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.     

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.     

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.     

Adjuvant solution for pandemic influenza vaccine production

Extensive preparation is underway to mitigate the next pandemic influenza outbreak. New vaccine technologies intended to supplant egg-based production methods are being developed, with recombinant hemagglutinin (rHA) as the most advanced program for preventing seasonal and avian H5N1 Influenza. Increased efforts are being focused on adjuvants that can broaden vaccine immunogenicity against emerging viruses and maximize vaccine supply on a worldwide scale. Here, we test protection against avian flu by using H5N1-derived rHA and GLA-SE, a two-part adjuvant system containing glucopyranosyl lipid adjuvant (GLA), a formulated synthetic Toll-like receptor 4 agonist, and a stable emulsion (SE) of oil in water, which is similar to the best-in-class adjuvants being developed for pandemic flu. Notably, a single submicrogram dose of rH5 adjuvanted with GLA-SE protects mice and ferrets against a high titer challenge with H5N1 virus. GLA-SE, relative to emulsion alone, accelerated induction of the primary immune response and broadened its durability against heterosubtypic H5N1 virus challenge. Mechanistically, GLA-SE augments protection via induction of a Th1-mediated antibody response. Innate signaling pathways that amplify priming of Th1 CD4 T cells will likely improve vaccine performance against future outbreaks of lethal pandemic flu.H5N1 is a highly pathogenic avian influenza virus that can cause severe disease and death in humans, and world health authorities agree that the potential for pandemic H5N1 infection is high. Vaccination remains the most effective mechanism for preventing influenza, but there are complex challenges in implementing a pandemic preparedness plan, including: an inability to rapidly deploy the vast numbers of safe and effective doses needed on a worldwide scale; the fact that the immunogenicity of current nonadjuvanted H5N1 vaccines are relatively weak and require large antigen doses; and the potency of stockpiled prepandemic vaccines may be severely limited given the anticipated antigenic drift/shift associated with the emergence of a novel strain of pandemic H5N1.The US government has outlined provisions for new technologies that maximize immunogenicity and manufacturing capacity of vaccines for influenza, including the use of recombinant protein-based vaccines and adjuvants, which augment immunity and dose-sparing capacity. The most advanced egg-free flu vaccine candidate is a recombinant multimeric H5 hemagglutinin protein (rH5) produced by using a baculovirus expression vector system in SF+ insect cells (1, 2). Previous clinical studies suggested that two 90-μg doses of rH5 induced modest responses equivalent to conventional subvirion-based H5N1 vaccines (3, 4). This finding has prompted efforts to test rH5 with an adjuvant. Currently, the leading H5N1 vaccine adjuvants are oil-in-water (o/w) emulsions, which augment neutralizing antibody titers, increase the breadth of cross-reactive antibodies, and possess significant dose-sparing activity (5, 6). Importantly, these adjuvants are particularly effective in priming naïve individuals in the absence of preexisting memory.Vaccine adjuvants regulate adaptive immunity by stimulating dendritic cell maturation and antigen presentation (7, 8). A leading adjuvant target on DC is the family of innate Toll-like receptors, particularly the LPS receptor, Toll-like receptor 4 (TLR4). Glucopyranosyl lipid adjuvant (GLA) is a formulated form of the synthetic TLR4 agonist PHAD (Avanti Polar Lipids), which is analogous to the detoxified LPS derivative monophosphoryl lipid A (MPL), a component of the human papillomavirus vaccine Cervarix (9). Experimental vaccines containing GLA demonstrate enhanced immunogenicity in a variety of disease models (8), and in the context of influenza, GLA formulated in a stable emulsion (GLA-SE) improved Fluzone-dependent antibody titers in mice and nonhuman primates, relative to an emulsion alone (10–13). Given the critical importance of immunological priming for pandemic vaccine preparedness, we set out to test whether adjuvanting a recombinant H5 antigen with GLA-SE would broaden protective immunity against H5N1.     

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.     

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.     

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.     

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.     

Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody

H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer’s formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.The initial steps in influenza virus infection involve sialic acid receptor binding and membrane fusion, both of which are functions of the hemagglutinin (HA) virus membrane glycoprotein. Anti-HA antibodies that block these functions neutralize virus infectivity. Such antibodies are induced by infection and by vaccination, and the immune pressure that they impose on subsequently infecting viruses is responsible for the antigenic drift for which influenza viruses are notorious. Zoonotic infections, which can lead to new pandemics, occur periodically, and H5N1, H7N9, and H10N8 avian viruses are recent examples of this sort. The threat that zoonotic infections present is based, in part, on the lack of immunity in the human population to the novel HAs that they contain. In attempts to substitute for this deficiency, human immune sera have been used successfully to treat severe infections (1), and monoclonal antibodies have been prepared from mice and from humans for potential use in immunotherapy.Analyses of antibodies produced by cloned immune cells derived from infected patients have revealed that antibodies are induced that are either subtype- or group-specific and others that cross-react with HAs of both groups (2). To date, cross-reactive antibodies have been shown to recognize both membrane-distal and membrane-proximal regions of HA (3). Subtype-specific antibodies, on the other hand, bind to the membrane-distal region, covering the receptor-binding site and, in some cases, inserting into it (4, 5).In the studies reported here, a human monoclonal antibody is described that recognizes the HAs of viruses of all three clades of the H5 subtype that have caused human infection and is shown to be effective in protecting mice from lethal challenge. EM and X-ray crystallography studies of HA-Fab complexes indicate that the antibody binds to a site containing residue 122, located on the membrane-distal surface of the HA trimer. We describe the antibody-binding site in detail to show that binding occurs at a distance from the receptor-binding site. Infectivity neutralization and receptor-binding experiments, together with these observations, lead to the conclusion that the antibody neutralizes viruses by blocking receptor binding in a way that is dependent on the Fc region of the bound antibody. We compare the site with similar sites reported by others (6–9) for antibodies that have not as yet given crystalline HA-Fab complexes.Under the conditions that we obtain crystals of the HA-Fab complex, the HA dissociates and reveals the structure of a monomeric HA. We consider the structure of the monomer in relation to the structure that HA has been shown to assume after exposure to the pH of membrane fusion.     

Molecular requirements for a pandemic influenza virus: An acid-stable hemagglutinin protein

Influenza pandemics require that a virus containing a hemagglutinin (HA) surface antigen previously unseen by a majority of the population becomes airborne-transmissible between humans. Although the HA protein is central to the emergence of a pandemic influenza virus, its required molecular properties for sustained transmission between humans are poorly defined. During virus entry, the HA protein binds receptors and is triggered by low pH in the endosome to cause membrane fusion; during egress, HA contributes to virus assembly and morphology. In 2009, a swine influenza virus (pH1N1) jumped to humans and spread globally. Here we link the pandemic potential of pH1N1 to its HA acid stability, or the pH at which this one-time-use nanomachine is either triggered to cause fusion or becomes inactivated in the absence of a target membrane. In surveillance isolates, our data show HA activation pH values decreased during the evolution of H1N1 from precursors in swine (pH 5.5–6.0), to early 2009 human cases (pH 5.5), and then to later human isolates (pH 5.2–5.4). A loss-of-function pH1N1 virus with a destabilizing HA1-Y17H mutation (pH 6.0) was less pathogenic in mice and ferrets, less transmissible by contact, and no longer airborne-transmissible. A ferret-adapted revertant (HA1-H17Y/HA2-R106K) regained airborne transmissibility by stabilizing HA to an activation pH of 5.3, similar to that of human-adapted isolates from late 2009–2014. Overall, these studies reveal that a stable HA (activation pH ≤ 5.5) is necessary for pH1N1 influenza virus pathogenicity and airborne transmissibility in ferrets and is associated with pandemic potential in humans.Wild aquatic birds are thought to be the natural reservoir of influenza A viruses (1). Influenza pandemics occur every few decades, and swine are widely believed to be a key factor in the genesis of pandemics by facilitating reassortment of the eight viral gene segments and replacing avian-like (α-2,3-linked) hemagglutinin (HA) sialic acid receptor-binding specificity with human-like (α-2,6-linked) (2). If the molecular adaptations that allow efficient human-to-human transmissibility are understood, then circulating viruses undergoing these changes (i.e., those with the greatest pandemic potential) could be identified.In 2009, pandemic (p) H1N1 emerged from swine and swiftly infected more than 60 million people, causing 12,000 US deaths in the first year (3). The pandemic strain originated by reassortment in swine, combining five genes (PB1, PB2, PA, NP, and NS) from North American triple-reassortant swine (TRS) viruses, two genes (NA and M) from Eurasian avian-like swine viruses, and an HA gene closely related to that of the classical swine lineage (4). pH1N1 viruses continue to circulate as seasonal H1N1 viruses. They retain several known pandemic traits, including α-2,6-linked sialic acid receptor-binding specificity of the HA, functional balance of HA and NA activity, and a polymerase adapted to the mammalian upper airway (5). Although these traits appear to be necessary for airborne transmissibility of influenza viruses, they do not appear to be sufficient. For example, H5N1 viruses engineered to have these traits were not air-transmissible among ferrets until a mutation increased HA thermostability and lowered the HA activation pH (6–8). The importance of HA stabilization in supporting the adaptation of influenza viruses to humans or enabling a human pandemic is not completely understood.After receptor binding and endocytosis, low pH triggers irreversible structural changes in the HA protein that fuse the viral envelope and host endosomal membrane (9). Measured HA activation pH values across all subtypes and species range from ∼5.0 to 6.0, trending higher in avian viruses (pH 5.6–6.0) and lower in human viruses (pH 5.0–5.5) (10).The goal of this study was to define the role of HA acid stability in pH1N1 pandemic capability. Our data show that HA activation pH decreased as H1N1 adapted from swine to humans. Complementary experiments in ferrets recapitulated this evolution, as we observed a loss-of-function pH1N1 virus acquired airborne transmissibility via stabilizing mutations. Overall, these studies link a fundamental molecular property, the barrier for activation of a membrane fusion protein (for influenza virus HA, its acid stability), to the interspecies adaptation of a ubiquitous respiratory virus.     

