breadth of binding and neutralization to highly divergent influenza virus strains and subtypes.Influenza virus is the etiologic agent responsible for seasonal flu and sporadic pandemics and remains a significant health and economic burden by infecting millions each year. Hemagglutinin (HA), the major surface glycoprotein on influenza virus, facilitates virus entry and infection of host cells by binding sialic acid receptors on the surface of endothelial cells, thereby promoting virus entry into endosomes (1, 2). HA exists in 17 distinct subtypes (primarily in birds), which can be split into two major groups by phylogeny (3, 4) and are classified (H1–H17) by their uniqueness of reactivity against polyclonal antisera. Group 1 is comprised of subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, and the recently identified H17 (5), whereas the H3, H4, H7, H10, H14, and H15 subtypes form group 2. Annual vaccines against HA are administered as a countermeasure against influenza and are composed of a mixture of representative H1, H3, and influenza B strains that are selected to match the prevailing or anticipated circulating strains. However, the effectiveness of vaccines heavily relies on the match of the dominant circulating virus to the vaccine strains (6). Additionally, the influenza virus rapidly mutates and can escape the host immune response if sufficient viable HA mutations are incorporated to mask the surface from previously elicited antibodies (7, 8). Thus, a vaccine that provides protection by eliciting an antibody response against multiple HA subtypes may potentially combat a much larger range of strains and subtypes of influenza viruses (9).The HA protein is trimeric in structure and is composed of three identical copies of a single HA0 polypeptide precursor, which upon proteolytic maturation, is cleaved to produce a pH-dependent, metastable intermediate, comprised of HA1 and HA2 subdomains that serve distinct roles in viral infection (10). The membrane distal “head” is composed entirely of HA1 residues and contains the receptor binding site that is used for recognition of sialic acid receptors on host cells (1, 2). The membrane proximal “stem” is assembled from HA2 and some HA1 residues and contains the fusion machinery that is triggered in the low pH environment of late endosomes (11, 12). To inhibit viral infection, antibodies can impede viral attachment to host cells by sterically blocking either receptor binding (13–16), preventing the low pH-induced conformational change (14, 17–19), or interfering with the maturation of HA0 to HA1 and HA2 (18, 20). The HA stem is highly conserved and antibody recognition against this region has been shown to be extremely broad, with neutralization reported against almost all strains within the subtypes from group 1 (17, 21–24), group 2 (18), or both (19, 20). However, eliciting high levels of these stem-directed antibodies by vaccination remains a challenge, either because of poor immunogenicity, mode of immunization, or more restricted access to the HA stem, but recent studies have suggested that such antibodies are produced in some individuals (25, 26) and can be enhanced by DNA prime-boost methods (27). In contrast, HA1 is usually highly immunogenic for most subtypes except H5 (28), although the breadth of neutralization of head-targeted antibodies has generally been poor because of the hypervariability of the residues that surround the receptor binding site (7, 8).Despite the overall sequence variability of HA1, the receptor binding site is relatively conserved as it is constrained to preserve its receptor-binding function. Broadly neutralizing antibodies that specifically target the receptor binding site have been rare, perhaps in part because of its relatively small footprint. S139/1 was the first antibody to be described with heterosubtypic reactivity, neutralizing strains from multiple subtypes, such as H1, H2, H3, and H13 (29), that cross the HA group barrier. A few other recent reports have described a number of broadly neutralizing antibodies that map to the apex of HA close to or at the receptor binding site (29–32), such as CH65, an antibody specific to the H1 subtype (16), as well as C05, which has activity against multiple subtypes (33). Here, we report the crystal structure of the S139/1 Fab in complex with A/Victoria/3/1975 (H3N2) (Vic75/H3) HA and show that S139/1 achieves heterosubtypic neutralization by targeting the receptor binding site on HA. Furthermore, we show that, although Fab is sufficient for neutralization of H3 isolates, S139/1 is unusually dependent upon avidity for heterosubtypic neutralization. Bivalent binding of the IgG significantly boosts the affinity compared with the Fab and is correlated closely with the antibody’s neutralization potential. These findings suggest that antibodies against the HA receptor binding site may possess much greater cross-reactivity than was previously appreciated, and the use of avidity to extend neutralization breadth may be a general feature of many antibodies targeting highly variable surface glycoproteins of viruses, such as influenza and HIV.     

Multiannual forecasting of seasonal influenza dynamics reveals climatic and evolutionary drivers

