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Although some adults infected with influenza 2009 A(H1N1)pdm09 viruses mounted high hemagglutination inhibition (HAI) antibody response, they still suffered from severe disease, or even death. Here, we analyzed antibody profiles in patients (n = 31, 17–65 years) admitted to intensive care units (ICUs) with lung failure and invasive mechanical ventilation use due to infection with A(H1N1)pdm09 viruses during 2009–2011. We performed a comprehensive analysis of the quality and quantity of antibody responses using HAI, virus neutralization, biolayer interferometry, enzyme-linked-lectin and enzyme-linked immunosorbent assays. At time of the ICU admission, 45% (14/31) of the patients had HAI antibody titers ≥ 80 in the first serum (S1), most (13/14) exhibited narrowly-focused HAI and/or anti-HA-head binding antibodies targeting single epitopes in or around the receptor binding site. In contrast, 42% (13/31) of the patients with HAI titers ≤ 10 in S1 had non-neutralizing anti-HA-stem antibodies against A(H1N1)pdm09 viruses. Only 19% (6/31) of the patients showed HA-specific IgG1-dominant antibody responses. Three of 5 fatal patients possessed highly focused cross-type HAI antibodies targeting the (K130 + Q223)-epitopes with extremely low avidity. Our findings suggest that narrowly-focused low-quality antibody responses targeting specific HA-epitopes may have contributed to severe infection of the lower respiratory tract.

Individuals with metabolic dysregulation of cellular glycosylation often experience severe influenza disease, with a poor immune response to the virus and low vaccine efficacy. Here, we investigate the consequences of aberrant cellular glycosylation for the glycome and the biology of influenza virus. We transiently induced aberrant N-linked glycosylation in cultured cells with an oligosaccharyltransferase inhibitor, NGI-1. Cells treated with NGI-1 produced morphologically unaltered viable influenza virus with sequence-neutral glycosylation changes (primarily reduced site occupancy) in the hemagglutinin and neuraminidase proteins. Hemagglutinin with reduced glycan occupancy required a higher concentration of surfactant protein D (an important innate immunity respiratory tract collectin) for inhibition compared to that with normal glycan occupancy. Immunization of mice with NGI-1-treated virus significantly reduced antihemagglutinin and antineuraminidase titers of total serum antibody and reduced hemagglutinin protective antibody responses. Our data suggest that aberrant cellular glycosylation may increase the risk of severe influenza as a result of the increased ability of glycome-modified influenza viruses to evade the immune response.

There is a high incidence of adenovirus (AdV) infection in humans due to the presence of more than 60 types of human adenoviruses (HAdVs). The majority of individuals are exposed to one or more HAdV types early in their lives, leading to the development of AdV type-specific neutralizing antibodies. Similarly, immunization or gene therapy with AdV vectors leads to immune responses to the AdV vector. This ‘vector immunity’ is a concern for AdV vector-based applications for vaccines or gene therapy, especially when the repeated administration of a vector is required. The objective of this investigation was to establish whether AdV neutralizing antibody titers decline sufficiently in a year to permit annual vaccination with the same AdV vector. Naive or human adenoviral vector group C, type 5 (HAdV-C5)-primed mice were mock-inoculated (with PBS) or inoculated i.m. with 108 PFU of either HAd-GFP [HAdV-C5 vector expressing the green fluorescent protein (GFP)] to mimic the conditions for the first inoculation with an AdV vector-based vaccine. At 1, 3, 6, and 10 months post-HAd-GFP inoculation, naive- or HAdV-primed animals were vaccinated i.m. with 108 PFU of HAd-H5HA [HAdV-C5 vector expressing hemagglutinin (HA) of H5N1 influenza virus]. There was a significant continual decrease in vector immunity titers with time, thereby leading to significant continual increases in the levels of HA-specific humoral and cell-mediated immune responses. In addition, significant improvement in protection efficacy against challenge with an antigenically heterologous H5N1 virus was observed in HAdV-primed animals at 6 months and onwards. These results indicate that the annual immunization with the same AdV vector may be effective due to a significant decline in vector immunity.