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.     

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.     

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.     

Physical detection of influenza A epitopes identifies a stealth subset on human lung epithelium evading natural CD8 immunity

Vaccines eliciting immunity against influenza A viruses (IAVs) are currently antibody-based with hemagglutinin-directed antibody titer the only universally accepted immune correlate of protection. To investigate the disconnection between observed CD8 T-cell responses and immunity to IAV, we used a Poisson liquid chromatography data-independent acquisition MS method to physically detect PR8/34 (H1N1), X31 (H3N2), and Victoria/75 (H3N2) epitopes bound to HLA-A*02:01 on human epithelial cells following in vitro infection. Among 32 PR8 peptides (8–10mers) with predicted IC₅₀ < 60 nM, 9 were present, whereas 23 were absent. At 18 h postinfection, epitope copies per cell varied from a low of 0.5 for M1_3–11 to a high of >500 for M1_58–66 with PA, HA, PB1, PB2, and NA epitopes also detected. However, aside from M1_58–66, natural CD8 memory responses against conserved presented epitopes were either absent or only weakly observed by blood Elispot. Moreover, the functional avidities of the immunodominant M1_58–66/HLA-A*02:01-specific T cells were so poor as to be unable to effectively recognize infected human epithelium. Analysis of T-cell responses to primary PR8 infection in HLA-A*02:01 transgenic B6 mice underscores the poor avidity of T cells recognizing M1_58–66. By maintaining high levels of surface expression of this epitope on epithelial and dendritic cells, the virus exploits the combination of immunodominance and functional inadequacy to evade HLA-A*02:01-restricted T-cell immunity. A rational approach to CD8 vaccines must characterize processing and presentation of pathogen-derived epitopes as well as resultant immune responses. Correspondingly, vaccines may be directed against “stealth” epitopes, overriding viral chicanery.Human influenza is an acute respiratory infection caused by Orthomyxoviridae, a family of single-stranded, negative sense RNA viruses containing a segmented genome. The influenza virion is enveloped by a lipid bilayer derived from the host cell with hemagglutinin (HA), neuraminidase (NA), and matrix 2 (M2) transmembrane proteins of the virus exposed and antibody (Ab) accessible. HA is the most abundant protein on the surface and is the principle antigen recognized by humoral immunity (1). RNA replication is error prone, with influenza A virus (IAV) averaging roughly one error for each replicated genome (2). Notably, the HA protein manifests a high functional tolerance to sequence variation not evident in some of the internal proteins (3). Current vaccines protect largely by inducing Abs against the HA and NA proteins but require continual reformulation based on inferring the dominant strains that will circulate in the upcoming flu season, an imperfect science at best. Recognizing the challenges for universal protection against influenza via antibodies, there has been extensive discussion about vaccines harnessing cellular immunity (4–6). Cytotoxic T lymphocytes (CTLs), primarily CD8⁺ T cells, can recognize and kill infected cells when their T-cell receptors (TCRs) recognize fragments of viral proteins that are in turn bound to major histocompatibility complex (MHC in general; for humans, HLA) proteins on the surface of infected cells. These peptides can derive from segments of internal proteins conserved among IAV strains so that CTLs targeting these sequence-constrained peptides would remain effective as surface HA and NA antigens, in contrast, change by genetic drift or shift. There is substantial evidence for T-cell–mediated protection in mice (7, 8), but qualified evidence in humans (9, 10). The limited CTL protection has been ascribed to slow cellular responses against IAV, a latency reflecting the expansion and activation of central memory T cells in lymph nodes and the subsequent recruitment of CTLs to infected lung (11). The recent identification of CD8⁺ effector resident memory T cells (T_RM) within barrier tissues postinfection (12, 13) could challenge this picture. However, vaccines for cellular immunity are at an early stage of development and what formulations and delivery methods are needed to harness T_RM immunity is not well understood. In particular, the pathogen-derived peptides bound to HLA molecules on the surface of IAV-infected lung epithelial cells are unknown. As a T cell monitors a single MHC-restricted antigen, surveillance is constrained by the numbers of antigen-specific cells and their motility in lung parenchyma under homeostatic conditions. Sentinel T_RM cell populations lodged in barrier tissues are limited, so that inducing irrelevant specificities will necessarily displace those with useful specificities. In contrast, the fluid volume surrounding a cell at the site of infection can contain a large set of antibodies recognizing diverse antigens. Rational T-cell vaccine development must focus on the peptides directly presented by infected lung epithelium and identify those peptides that can be recognized by high avidity T_RM cells.IAV peptides displayed by infected cells are conventionally identified by T-cell functional assays. In principle this “reverse immunology” only identifies what epitopes the antigen-experienced host has recognized, not what can or should be recognized. As such, reverse immunology is a poor guide for vaccine development. Here new acquisition and analysis methods in mass spectrometry (MS) are applied to directly identify these peptides. New methodology is necessary as conventional data-dependent acquisition (DDA) MS, identifying ions largely in order of signal intensity, cannot plow deep enough into the sample to uncover the IAV peptides underneath an ocean of “self” peptides. Reflecting the prevalence of the HLA-A2 allele in the population and the large number of studies of IAV infection and CD8⁺ T-cell responses restricted by this allele, this study focuses on antigen presentation by HLA-A*02:01 (A2). Our results identify previously unrecognized IAV epitopes that may be useful in vaccine formulations, question the veracity of functionally ascribed epitope display, and show via stable isotope labeling by amino acids in culture (SILAC) that positive strand RNA-derived translation and not protein cross-presentation is the basis of A2 IAV peptidome array on dendritic cells (DCs) that phagocytose non-A2 IAV-infected UV-irradiated cells. In addition, our data rationalize why an immunodominant CD8 T-cell response to the highly conserved M1_58–66 peptide GILGFVFTL does not provide sterilizing immunity to IAV.     