Human influenza occurs annually in most temperate climatic zones of the world, with epidemics peaking in the cold winter months. Considerable debate surrounds the relative role of epidemic dynamics, viral evolution, and climatic drivers in driving year-to-year variability of outbreaks. The ultimate test of understanding is prediction; however, existing influenza models rarely forecast beyond a single year at best. Here, we use a simple epidemiological model to reveal multiannual predictability based on high-quality influenza surveillance data for Israel; the model fit is corroborated by simple metapopulation comparisons within Israel. Successful forecasts are driven by temperature, humidity, antigenic drift, and immunity loss. Essentially, influenza dynamics are a balance between large perturbations following significant antigenic jumps, interspersed with nonlinear epidemic dynamics tuned by climatic forcing.Influenza outbreaks have been documented in the scientific literature in records that extend back to at least 1650 (1), making it an exceptional example of a persisting, recurrent disease. Being a respiratory infection, influenza spreads rapidly from person to person through a population in the form of virus particles airborne as respiratory droplets or aerosols. Depending on the circumstances, influenza typically infects between 10% and 50% of a given population and has become a source of considerable human morbidity and mortality (2). There is much controversy in identifying the seasonal drivers that generate annual influenza epidemics and the processes that give rise to their large variability (3–12). This is an outstanding problem of influenza research today. Using long-term modeling, a recent study (9) gave support to the possibility that absolute humidity is the predominant determinant of influenza seasonality in temperate zones, driving disease transmission and controlling the timing of individual wintertime outbreaks. Another study investigated the physical properties of absolute humidity on influenza virus transmission and influenza virus survival (3). However, a general understanding of the mechanisms underlying influenza seasonal variation remains quite limited (8). Here, we use a simple mathematical model to unravel the interplay between climate and evolution to predict long-term influenza dynamics correctly for the years since June 2010.A requirement for the generation of recurrent epidemics is a sufficient and continuous source of new susceptible individuals arising in the population, enough to fuel each new outbreak (13). In the case of influenza, infected individuals recover with immunity but eventually become susceptible again because of the rapidly evolving nature of the influenza virus (7, 14). Positive selection exerted by the host immune system leads to a continual antigenic drift of the influenza virus’s glycoproteins, particularly the main antigen, hemagglutinin, thus allowing the virus to eventually evade the immune system (15). The process of antigenic drift thereby creates an important renewed source of susceptible individuals. Hence, evolutionary forces are considered tremendously important in shaping complex recurrent patterns of infectious diseases and explain why influenza is regarded as “an invariable disease caused by a variable virus” (1).The changing rate of antigenic drift also has a significant impact on the timing and amplitude of influenza outbreaks (16). Recent studies reveal that the evolution of influenza A H3N2’s main antigen is punctuated in character such that the drift occurs within discrete antigenic clusters (neutral periods), but with jumps to newly arising clusters after irregular periods (17, 18). A significant jump for the A H3N2 lineage last occurred during the 2003–4 season with the appearance of the A/Fujian virus strain coinciding with a sharp influenza outbreak approximately 2 mo earlier than usual, with a normal attack rate. Nevertheless, it is difficult to demonstrate a consistent and conclusive direct link between the size of antigenic jumps and changes in influenza dynamics at the population level (6, 19–21).     

Critical role of segment-specific packaging signals in genetic reassortment of influenza A viruses