Several human and nonhuman adenovirus (AdV) vectors including bovine AdV type 3 (BAdV-3) were developed as gene delivery vectors to supplement and/or elude human AdV (HAdV)-specific neutralizing antibodies (vector immunity). Here we evaluated the vaccine immunogenicity and efficacy of BAdV-3 vector (BAd-H5HA) expressing hemagglutinin (HA) of a H5N1 influenza virus in a dose escalation study in mice with the intranasal (IN) or intramuscular (IM) route of inoculation in comparison with the HAdV type C5 (HAdV-C5) vector (HAd-H5HA) expressing HA of a H5N1 influenza virus. Dose-related increases in the immune responses were clearly noticeable. A single IM inoculation with BAd-H5HA resulted in enhanced cellular immune responses compared with that of HAd-H5HA and conferred complete protection following challenge with a heterologous H5N1 virus at the dose of 3 × 107 plaque-forming units (PFUs), whereas a significant amount of influenza virus was detected in the lungs of mice immunized with 1 × 108 PFUs of HAd-H5HA. Similarly, compared with that of HAd-H5HA, a single IN inoculation with BAd-H5HA produced significantly enhanced humoral (HA-specific immunoglobulin [IgG] and its subclasses, as well as HA-specific IgA) and cellular immune responses, and conferred complete protection following challenge with a heterologous H5N1 virus. Complete protection with BAd-H5HA was observed with the lowest vaccine dose (1 × 106 PFUs), but similar protection with HAd-H5HA was observed at the highest vaccine dose (1 × 108 PFUs). These results suggest that at least 30-fold dose sparing can be achieved with BAd-H5HA vector compared with HAd-H5HA vaccine vector. Keywords: adenoviral vector, bovine adenoviral vector, human adenoviral vector, enhanced immunogenicity and protection, avian influenza, route of immunization, dose sparing, universal influenza vaccine

Background Although ferret antisera used in influenza surveillance did not detect antigenic drift of A(H1N1)pdm09 viruses during the 2015-2016 season, low vaccine effectiveness was reported in adults. We investigated the immune basis of low responses to circulating A(H1N1)pdm09 viruses after vaccination. Methods Prevaccination and postvaccination serum samples collected from >300 adults (aged 18-49 years) in 6 seasons (2010-2011 to 2015-2016) were analyzed using hemagglutination inhibition assays to evaluate the antibody responses to 13 A(H1N1) viruses circulated from 1977 to 2016. Microneutralization and serum adsorption assays were used to verify the 163K and 223R specificity of antibodies. Results Individual antibody profiles to A(H1N1) viruses revealed 3 priming patterns: USSR/77, TW/86, or NC/99 priming. More than 20% of adults had reduced titers to cell-propagated circulating 6B.1 and 6B.2 A(H1N1)pdm09 viruses compared with the A/California/07/2009 vaccine virus X-179A. Significantly reduced antibody reactivity to circulating viruses bearing K163Q was observed only in the USSR/77-primed cohort, whereas significantly lower reactivity caused by egg-adapted Q223R change was detected across all 3 cohorts. Conclusion Both 163K specificity driven by immune priming and 223R specificity from egg-adapted changes in the vaccine contributed to low responses to circulating A(H1N1)pdm09 viruses after vaccination. Our study highlights the need to incorporate human serology in influenza surveillance and vaccine strain selection.