Human mesenchymal stromal cells reduce influenza A H5N1-associated acute lung injury in vitro and in vivo

Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium’s protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation.Acute lung injury is a continuum of clinical and radiographic changes, terminating at its most severe, with acute respiratory distress syndrome. Infection with highly pathogenic avian influenza (HPAI) viruses of the H5N1 and more recent H7N9 subtypes often leads to acute lung injury whereas seasonal influenza viruses and the 2009 pandemic H1N1 influenza viruses do so more rarely. The underlying mechanisms of influenza-related acute lung injury remain unclear, and effective therapies are lacking. Viruses that are highly pathogenic to humans (e.g., H5N1 viruses) may differ intrinsically from the less pathogenic (LP) (e.g., seasonal H1N1) viruses in their replication competence, cell tropism, and/or cytokine dysregulation (1, 2). Early treatment of H5N1 disease with the antiinfluenza drug oseltamivir is helpful but does not ensure a favorable outcome (3). Thus, effective adjunctive therapies that do not compromise beneficial host defenses are needed (4).H5N1 (5) and H7N9 (6) influenza viruses target alveolar epithelial cells, which form the crucial gas exchange interface in the lung. These cells also help to maintain intraalveolar and intravascular fluid homeostasis by vectorial transport of sodium, chloride, and water from the apical to the basolateral surface of the alveolar epithelium [alveolar fluid clearance (AFC)]. Impaired AFC and increased alveolar protein permeability (APP) contribute to acute lung injury (7). Therapies that normalize alveolar fluid clearance are likely to be free of off-target effects, unlike immunomodulation, that may promote virus replication.Human bone marrow-derived multipotent mesenchymal stromal cells (MSCs) have applications in multiple clinical disorders, including sepsis, myocardial infarction, diabetes, and acute renal failure (8). Allogeneic MSC therapy has beneficial preclinical effects on endotoxin-, bacteria-, and ventilator-induced acute lung injury (9) via MSC secretion of the soluble paracrine growth factors angiopoietin-1 (Ang1) and keratinocyte growth factor (KGF) (9, 10). MSCs can also transfer mitochondria and microvesicles that modulate immunity and epithelial response to injury (11). Current clinical trials are testing MSCs as a therapy for sepsis and acute respiratory distress syndrome (12). However, little is known about the impact of MSCs on acute respiratory viral infections, including influenza, with the exception of a study in which MSCs failed to reduce influenza-induced lung injury in mice (13). Here, we showed that influenza A/H5N1 virus infection dysregulates AFC and APP in vitro by inducing infected cells to release soluble mediators that down-regulate alveolar sodium and chloride transporters. When we cocultured alveolar epithelium with MSCs, these injury mechanisms were prevented or reduced. We then treated mice infected with influenza A/H5N1 with MSCs and demonstrated a clinically significant reduction in lung pathology and increased survival in association with a modulation of these pathogenic mechanisms in vivo.     

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.     