The fragmented nature of the influenza A genome allows the exchange of gene segments when two or more influenza viruses infect the same cell, but little is known about the rules underlying this process. Here, we studied genetic reassortment between the A/Moscow/10/99 (H3N2, MO) virus originally isolated from human and the avian A/Finch/England/2051/91 (H5N2, EN) virus and found that this process is strongly biased. Importantly, the avian HA segment never entered the MO genetic background alone but always was accompanied by the avian PA and M fragments. Introduction of the 5′ and 3′ packaging sequences of HA_MO into an otherwise HA_EN backbone allowed efficient incorporation of the chimerical viral RNA (vRNA) into the MO genetic background. Furthermore, forcing the incorporation of the avian M segment or introducing five silent mutations into the human M segment was sufficient to drive coincorporation of the avian HA segment into the MO genetic background. These silent mutations also strongly affected the genotype of reassortant viruses. Taken together, our results indicate that packaging signals are crucial for genetic reassortment and that suboptimal compatibility between the vRNA packaging signals, which are detected only when vRNAs compete for packaging, limit this process.The mechanisms by which animal viruses are introduced into and are disseminated through the human population remain to be addressed. In particular, emerging pathogenic influenza viruses, such as the highly pathogenic avian H5N1 virus and the 2009 “swine” H1N1 virus (H1N1pdm2009), pose major public health and scientific challenges (1, 2). Even though the natural reservoirs of influenza A viruses are wild aquatic birds, influenza A viruses exhibit a broad host range and a wide antigenic diversity, represented by combinations of 17 hemagglutinin (HA) and nine neuraminidase (NA) subtypes (3). Two subtypes of influenza A viruses, H1N1 and H3N2, currently are circulating in the human population.The genome of influenza A viruses is composed of eight single-stranded, negative-sense viral RNA (vRNA) segments. Each segment is associated with the heterotrimeric polymerase complex consisting of polymerase basic proteins 1 and 2 and polymeric acid (PB1/PB2/PA) and is covered by the viral nucleoprotein (NP) to form a viral ribonucleoparticle (vRNP). The fragmented nature of the genome allows the exchange of gene segments when two or more influenza viruses coinfect the same cell, in a process named “genetic reassortment” (4). Genetic reassortment is a major feature of influenza evolution and cross-species transmission and also is important for the generation of antigenically novel isolates by introducing novel HA segments in compatible genetic backgrounds (5–7). Future pandemic viruses most likely will carry different HA genes to which human populations are immunologically naive. The strains giving rise to the 1918 Spanish, 1957 Asian, and 1968 Hong Kong influenza pandemics all harbored an HA segment derived from an avian virus. The avian viruses circulating in the waterfowl are the source of the HA genes most likely to be introduced into the human population (8). Phylogenetic, epidemic, epizootic, and virology studies suggest that swine serve as “mixing vessels” for the generation of human–avian–swine reassortant viruses.When the reassortment process takes place between a human and an avian influenza virus, there are in theory 127 possible reassortant viruses harboring the avian HA segment. Two studies used forced reverse genetics (i.e., a minimal set of reverse genetic plasmids allowing no competition between segments) to generate all 127 reassortant viruses carrying the HA segment from an avian H5N1 virus in the genetic background of a human H3N2 or the HA segment from an avian H9N2 virus in the genetic background of the human 2009 pandemic H1N1 virus (9, 10). They showed that 49% (H5N1/H3N2) and 58% (H9N2/H1N1) of these reassortant viruses replicated efficiently in Madin–Darby canine kidney (MDCK) cells (9, 10). However, several reports indicated that the number of observed natural or experimental reassortant viruses is much smaller than 127, suggesting that reassortment is somehow restricted (4, 11, 12). When analyzing viruses from the nasal secretions of ferrets coinfected with human H3N2 and avian H5N1 viruses, Jackson et al. (13) observed that only 3.1% were reassortant viruses possessing the HA H5 gene, and they corresponded to only five distinct genotypes. Genetic reassortment between human H3N2 and an equine H7N7 virus has been studied using cotransfection (4). Only 1.6% of purified viruses, corresponding to two genotypes, were reassortant viruses possessing the HA H7 gene (4). In contrast, a high frequency of genetic reassortment was observed recently between swine-origin H1N1 and avian H5N1 viruses: 64% of purified viruses, corresponding to 20 different genotypes, were reassortant viruses possessing the HA H5 gene (14). In this case, the high reassortment rate was attributed to the triple reassortant internal gene cassette, consisting of the avian PA and PB2 genes, the nonstructural (NS), NP, and matrix (M) swine genes, and the human PB1 gene (15).The low number of reassortant genotypes usually generated from genetically diverse influenza viruses suggests incompatibilities at the protein and/or genomic level. Accumulating evidence indicates that protein incompatibility among the vRNP components is a limiting factor for reassortment between two viruses (4, 16–18), but little is known about genetic incompatibilities between the vRNA segments. Although incompatibility between proteins is expected to have similar effects in cotransfection or coinfection experiments and in forced reverse genetic experiments, genomic incompatibilities may have several possible effects, especially at the level of the vRNA-packaging signals. Some incompatibilities between packaging signals might reduce viral replication in the absence of competition (absolute incompatibility), whereas more subtle ones might be revealed only when vRNA segments from the two parental viruses compete for packaging (suboptimal compatibility). Reverse genetics-derived reassortant viruses (RGd-RV) that possess the H5N1 (H5) HA in an otherwise H3N2 genetic background show high replicative capacities in MDCK cells (10). Similarly, RGd-RV with the HA gene from H5N1 virus in the H1N1pdm2009 genetic background replicated efficiently in primary human respiratory epithelial cells and caused 100% mortality in mice (19). However, phylogenetic analyses of natural or experimental reassortant viruses have shown that the HA segment from avian, swine, or equine viruses was never incorporated alone in the genetic background of a human virus (13, 14, 20): The HA segment is packaged with additional groups of gene segments depending on the viral subtypes involved in the coinfection process (13, 14).The inability to obtain a virus containing a nonhuman HA gene in an otherwise human genetic background, in contrast with the ability to produce “7+1” RGd-RV with a high yield of replication, suggests that the reassortment process might be restricted by suboptimal compatibility between the vRNA-packaging signals (10).To predict how pandemic influenza viruses can emerge, the complex molecular mechanisms limiting or facilitating genetic reassortment must be deciphered. Using reverse genetics, cis-packaging signals of the human H1N1 WSN and PR8 strains were found to reside at both ends of each vRNA, including the UTRs, along with up to 80 bases of adjacent coding sequences (21–28). In this study, we generated reassortant viruses in vitro from avian H5N2 and human H3N2 viruses to identify incompatibilities between the two parental viruses arising at the vRNA level. Our experiments focusing on the generation of reassortant viruses containing the HA H5 gene segment in an H3N2 genetic background indicate that genomic suboptimal compatibility driven by the selective packaging mechanism limits the generation of HA H5 reassortant viruses in vitro.     

Chromatographically isolated CD63+CD81+ extracellular vesicles from mesenchymal stromal cells rescue cognitive impairments after TBI

Extracellular vesicles (EVs) secreted by cells present an attractive strategy for developing new therapies, but progress in the field is limited by several issues: The quality of the EVs varies with the type and physiological status of the producer cells; protocols used to isolate the EVs are difficult to scale up; and assays for efficacy are difficult to develop. In the present report, we have addressed these issues by using human mesenchymal stem/stromal cells (MSCs) that produce EVs when incubated in a protein-free medium, preselecting the preparations of MSCs with a biomarker for their potency in modulating inflammation, incubating the cells in a chemically defined protein-free medium that provided a stable environment, isolating the EVs with a scalable chromatographic procedure, and developing an in vivo assay for efficacy of the cells in suppressing neuroinflammation after traumatic brain injury (TBI) in mice. In addition, we demonstrate that i.v. infusion of the isolated EVs shortly after induction of TBI rescued pattern separation and spatial learning impairments 1 mo later.Traumatic brain injury (TBI) has devastating effects on the victims and creates a large burden on the healthcare system (1). TBI was originally considered an acute injury syndrome, but it is now recognized to have chronic effects similar to those found in neurodegenerative disorders (2–5). In the acute phase, the trauma destroys tissue, and it also triggers a cascade of events that include excessive neural excitability, oxidative stress, disruption of the blood–brain barrier, and inflammation. The cascade causes additional cell death that occurs through necrosis, apoptosis, and excessive autophagy. The cascade involves astrocytes and microglia, in addition to invading neutrophils, monocytes/macrophages, and T cells. The sequence of events is similar to the sequence seen with sterile injuries to other tissues. Initially, proinflammatory effects predominate and are useful in clearing tissue debris. Thereafter, there is a transition to an antiinflammatory phase, with the microglia and macrophages transiting from “classical” proinflammatory M1 phenotype to multiple alternative M2 phenotypes that suppress the M1 proinflammatory mediators and enhance tissue repair. The chronic effects of TBI occur because the inflammatory phase is not fully suppressed. Instead, the inflammatory responses persist, and they initiate a self-perpetuating cycle of tissue destruction, followed by further inflammation. A similar cycle is now recognized to contribute to the pathology of many chronic diseases.Multiple strategies have been tested to modulate inflammation in TBI and other CNS disorders (2–4). Among these strategies is the use of mesenchymal stem/stromal cells (MSCs) from bone marrow and other tissues (6–19). The beneficial effects of the MSCs are probably explained by their normal roles as perivascular cells that are among the first responders to tissue injury. One of their responses is to act in concert with other cells as guardians of excessive inflammation because they are activated by proinflammatory cytokines such as TNF-α to secrete modulators of inflammation that include TNF-alpha stimulated gene/protein 6 (TSG-6), PGE-2, STC-1, IL-1 receptor antagonist, and TIMP3 (18, 20–25).Recently, we have explored the hypothesis that extracellular vesicles (EVs) produced by MSCs may be an effective therapy for TBI because extensive recent reports indicate that EVs may provide a highly efficient means of delivering therapeutic factors to target cells (26–29). As noted by György et al. (29), there are several issues that currently limit therapeutic applications of EVs. In the present report, we have addressed most of these issues. In addition, we demonstrate the efficacy of EVs isolated from MSCs in a mouse model for TBI. As this work was in progress, Zhang et al. (30) reported that exosomes isolated from MSCs improved functional recovery in a rat model for TBI, but they did not characterize the exosomes.     