Specific residues of influenza A virus (IAV) PB1-F2 proteins may enhance inflammation or cytotoxicity. In a series of studies, we evaluated the function of these virulence-associated residues in the context of different IAV subtypes in mice. Here, we demonstrate that, as with the previously assessed pandemic 1968 (H3N2) IAV, PB1-F2 inflammatory residues increase the virulence of H1N1 IAV, suggesting that this effect might be a universal feature. Combining both inflammatory and cytotoxic residues in PB1-F2 enhanced virulence further, compared to either motif alone. Residues from these virulent motifs have been present in natural isolates from human seasonal IAV of all subtypes, but there has been a trend toward a gradual reduction in the number of virulent residues over time. However, human IAV of swine and avian origin tend to have more virulent residues than do the human-adapted seasonal strains, raising the possibility that donation of PB1 segments from these zoonotic viruses may increase the severity of some seasonal human strains. Our data suggest the value of surveillance of virulent residues in both human and animal IAV to predict the severity of influenza season.

Avian influenza viruses, notably H5 subtype viruses, pose a continuous threat to public health due to their pandemic potential. In recent years, influenza virus H5 subtype split vaccines with novel oil-in-water emulsion based adjuvants (e.g. AS03, MF59) have been shown to be safe, immunogenic, and able to induce broad immune responses in clinical trials, providing strong scientific support for vaccine stockpiling. However, whether such vaccines can provide protection from infection with emerging, antigenically distinct clades of H5 viruses has not been adequately addressed. Here, we selected two AS03-adjuvanted H5N1 vaccines from the US national pre-pandemic influenza vaccine stockpile and assessed whether the 2004-05 vaccines could provide protection against a 2014 highly pathogenic avian influenza (HPAI) H5N2 virus (A/northern pintail/Washington/40964/2014), a clade 2.3.4.4 virus responsible for mass culling of poultry in North America. Ferrets received two doses of adjuvanted vaccine containing 7.5µg of hemagglutinin (HA) from A/Vietnam/1203/2004 (clade 1) or A/Anhui/1/2005 (clade 2.3.4) virus either in a homologous or heterologous prime-boost vaccination regime. We found that both vaccination regimens elicited robust antibody responses against the 2004-05 vaccine viruses and could reduce virus-induced morbidity and viral replication in the lower respiratory tract upon heterologous challenge despite the low level of cross-reactive antibody titers to the challenge H5N2 virus. This study supports the value of existing stockpiled 2004-05 influenza H5N1 vaccines, combined with AS03-adjuvant for early use in the event of an emerging pandemic with H5N2-like clade 2.3.4.4 viruses.

Epidemiological studies suggest that humans who receive repeated annual immunization with influenza vaccine are less well protected against influenza than those who receive vaccine in the current season only. To better understand potential mechanisms underlying these observations, we vaccinated influenza-naive ferrets either twice, 10 months apart (repeated vaccination group; RV), or once (current season only group; CS), using a prime-boost regimen, and then challenged the ferrets with A/Hong Kong/4801/2014(H3N2). Ferrets that received either vaccine regimen were protected against influenza disease and infection relative to naive unvaccinated ferrets, but the RV group shed more virus, especially at the peak of virus shedding 2 days post infection (p, Influenza: Repeat vaccination impairs protection Epidemiological evidence suggests that influenza vaccination in prior years can reduce the efficacy of influenza vaccination compared to vaccine given in the current season alone. Ian A. York and colleagues of the Centers for Disease Control and Prevention, USA investigate this phenomenon using the human relevant ferret model of influenza infection. Ferrets undergoing repeat vaccination — twice 10 months apart — show quantitatively similar antibody and cell-mediated immune responses as those given a single current-season vaccination. However, qualitatively the responses elicited by the current season vaccine alone are superior — with animals showing less dramatic loss of body weight, milder fever, reduced virus shedding and quicker recovery of lymphocyte numbers. Although less protective than current-season vaccination alone, repeated vaccination offers better protection than mock vaccination.