Androgens alter T-cell immunity by inhibiting T-helper 1 differentiation

The hormonal milieu influences immune tolerance and the immune response against viruses and cancer, but the direct effect of androgens on cellular immunity remains largely uncharacterized. We therefore sought to evaluate the effect of androgens on murine and human T cells in vivo and in vitro. We found that murine androgen deprivation in vivo elicited RNA expression patterns conducive to IFN signaling and T-cell differentiation. Interrogation of mechanism showed that testosterone regulates T-helper 1 (Th1) differentiation by inhibiting IL-12–induced Stat4 phosphorylation: in murine models, we determined that androgen receptor binds a conserved region within the phosphatase, Ptpn1, and consequent up-regulation of Ptpn1 then inhibits IL-12 signaling in CD4 T cells. The clinical relevance of this mechanism, whereby the androgen milieu modulates CD4 T-cell differentiation, was ascertained as we found that androgen deprivation reduced expression of Ptpn1 in CD4 cells from patients undergoing androgen deprivation therapy for prostate cancer. Our findings, which demonstrate a clinically relevant mechanism by which androgens inhibit Th1 differentiation of CD4 T cells, provide rationale for targeting androgens to enhance CD4-mediated immune responses in cancer or, conversely, for modulating androgens to mitigate CD4 responses in disorders of autoimmunity.The sex-specific hormones, testosterone and estrogen, have a number of immuno-modulatory effects. Women almost universally respond as well or better than men to antibody-inducing vaccinations (1). For example, healthy women treated with the trivalent inactivated influenza vaccine generate a greater antibody titer than men (2). Findings like these have led to the suggestion that estrogen promotes T-helper 2 (Th2) differentiation and antibody production (3). Further supporting an increased antibody response caused by estrogen, more than 80% of patients suffering from antibody-driven autoimmunities such as systemic lupus erythematosus, Sjörgren syndrome, and Hashimoto thyroiditis are women (4). In contrast to estrogen, how testosterone affects the immune system is less clear, but its role in immunity against viruses and host antigens is certainly immunosuppressive.Recently, it was reported that testosterone levels negatively correlated with the antibody response to the trivalent inactivated seasonal influenza vaccine by interfering with lipid metabolism (5). Testosterone levels are also positively correlated with the viral load of Venezuelan equine encephalitis virus in macaques (6). In addition to the response to viruses, testosterone regulates the response to host antigens in many biological systems. Elevated levels of testosterone following colonization with commensal microbes correlated with reduced islet inflammation and protection from type 1 diabetes in nonobese diabetic mice (7). Also, tolerance to tumor antigens is regulated by testosterone, as androgen ablation in a mouse model of prostate cancer reversed CD4 T-cell tolerance to a prostate restricted tumor antigen (8). Similarly, castration of male mice before vaccination with prostate-specific antigens enhanced CD8 T-cell vaccine response in many studies (9, 10). Patients undergoing androgen deprivation in prostate cancer had increased infiltration of T cells into benign and malignant prostate tissue (11). Based on these findings, clinical trials are currently underway to test the combinatorial efficacy of androgen deprivation and immunotherapy in prostate cancer patients (12). Together, these observations suggest a critical role for testosterone in maintaining T-cell tolerance toward not only viruses, but also host and tumor antigens.Despite these observations that testosterone inhibits immunity, the precise molecular mechanisms by which testosterone achieves this effect are poorly understood. Here, we sought to address this question by performing gene expression profiling of CD4 T cells isolated from castrated mice. Gene expression analysis revealed a critical effect of testosterone on CD4 T-cell differentiation and identified protein tyrosine phosphatase nonreceptor type 1 (Ptpn1) as a mediator of androgen-induced suppression of CD4 T-cell differentiation. The research presented here highlights a previously unreported molecular mechanism by which testosterone suppresses immunity and allows a better understanding of sex differences in the response to viruses, autoimmunity, and immune escape in prostate cancer.     

Vaccine-elicited antibody that neutralizes H5N1 influenza and variants binds the receptor site and polymorphic sites