Induction of broadly cross-reactive antibody responses to the influenza HA stem region following H5N1 vaccination in humans

The emergence of pandemic influenza viruses poses a major public health threat. Therefore, there is a need for a vaccine that can induce broadly cross-reactive antibodies that protect against seasonal as well as pandemic influenza strains. Human broadly neutralizing antibodies directed against highly conserved epitopes in the stem region of influenza virus HA have been recently characterized. However, it remains unknown what the baseline levels are of antibodies and memory B cells that are directed against these conserved epitopes. More importantly, it is also not known to what extent anti-HA stem B-cell responses get boosted in humans after seasonal influenza vaccination. In this study, we have addressed these two outstanding questions. Our data show that: (i) antibodies and memory B cells directed against the conserved HA stem region are prevalent in humans, but their levels are much lower than B-cell responses directed to variable epitopes in the HA head; (ii) current seasonal influenza vaccines are efficient in inducing B-cell responses to the variable HA head region but they fail to boost responses to the conserved HA stem region; and (iii) in striking contrast, immunization of humans with the avian influenza virus H5N1 induced broadly cross-reactive HA stem-specific antibodies. Taken together, our findings provide a potential vaccination strategy where heterologous influenza immunization could be used for increasing the levels of broadly neutralizing antibodies and for priming the human population to respond quickly to emerging pandemic influenza threats.The emergence of novel influenza virus strains poses a continuous public health threat (1, 2). The World Health Organization estimates that influenza viruses infect one-billion people annually, with three- to five-million cases of severe illness, and up to 500,000 deaths worldwide (3). Following influenza virus infection, humoral immune responses against the viral hemagglutinin (HA) protein may persist for decades in humans (4). These anti-HA responses correlate strongly with protection against influenza infection (5). Serological memory is maintained by antibody-secreting long-lived plasma cells and reinforced by memory B cells, which can rapidly differentiate into antibody-secreting cells upon antigen reexposure (6).Influenza vaccine efficacy is constantly undermined by antigenic variation in the circulating viral strains, particularly in the HA and neuraminidase (NA) proteins. Current influenza vaccination strategies rely on changing the HA and NA components of the annual human vaccine to ensure that they antigenically match circulating influenza strains (7, 8). Developing an influenza vaccine that is capable of providing broad and long-lasting protective antibody responses remains the central challenge for influenza virus research.HA is a trimer, with each monomer comprised of two subunits: HA1, which includes the HA globular head, and HA2, whose ectodomain together with the N- and C-terminal parts of HA1 constitute the HA stem region (9). Phylogenetically, the 18 HA subtypes characterized so far are divided into two groups. Among strains that have recently caused disease in humans, H1 and H5 HAs belong to group 1, whereas H3 and H7 HAs belong to group 2 (10). Conventional anti-HA neutralizing antibodies primarily target a few immunodominant epitopes located in proximity to the receptor-binding domain within the globular head region of the molecule (11, 12). Although these antibodies are potentially protective, they are strain-specific because of the high variability of such epitopes, and thus lack, in general, the much-desired broad neutralizing activity. Recently, broadly neutralizing human (13–18) and murine (19) monoclonal antibodies (mAbs) directed against distinct epitopes within the HA stem region have been extensively characterized. These mAbs were shown to interfere with the influenza viruses’ life cycle in different ways (20). By generating monoclonal antibodies from plasmablasts isolated ex vivo, we demonstrated that these broadly neutralizing antibodies could be retrieved from patients infected with or vaccinated against the pandemic H1N1 2009 influenza virus (18, 21). Recent observations that HA stem epitopes are accessible on the majority of HA trimers on intact virions (22), and that a stable HA stem protein that is immunologically intact could be produced (23), provided further hope for the feasibility of a stem-based universal influenza vaccine (24).Notably, HA stem-specific mAbs isolated from humans showed a high degree of affinity maturation, suggesting a memory B-cell origin. These results raised two important questions that we address in the current study. First, what are the baseline levels of broadly cross-reactive stem-binding antibodies and memory B cells? Second, using current influenza vaccines, to what extent can HA stem-specific responses be boosted in comparison with those directed against the HA globular head?Structural studies have clearly demonstrated that the main neutralizing antibody epitopes within the HA stem region are conformation-dependent, and that the integrity of these epitopes requires the presence of the HA1 subunit in addition to the HA2 subunit, which constitute the bulk of the HA stem (16, 17). To be able to directly measure HA stem-reactive antibodies and memory B cells, we used a chimeric HA molecule that expresses the globular head of H9 HA on H1 backbone (25). Our data demonstrate that post-2009 trivalent inactivated vaccines (TIV) induced minimal stem-specific responses in comparison with head-specific responses. On the other hand, immunization with H5N1 generated relatively strong anti-HA stem responses, demonstrating that it is feasible to elicit broadly neutralizing responses in humans given the right immunogen design.     

Relationship of the quaternary structure of human secretory IgA to neutralization of influenza virus

Secretory IgA (S-IgA) antibodies, the major contributors to humoral mucosal immunity to influenza virus infection, are polymeric Igs present in many external secretions. In the present study, the quaternary structures of human S-IgA induced in nasal mucosa after administration of intranasal inactivated influenza vaccines were characterized in relation to neutralization potency against influenza A viruses. Human nasal IgA antibodies have been shown to contain at least five quaternary structures. Direct and real-time visualization of S-IgA using high-speed atomic force microscopy (AFM) demonstrated that trimeric and tetrameric S-IgA had six and eight antigen-binding sites, respectively, and that these structures exhibited large-scale asynchronous conformational changes while capturing influenza HA antigens in solution. Furthermore, trimeric, tetrameric, and larger polymeric structures, which are minor fractions in human nasal IgA, displayed increased neutralizing potency against influenza A viruses compared with dimeric S-IgA, suggesting that the larger polymeric than dimeric forms of S-IgA play some important roles in protection against influenza A virus infection in the human upper respiratory tract.Antibodies in respiratory mucosa are primary mediators of protective immunity against influenza. Notably, preexisting secretory IgA (S-IgA) antibodies can provide immediate immunity by eliminating a pathogen before the virus passes the mucosal barrier (1–3). Parenteral vaccination induces serum IgG but not S-IgA, so vaccine efficacy is limited. In contrast, intranasal administration of an inactivated influenza vaccine elicits both S-IgA and IgG responses, thus improving the protective efficacy of current vaccination procedures (4–8).IgA in human serum exists predominantly in the form of monomers, whereas the majority of IgA in external secretions is present in the form of polymers. These polymeric IgA forms are associated with the extracellular portion of the polymeric Ig receptor, generating a complex (receptor + polymeric IgA) called S-IgA (9). S-IgA primarily corresponds to dimeric IgA, although low levels of some larger polymeric forms, particularly tetramers, are also present (9–15). Polymeric S-IgA has been shown (both in vitro and in experimental animal models) to be more effective than monomeric IgA or IgG for the neutralization of influenza viruses (16–19). However, little is known of the quaternary structures and neutralizing potencies in viral infection of the various forms of polymeric S-IgA in the human nasal mucosa. In this study, the quaternary structures and neutralizing potencies of nasal antibodies against influenza virus were examined using nasal wash samples from healthy adults who had received intranasally administered inactivated influenza vaccines. These nasal wash samples, containing variously sized Igs, were separated by gel filtration chromatography (GFC) and assessed for neutralization activity against influenza virus. The quaternary structures of the nasal IgA induced by intranasally administered inactivated influenza vaccines then were determined using biochemical techniques and high-speed atomic force microscopy (AFM). We found that human nasal IgA comprised at least five quaternary structures, including monomer, dimer, trimer, and tetramer structures, as well as a polymeric form larger than the tetramer structure. Among these forms, the polymeric structure demonstrated higher neutralizing potency against seasonal influenza viruses (H3N2) and highly pathogenic avian influenza virus (H5N1) compared with the dimeric form, suggesting that large polymeric S-IgA antibodies play crucial roles in protective immunity against influenza virus infection of the human upper respiratory tract.     