Antigenic drift of circulating seasonal influenza viruses necessitates an international vaccine effort to reduce the impact on human health. A critical feature of the seasonal vaccine is that it stimulates an already primed immune system to diversify memory B cells to recognize closely related, but antigenically distinct, influenza glycoproteins (hemagglutinins). Influenza pandemics arise when hemagglutinins to which no preexisting adaptive immunity exists acquire the capacity to infect humans. Hemagglutinin 5 is one subtype to which little preexisting immunity exists and is only a few acquired mutations away from the ability to transmit efficiently between ferrets, and possibly humans. Here, we describe the structure and molecular mechanism of neutralization by H5.3, a vaccine-elicited antibody that neutralizes hemagglutinin 5 viruses and variants with expanded host range. H5.3 binds in the receptor-binding site, forming contacts that recapitulate many of the sialic acid interactions, as well as multiple peripheral interactions, yet is not sensitive to mutations that alter sialic acid binding. H5.3 is highly specific for a subset of H5 strains, and this specificity arises from interactions to the periphery of the receptor-binding site. H5.3 is also extremely potent, despite retaining germ line-like conformational flexibility.Influenza remains a major public health concern because seasonal influenza infects 600 million to 1.1 billion people annually, resulting in 3–5 million cases of severe disease, and 250,000–500,000 deaths (1). By comparison, the four influenza pandemics of the 20th century, caused by novel influenza strains infecting the immunologically naive human population, resulted in 50–100 million deaths (1–4). Influenza A immunity is principally mediated by the antibody response to the viral glycoprotein, hemagglutinin (HA) (5). HA is expressed as a preprotein, HA0, assembled as a trimer on the viral envelope, and cleaved by host proteases into HA1 and HA2. HA1 is a largely globular domain responsible for receptor binding, and HA2 is a rod-shaped helical bundle responsible for membrane fusion (Fig. 1A) (5). There are 18 genetically distinct subtypes of influenza A HA (H1–H18), of which only H1 and H3 currently circulate among humans (1, 6–9).Open in a separate windowFig. 1.Human monoclonal antibody H5.3 recognizes the H5 receptor-binding site. (A) H5.3-wt_H5hd complex overlaid on the VN/1203 H5 trimer (PDB ID code 2FK0) showing H5hd in gold, the H5.3 light chain in purple, the H5.3 heavy chain in teal, and the H5 trimer (2FK0) in gray. (B) A cartoon diagram of H5hd showing HA residues contacted by H5.3 as sticks. The structural elements of the RBS are highlighted: the 130 loop is cyan, the 140 loop is pink, the 150 loop is orange, the 190 helix is blue, and the 220 loop is green. Trp153 forms the base and denotes the approximate center of the receptor-binding site. (C) A surface representation of H5hd in the same orientation as in B, with the solvent inaccessible interface shown in gray. H5.3 contact residues are labeled and shown as sticks and colored by CDR, with CDRH1 in light blue, CDRH2 in blue, CDRH3 in teal, CDRL1 in light pink, CDRL2 in dark pink, and CDRL3 in purple.Despite the widespread presence of H5N1 influenza viruses in wild birds, the virus is not currently transmissible within the human population. Human-to-human transmission is inefficient and is partially restricted by the receptor specificity of the virus; human-type HAs preferentially recognize α2,6-linked sialic acid whereas avian-type HAs prefer α2,3-linked sialic acid (1, 10–12). However, there have been >600 human cases of H5N1 infection since 2004, resulting from the direct transmission of the virus from birds to humans, associated with an ∼60% mortality rate. There is the potential for a significant pandemic if H5 viruses develop the ability to spread efficiently between humans, which would necessitate specificity for α2,6-linked sialic acid (1–4, 13).Receptor binding occurs in a shallow depression on the HA globular head domain, the edges of which are formed by four structural elements, the 190 helix and the 130, 150, and 220 loops (Fig. 1B), and the receptor binding site (RBS) base, which includes invariant hydrophobic residues Tyr98, Trp153, and Leu194 (5, 14, 15). Receptor specificity is critically influenced by position 226 on HA; Gln226-containing H3 strains are specific for α2,3 sialic acid linkages, and Leu226-containing H3 strains are specific for α2,6 sialic acid linkages (5, 16). In H5 strains, Leu226 enhances binding to α2,6-linked sialic acid receptors, but H5 viruses isolated from humans contain mutations at other sites that also promote use of α2,6-linked sialic acid receptors (11, 17, 18). Recent influenza pandemics have been caused by the acquisition of mutations that change the receptor preference to α2,6 sialic acid linkages, and recent studies with multiply passaged laboratory strains indicated that only a small number of mutations are necessary to introduce preference for α2,6 linkages into H5 strains (1, 6–9, 18–23). These viruses, termed respiratory droplet transmissible (rdt), typically have three mutations in or near the receptor binding site on HA (21, 22).The most frequent potent neutralizing antibody response to HA arises from antibodies that target the receptor binding site and prevent virus attachment (5). Recent studies indicate that among RBS-directed antibodies, broad neutralization (across multiple isolates within a subtype or across subtypes) is achieved by insertion of a single complementary determining region (CDR) into the RBS to inhibit receptor binding (8). These broadly neutralizing antibodies (bnAbs) target conserved amino acids within the RBS and simultaneously avoid polymorphic sites on the ridges of the RBS. BnAbs may be relatively rare in human repertoires, and, as a consequence, current seasonal vaccine efforts focus on developing or boosting strain-specific responses to three or four currently circulating (“seasonal”) variants (2). Such strategies do not directly address the threat posed by noncirculating viruses with pandemic potential, such as H5 strains that circulate widely in wild bird populations and sporadically infect humans where they acquire mutations that enhance binding to human receptors (17). Instead, H5N1 vaccines against “prepandemic” strains have been developed commercially for future use in the case of a pandemic, illustrating that a prepandemic immunization program is feasible (24, 25).The immune response against H5N1 vaccines in healthy adults is less robust than for most seasonal influenza strains, typically resulting in a response restricted to the strain used in the vaccine and to closely related variants (26–29). Notwithstanding this observation, we recently described a panel of human anti-H5 antibodies induced in response to vaccination of volunteers with an experimental H5N1 subunit vaccine (30) and here describe the structure and characterization of a human monoclonal antibody, H5.3, bound to A/Vietnam/1203/2004 (VN/1203) H5 and to two H5 rdt variants. H5.3 is an RBS-directed antibody that recapitulates many of the electrostatic interactions of the natural receptor, sialic acid, as well as forming additional interactions to the periphery of the RBS that provide specificity. H5.3 is potent and specific despite containing only 11 mutations from its unmutated common ancestor (UCA) and maintaining the structural flexibility typically associated with unmutated antibodies, as evidenced by significant rearrangement of CDRH3 and CDRL3. The structures determined here offer a chemical explanation for the evident trade-off between breadth and potency, and the germ-line characteristics highlight the role of lightly mutated antibodies in neutralization of new viral strains.     