Prevention of influenza by targeting host receptors using engineered proteins

There is a need for new approaches for the control of influenza given the burden caused by annual seasonal outbreaks, the emergence of viruses with pandemic potential, and the development of resistance to current antiviral drugs. We show that multivalent biologics, engineered using carbohydrate-binding modules specific for sialic acid, mask the cell-surface receptor recognized by the influenza virus and protect mice from a lethal challenge with 2009 pandemic H1N1 influenza virus. The most promising biologic protects mice when given as a single 1-μg intranasal dose 7 d in advance of viral challenge. There also is sufficient virus replication to establish an immune response, potentially protecting the animal from future exposure to the virus. Furthermore, the biologics appear to stimulate inflammatory mediators, and this stimulation may contribute to their protective ability. Our results suggest that this host-targeted approach could provide a front-line prophylactic that has the potential to protect against any current and future influenza virus and possibly against other respiratory pathogens that use sialic acid as a receptor.Influenza viruses continue to be a threat to human health and a burden on health services (1). The emergence of highly pathogenic H5N1 viruses and recent introductions of H7N9 viruses from avian sources (2), and their potential to acquire human transmissibility, increase the threat (3–5). Although vaccines remain a cornerstone of prevention, significant time is required to develop an effective vaccine against a new virus strain. Anti-influenza drugs approved by the Food and Drug Administration, such as the viral neuraminidase inhibitors oseltamivir (Tamiflu) and zanamivir (Relenza) and the M2 ion-channel blocker adamantanes (amantadine and rimantadine), are available, but their effectiveness can be compromised by the virus’s ability to mutate and become drug resistant (6, 7).The influenza virus binds to sialic acid receptors present on the respiratory tract epithelium via its surface HA glycoprotein, an event that triggers viral endocytosis (8). Other respiratory pathogens, such as parainfluenza viruses (9), some coronaviruses (10), and Streptococcus pneumoniae (11), also use sialic acid as a receptor. Human influenza viruses such as the 2009 pandemic H1N1 virus recognize α-2,6–linked sialic acid receptors present in the upper respiratory tract, whereas avian influenza viruses such as H5N1 predominantly recognize α-2,3–linked sialic acid receptors, which are present in the human lower respiratory tract as well (12, 13). The recently emerged human H7N9 influenza virus is unusual in recognizing both types of receptors and therefore has the possibility of sustained human-to-human transmission and pandemic potential (14, 15).We hypothesized that masking such receptors in the respiratory tract with proteins specific for sialic acid could provide a novel host-targeted therapeutic route to prevent infection. Numerous sialic acid-binding proteins are known, but most have low affinity for sialic acid (e.g., the HA monomer that has ∼2.5 mM affinity for its receptor but gains affinity by being present in high copy number on the virus surface) (16). We have shown previously that engineered multivalent polypeptides containing up to four tandemly linked copies of the sialic acid-recognizing carbohydrate-binding module (CBM) from Vibrio cholerae nanH sialidase display low (nanomolar) binding affinity compared with the 30-μM affinity of the single domain (17).Here we report the engineering and characterization of further sialic acid-recognizing multivalent CBMs (mCBMs) together with in vitro and in vivo evidence of their potential in preventing influenza infection. Significantly, our lead mCBM demonstrates protective in vivo efficacy when given to mice as a single 1-μg dose 7 d in advance of a lethal virus challenge with pandemic 2009 H1N1 influenza virus, indicating that these mCBMs show great promise as biologics for the prophylaxis of influenza and potentially other respiratory pathogens that recognize sialic acid receptors.     

Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection

Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks.Seasonal influenza outbreaks across the globe cause an estimated 250,000–500,000 deaths annually (1). Current influenza vaccines need to be updated every few years because of antigenic drift (2). Despite intensive monitoring, strain mismatch between vaccine formulation and influenza viruses circulating within the population has occurred in the past (2). Public health is further compromised when an unpredictable mixing event among influenza virus genomes leads to antigenic shift facilitating a potential pandemic outbreak. These concerns have expedited efforts toward developing a universal influenza vaccine.Neutralizing antibodies (nAbs) against hemagglutinin (HA) are the primary correlate for protection in humans and hence HA is an attractive target for vaccine development (3). The precursor polypeptide, HA0, is assembled into a trimer along the secretory pathway and transported to the cell surface. Cleavage of HA0 generates the disulfide-linked HA1 and HA2 subunits. Mature HA has a globular head domain which mediates receptor binding and is primarily composed of the HA1 subunit, whereas the stem domain predominantly comprises the HA2 subunit. The HA stem is trapped in a metastable state and undergoes an extensive low-pH-induced conformational rearrangement in the host-cell endosomes to adopt the virus–host membrane fusion-competent state (4, 5).The antigenic sites on the globular head of HA are subjected to heightened immune pressure resulting in escape variants, thereby limiting the breadth of head-directed nAbs (6). However, extensive efforts have resulted in the isolation of monoclonal antibodies (mAbs) that bind within the globular head and inhibit receptor attachment, which neutralize drifted variants of an HA subtype or heterosubtypic HA (7–16). The HA stem is targeted by several broadly neutralizing antibodies (bnAbs) with neutralizing activity against diverse influenza A virus subtypes (17). The epitopes of these bnAbs in the HA stem are more conserved across different influenza HA subtypes compared with the antigenic sites in the HA globular head (18).During a primary infection, the immunodominant globular head domain suppresses the response toward the conserved stem. Several efforts have been made to circumvent this problem. Repeated immunizations with full-length, chimeric HAs (cHAs) in a protracted vaccination regimen have been shown to boost stem-directed responses in mice (19). Alternatively, full-length HA presented on nanoparticles (np) has been shown to elicit stem-directed nAbs (20). Attempts have also been made to steer the immune response toward the conserved HA stem by hyperglycosylating the head domain (21). Although the aforementioned strategies need to be further evaluated and provide novel alternatives, detrimental interference from the highly variable immunodominant head domain in eliciting a broad functional response cannot be completely evaded. A “headless” stem domain immunogen offers an attractive solution. However, early attempts at expressing the HA2 subunit independently in a native, prefusion conformation were unsuccessful. In the absence of the head domain, the HA2 subunit expressed in Escherichia coli spontaneously adopted the low-pH conformation (22) in which the functional epitopes of stem-directed bnAbs are disrupted. More recently, the entire HA stem region has been expressed in a prefusion, native-like conformation in both prokaryotic and eukaryotic systems adopting multiple strategies (23–26).Design of independently folding HA stem fragments which adopt the prefusion HA conformation presents another approach to elicit bnAbs against influenza (27, 28). The A helix of the HA2 subunit contributes substantial contact surface to the epitope of stem-directed bnAbs such as CR6261, F10, and others. Although multivalent display of A helix on the flock house virus as a virus-like particle platform elicited cross-reactive antibodies, it conferred only minimal protection (20%) against virus challenge in mice (29).We report the design and characterization of engineered headless HA stem immunogens based on the influenza A/Puerto Rico/8/34 (H1N1) subtype. H1HA10-Foldon, a trimeric derivative of our parent construct (H1HA10), bound conformation-sensitive, stem-directed bnAbs such as CR6261 (30), F10 (31), and FI6v3 (32) with a high-affinity [equilibrium dissociation constant (K_D) of 10–50 nM]. The designed immunogens elicited broadly cross-reactive antiviral antibodies which neutralized highly drifted influenza virus strains belonging to both group 1 (H1 and H5 subtypes) and 2 (H3 subtype) in vitro. Significantly, stem immunogens designed from unmatched, highly drifted influenza strains conferred protection against a lethal (2LD₉₀) heterologous A/Puerto Rico/8/34 virus challenge in mice. Our immunogens confer robust subtype-specific and modest heterosubtypic protection in vivo. In contrast to previous stem domain immunogens (23–25), the designed immunogens were purified from the soluble fraction in E. coli. The HA stem-fragment immunogens do not aggregate even at high concentrations and are cysteine-free, which eliminates the complications arising from incorrect disulfide-linked, misfolded conformations. The aforementioned properties of the HA stem-fragment immunogens make it amenable for scalability at short notice which is vital during pandemic outbreaks.     