Evolution of the receptor binding properties of the influenza A(H3N2) hemagglutinin

The hemagglutinin (HA) of influenza A(H3N2) virus responsible for the 1968 influenza pandemic derived from an avian virus. On introduction into humans, its receptor binding properties had changed from a preference for avian receptors (α2,3-linked sialic acid) to a preference for human receptors (α2,6-linked sialic acid). By 2001, the avidity of human H3 viruses for avian receptors had declined, and since then the affinity for human receptors has also decreased significantly. These changes in receptor binding, which correlate with increased difficulties in virus propagation in vitro and in antigenic analysis, have been assessed by virus hemagglutination of erythrocytes from different species and quantified by measuring virus binding to receptor analogs using surface biolayer interferometry. Crystal structures of HA–receptor analog complexes formed with HAs from viruses isolated in 2004 and 2005 reveal significant differences in the conformation of the 220-loop of HA1, relative to the 1968 structure, resulting in altered interactions between the HA and the receptor analog that explain the changes in receptor affinity. Site-specific mutagenesis shows the HA1 Asp-225→Asn substitution to be the key determinant of the decreased receptor binding in viruses circulating since 2005. Our results indicate that the evolution of human influenza A(H3N2) viruses since 1968 has produced a virus with a low propensity to bind human receptor analogs, and this loss of avidity correlates with the marked reduction in A(H3N2) virus disease impact in the last 10 y.Surveillance of influenza viruses is essential for updating vaccines, for tracking the emergence of drug resistant viruses, and for monitoring zoonotic infections. It also gives important insights into the mechanisms of virus evolution. This is particularly the case for interpreting the correlation between antigenic differences and changes in the sialic acid receptor binding properties of the HA glycoprotein. The correlation in these two properties arises because of the close proximity on HA of binding sites for antibodies that neutralize virus infectivity and the sialic acid receptor binding pocket (1), and accounts for the observations that mutations that prevent antibody binding can also result in changes in receptor binding (2–7). Reduction in affinity of human H3N2 viruses for avian receptors since the beginning of the pandemic in 1968 has meant that by the 1990s viruses with reduced ability to agglutinate chicken erythrocytes had emerged (8, 9). Moreover, viruses isolated after 1999 were shown to have reduced affinity for both human and avian receptors, a feature that correlated with their poor growth properties in eggs and different cells in culture (9–14). The evolution of the HA has resulted in at least three key changes that influence receptor binding. Two sequential substitutions occurred at residue 225: in 2001–2002, a substitution Gly-225→Asp was accompanied by a Trp-222→Arg substitution, and in 2004–2005, an Asp-225→Asn substitution was accompanied by the substitution Ser-193→Phe (while maintaining arginine at position 222). Residue 226, a key amino acid in determining receptor specificity (15), also changed twice: before 2001, Leu-226→Val, and in 2004, Val-226→Ile (Fig. S1).To correlate these amino acid substitutions with the biological properties of the viruses, we have analyzed the receptor binding characteristics of H3N2 viruses isolated between 2001 and 2010, examined changes in their ability to infect cells in culture, and determined the structures of two HAs of virus isolates from 2004 and 2005 in the absence of receptor and complexed with a human receptor analog. The data show that the progressive decrease in binding of these viruses to human receptors from 2000 onward correlates with changes in the efficiencies of infection of cultured cells. Comparison of structural data for HAs of viruses from 1968, 2004, and 2005 explain how particular mutations that affect the conformation of the HA1 220-loop component of the receptor binding site define the receptor binding phenotype of recent H3N2 human influenza viruses.     

Preemptive priming readily overcomes structure-based mechanisms of virus escape

A reverse-genetics approach has been used to probe the mechanism underlying immune escape for influenza A virus-specific CD8⁺ T cells responding to the immunodominant D^bNP₃₆₆ epitope. Engineered viruses with a substitution at a critical residue (position 6, P6M) all evaded recognition by WT D^bNP₃₆₆-specific CD8⁺ T cells, but only the NPM6I and NPM6T mutants altered the topography of a key residue (His155) in the MHC class I binding site. Following infection with the engineered NPM6I and NPM6T influenza viruses, both mutations were associated with a substantial “hole” in the naïve T-cell receptor repertoire, characterized by very limited T-cell receptor diversity and minimal primary responses to the NPM6I and NPM6T epitopes. Surprisingly, following respiratory challenge with a serologically distinct influenza virus carrying the same mutation, preemptive immunization against these escape variants led to the generation of secondary CD8⁺ T-cell responses that were comparable in magnitude to those found for the WT NP epitope. Consequently, it might be possible to generate broadly protective T-cell immunity against commonly occurring virus escape mutants. If this is generally true for RNA viruses (like HIV, hepatitis C virus, and influenza) that show high mutation rates, priming against predicted mutants before an initial encounter could function to prevent the emergence of escape variants in infected hosts. That process could be a step toward preserving immune control of particularly persistent RNA viruses and may be worth considering for future vaccine strategies.Virus-specific CD8⁺ T cells recognize peptide and class I MHC (pMHCI) epitopes derived predominantly from more conserved, internal proteins, offering a promising target for vaccine development. However, some RNA viruses, particularly the influenza A viruses, hepatitis C virus (HCV) and HIV, are a major challenge for preventive immunization as a low-fidelity RNA polymerase allows the rapid emergence of escape mutants. The question asked here is whether it is possible to design an immunization strategy that might minimize the likelihood that such virus variants will escape from immune control and survive.The influenza A viruses elicit robust and broad CD8⁺ T-cell immunity (1, 2) that can provide protection against serologically distinct strains, including newly emerged pandemic variants (3, 4). The experimental evidence is that influenza-specific CD8⁺ T cells, operating in either a primary response or following recall from memory, promote virus elimination and host recovery via the production of proinflammatory cytokines and the killing of virus-infected cells. At the stage of initial priming, the responding CD8⁺ cytotoxic T lymphocytes (CTLs) are selected as a consequence of the interaction between their clonotypic T-cell receptor (TCR) and pMHCI epitopes expressed on the surface of infected cells. The key to immunogenicity for CD8⁺ CTLs rests in both the nature of the TCR repertoire and the sequence, or structural complementarity, of peptides targeted in the MHCI groove (5, 6).Influenza virus-specific CD8⁺ CTLs can exert selective pressure that leads to the emergence of escape mutations in immunogenic peptides (7). This is a more familiar problem for chronic virus infections (HIV and HCV), with at least some of the variants that persist in the circulation being readily transmissible (8–10). Apart from subverting CD8⁺ T-cell–mediated control within the infected individual, this constitutes a major barrier to effective vaccine design. With influenza, although mutations have been found for >70% of immunogenic T-cell peptides (7), this finding has received little attention because of the acute nature of the disease. Even so, such escape mutants are readily generated using TCR transgenic mice (11), and “natural” variants occur within the influenza nucleoprotein (NP)₃₈₀ (HLA-B8), NP₃₈₃ (HLA-B27), and NP₄₁₈ (HLA-B35) viral peptides (12–14). Furthermore, given sufficient immune pressure and relative fitness, such mutated viruses can become fixed in the population, leading to the disappearance of WT T-cell specificities (15). For “seasonal” influenza infections, such escape from CD8⁺ T-cell–mediated immunity can also be relevant to the persistence of variants within the population (longevity and severity of influenza season). Moreover, in the face of a rapidly spreading, novel pandemic strain, established CD8⁺ T-cell memory constitutes the best protective mechanism. Clearly, any vaccine strategy that focuses on priming the CTLs needs to deal with emerging escape variants.The immunogenicity of a given epitope can be compromised in a variety of ways. Amino acid variation at an MHCI anchor residue can lead to the failure of pMHCI binding (12). Alternatively, changes at a TCR contact site (14, 16, 17) can prevent or decrease T-cell recognition, although cross-reactive T-cell immunity may still be retained between some influenza variants (18). The present study targets the mechanisms underlying virus escape at TCR contact sites and probes possible compensatory strategies using a readily manipulated C57BL/6J (B6, H2^b) influenza mouse model (17). The study focuses principally on escape variants selected in transgenic mice expressing a TCR specific for the immunodominant D^bNP₃₆₆ (ASNENMETM) epitope that reemerged from day 18 after infection and caused lethal disease within a month (11). Sequencing viral RNA recovered from the lungs of these TCR transgenic mice (11) established that NP₃₆₆ had mutated, especially at position (P) 6, and that the mice were no longer infected with the wt virus. The P6 methionine (M) is the most solvent-exposed residue for the D^bNP₃₆₆ complex (19, 20) and the change from P6M to P6A leads to loss in recognition by CD8⁺ T cells specific for the wt-D^bNP₃₆₆ epitope (17, 20, 21). The present analysis of the in vivo CTL responses to a panel of P6 mutants indicates that preemptive immunization against the escape variants can generate a good measure of protection.