Prevalence,genetics, and transmissibility in ferrets of Eurasian avian-like H1N1 swine influenza viruses

Pigs are important intermediate hosts for generating novel influenza viruses. The Eurasian avian-like H1N1 (EAH1N1) swine influenza viruses (SIVs) have circulated in pigs since 1979, and human cases associated with EAH1N1 SIVs have been reported in several countries. However, the biologic properties of EAH1N1 SIVs are largely unknown. Here, we performed extensive influenza surveillance in pigs in China and isolated 228 influenza viruses from 36,417 pigs. We found that 139 of the 228 strains from pigs in 10 provinces in China belong to the EAH1N1 lineage. These viruses formed five genotypes, with two distinct antigenic groups, represented by A/swine/Guangxi/18/2011 and A/swine/Guangdong/104/2013, both of which are antigenically and genetically distinct from the current human H1N1 viruses. Importantly, the EAH1N1 SIVs preferentially bound to human-type receptors, and 9 of the 10 tested viruses transmitted in ferrets by respiratory droplet. We found that 3.6% of children (≤10 y old), 0% of adults, and 13.4% of elderly adults (≥60 y old) had neutralization antibodies (titers ≥40 in children and ≥80 in adults) against the EAH1N1 A/swine/Guangxi/18/2011 virus, but none of them had such neutralization antibodies against the EAH1N1 A/swine/Guangdong/104/2013 virus. Our study shows the potential of EAH1N1 SIVs to transmit efficiently in humans and suggests that immediate action is needed to prevent the efficient transmission of EAH1N1 SIVs to humans.Pigs play a pivotal role in the ecology of influenza A viruses, being regarded as intermediate hosts for the generation of novel and potentially dangerous influenza viruses for humans. Cellular receptors containing α-2,3–linked sialic acids (Sias) (avian-like receptors) and α-2,6–linked Sias (human-like receptors) in the pig trachea favor the productive replication of viruses from both the avian and mammalian lineages (1). Influenza viruses of the subtypes H1N1, H1N2, and H3N2 are circulating in pigs globally (2). Two lineages of H1N1 swine influenza viruses (SIVs), classical H1N1 SIVs and Eurasian avian-like H1N1 (EAH1N1) SIVs, have been circulating in pigs since 1918 and 1979, respectively (3, 4). The classical H1N1 SIVs emerged in humans as a reassortant (2009/H1N1) and caused the 2009 H1N1 influenza pandemic (5). The EAH1N1 SIVs have been detected in pigs in many Eurasian countries (6) and have caused several human infections in European countries and also in China (7–11), where a fatal case was reported (11). EAH1N1 SIVs are reported to be most prevalent in pigs that have been brought into Hong Kong since 2005 (12). However, the evolution and biologic properties of the EAH1N1 SIVs are largely unknown.China is the largest pork-producing country in the world. Pigs in China are not vaccinated against influenza, and therefore, influenza viruses can spread freely once they are introduced into pig herds. In this study, we performed active surveillance in pigs and found that the EAH1N1 SIVs are predominant in the pig population in China; we further found that the EAH1N1 SIVs pose an imminent threat with regard to their ability to cause a human influenza pandemic.     

MSCs derived from iPSCs with a modified protocol are tumor-tropic but have much less potential to promote tumors than bone marrow MSCs

Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However, tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs, especially those in cancer patients. To circumvent these issues, we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable, but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial–mesenchymal transition, invasion, stemness, and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFβ, downstream protumor factors, and hyaluronan and its cofactor TSG6, which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for “off-the-shelf” therapies and bioengineering applications.The use of mesenchymal stromal or stem cells (MSCs) in cancer patients or cancer survivors is a promising strategy to improve treatment of advanced cancer (1) and to repair tissues damaged by cancers or by radical cancer therapies (2). Based on the unique homing capability of tissue-derived MSCs to stroma of various primary and metastatic cancers (3–6), MSCs have the potential to treat or even eliminate various cancers by delivering various anticancer agents (7–9). Because of their potential for differentiation (10, 11) and production of immunomodulatory, angiogenic, anti-apoptotic, anti-scarring, and prosurvival factors (12), MSCs have shown promising regeneration potential after radical cancer treatment in animal models, such as soft tissue reconstruction after disfiguring surgeries for head, neck, or breast cancers (13) and salivary gland regeneration for head and neck cancer patients treated with radiotherapy (14, 15). As one example, the combination of osteogenic potential and targeted delivery of anticancer agents make MSCs a promising option to treat tumor-induced osteolysis (16, 17). However, exogenous tissue-derived MSCs, including those from bone marrow, adipose tissues, and umbilical cord, have all shown a tendency to promote rather than inhibit cancers in many circumstances (18–23). Also, endogenous MSCs are a major source of reactive stromal cells that promote growth and metastasis of cancers (4, 24).Moreover, MSCs have a limited proliferation potential and lose some of their important biological functions as they are expanded (25). Therefore, it is difficult to prepare large banks of the cells with uniform biological activities and/or transgene expression required for experiments in large animals and for potential clinical therapies. Another problem is that MSCs are being prepared with a variety of protocols in different laboratories from different donors. As a result, standardization of the cells has been extremely difficult and the data presented in different publications are difficult to compare. Hence large banks of reference cells are needed to advance the MSC research (26).To address the limitations of expandability and standardization, we derived MSCs from induced pluripotent stem cells (iPSCs) with a modified protocol that can be expanded to provide large cell banks from a single cell clone. The protocol produces highly enriched MSC-like cells from iPSCs with high efficiency. The iPSC-derived MSCs (iPSC-MSCs) express the classical surface markers of MSCs, are capable of multilineage mesodermal differentiation and cancer homing, and can be expanded extensively, but do not preserve the pluoripotency of iPSCs. Surprisingly, iPSC-MSCs do not promote epithelial–mesenchymal transition (EMT), invasion, and stemness of cancer cells as is seen with bone marrow-derived MSCs (BM-MSCs). Consistent with these observations, the iPSC-MSCs express much lower levels than BM-MSCs of protumor factors including interleukine-6, prostaglandin E2, SDF1, and hyaluronan before and after exposure to tumor microenvironment. Our data indicated that iPSC-MSCs are a safe alternative to BM-MSCs for cancer therapy and other applications with better expandability and potential for genetic engineering.     

A functional sequence-specific interaction between influenza A virus genomic RNA segments

Influenza A viruses cause annual influenza epidemics and occasional severe pandemics. Their genome is segmented into eight fragments, which offers evolutionary advantages but complicates genomic packaging. The existence of a selective packaging mechanism, in which one copy of each viral RNA is specifically packaged into each virion, is suspected, but its molecular details remain unknown. Here, we identified a direct intermolecular interaction between two viral genomic RNA segments of an avian influenza A virus using in vitro experiments. Using silent trans-complementary mutants, we then demonstrated that this interaction takes place in infected cells and is required for optimal viral replication. Disruption of this interaction did not affect the HA titer of the mutant viruses, suggesting that the same amount of viral particles was produced. However, it nonspecifically decreased the amount of viral RNA in the viral particles, resulting in an eightfold increase in empty viral particles. Competition experiments indicated that this interaction favored copackaging of the interacting viral RNA segments. The interaction we identified involves regions not previously designated as packaging signals and is not widely conserved among influenza A virus. Combined with previous studies, our experiments indicate that viral RNA segments can promote the selective packaging of the influenza A virus genome by forming a sequence-dependent supramolecular network of interactions. The lack of conservation of these interactions might limit genetic reassortment between divergent influenza A viruses.Influenza A viruses (IAVs) belong to the Orthomyxoviridae family and cause annual influenza epidemics and occasional pandemics that represent a major threat for human health (1). The IAV genome consists of eight single-stranded negative-sense RNA segments (vRNAs), ranging from 890 to 2,341 nucleotides (nts) and packaged as viral ribonucleoproteins (vRNPs) containing multiple copies of nucleoprotein (NP) and a RNA-dependent RNA polymerase complex (2–4). The central coding region (in antisense orientation) of the vRNAs is flanked by short, segment-specific untranslated regions and conserved, partially complementary, terminal sequences that constitute the viral polymerase promoter and impose a panhandle structure to the vRNPs (4–9). The segmented nature of the IAV genome favors viral evolution by genetic reassortment. This process, which takes place when a single cell is coinfected by different IAVs, can generate pandemic viruses that represent a major threat for human health (1). However, segmentation complicates packaging of the viral genome into progeny virions.Although it had initially been proposed that the vRNAs are randomly packaged into budding viral particles, several lines of experiment suggest that IAVs specifically package one copy of each vRNA during viral assembly (7). First, electron microscopy and tomography revealed that the relative disposition of the eight vRNPs within viral particles is not random, even though some variability is tolerated, and they adopt a typical arrangement, with seven vRNPs surrounding a central one (10–12). Second, genetic and biochemical analysis revealed that the vast majority of IAV particles contain exactly one copy of each vRNA (7, 13, 14). Third, analysis of defective interfering RNAs (7, 15–17) and reverse genetic experiments (7, 18–25) identified specific bipartite packaging signals, most often located within the ends of the coding regions, in each segment. Of note, the terminal promoters are crucial for RNA packaging (8), but they cannot confer specificity to the packaging process (7).A selective packaging mechanism requires the existence of direct RNA–RNA or indirect RNA–protein interactions between vRNAs (7). Because all vRNAs associate with the same viral proteins to form vRNPs and no cellular protein has been identified that would specifically recognize an IAV packaging signal, we (10) and others (7, 12, 19) hypothesized that direct interactions between vRNAs might ensure selective packaging. However, these interactions remain elusive. We recently showed that the eight vRNAs of both a human H3N2 IAV (10) and an avian H5N2 IAV (26) form specific networks of intermolecular interactions in vitro, but the functional relevance of these interactions was not demonstrated. Here, we used a biochemical approach to identify, at the nt level, an interaction between two in vitro transcribed vRNAs. Unexpectedly, this interaction occurs between regions not previously identified as packaging signals. We then demonstrated that this interaction is important for infectivity and packaging of the viral genome.