Zoonotic A(H7N9) avian influenza viruses emerged in China in 2013 and continue to be a threat to human public health, having infected over 800 individuals with a mortality rate approaching 40%. Treatment options for people infected with A(H7N9) include the use of neuraminidase (NA) inhibitors. However, like other influenza viruses, A(H7N9) can become resistant to these drugs. The use of monoclonal antibodies is a rapidly developing strategy for controlling influenza virus infection. Here we generated a murine monoclonal antibody (3c10-3) directed against the NA of A(H7N9) and show that prophylactic systemic administration of 3c10-3 fully protected mice from lethal challenge with wild-type A/Anhui/1/2013 (H7N9). Further, post-infection treatment with a single systemic dose of 3c10-3 at either 24, 48 or 72 h post A(H7N9) challenge resulted in both dose- and time-dependent protection of up to 100% of mice, demonstrating therapeutic potential for 3c10-3. Epitope mapping revealed that 3c10-3 binds near the enzyme active site of NA, and functional characterization showed that 3c10-3 inhibits the enzyme activity of NA and restricts the cell-to-cell spread of the virus in cultured cells. Affinity analysis also revealed that 3c10-3 binds equally well to recombinant NA of wild-type A/Anhui/1/2013 and to a variant NA carrying a R289K mutation known to infer NAI resistance. These results suggest that 3c10-3 has the potential to be used as a therapeutic to treat A(H7N9) infections either as an alternative to, or in combination with, current NA antiviral inhibitors.

There is controversy in the literature whether consecutive year influenza vaccinations lead to decreased vaccine efficacy. Evidence for this effect is strongest in the context of repeated H3N2 vaccinations. As recently reported (NPJ Vaccines 4:28 (2019)), we recreated this phenomenon in the ferret model of influenza. Ferrets exposed to consecutive year vaccinations had significantly increased weight loss, viral shedding and fever after A/Hong Kong/4801/2014 (HK4801) virus challenge than ferrets only exposed to the 2017–2018 vaccine. One possible explanation for this phenomenon is that the repeated vaccination induces non-protective antibodies against influenza virus, which might include low avidity antibodies, non-neutralizing antibodies, and antibodies that otherwise do not elicit a protective immune response. Ferrets that received repeated vaccination had higher avidity antibodies to HK4801 HA at Day 2 and 14 post-infection than did the other two groups. Preliminary data do not support increased levels of HA stem-binding antibodies in repeat vaccinees’ serum. NA and NP are two additional viral proteins expressed on the cell surface, with greater or lesser evidence for their role in protective antibody responses, respectively. Serum reactivity to both proteins correlated with the number of vaccinations. Together, these results do not support a model of increased non-protective antibodies after repeat vaccination. Furthermore, the breadth of the T cell response against H3N2 viruses from different clades was similar between both vaccinated groups. Ongoing studies continue to explore the mechanism for the decreased protection observed in the repeat vaccination group compared with the single year vaccination group.

Influenza viruses cause seasonal epidemics and sporadic pandemics in humans. The virus’s ability to change its antigenic nature through mutation and recombination, and the difficulty in developing highly effective universal vaccines against it, make it a serious global public health challenge. Influenza virus’s surface glycoproteins, hemagglutinin and neuraminidase, are all modified by the host cell’s N-linked glycosylation pathways. Host innate immune responses are the first line of defense against infection, and glycosylation of these major antigens plays an important role in the generation of host innate responses toward the virus. Here, we review the principal findings in the analytical techniques used to study influenza N-linked glycosylation, the evolutionary dynamics of N-linked glycosylation in seasonal versus pandemic and zoonotic strains, its role in host innate immune responses, and the prospects for lectin-based therapies. As the efficiency of innate immune responses is a critical determinant of disease severity and adaptive immunity, the study of influenza glycobiology is of clinical as well as research interest.

Characterization of the epitopes on antigen recognized by monoclonal antibodies (mAb) is useful for the development of therapeutic antibodies, diagnostic tools, and vaccines. Epitope mapping also provides functional information for sequence-based repertoire analysis of antibody response to pathogen infection and/or vaccination. However, development of mapping strategies has lagged behind mAb discovery. We have developed a site-directed mutagenesis approach that can be used in conjunction with bio-layer interferometry (BLI) biosensors to map mAb epitopes. By generating a panel of single point mutants in the recombinant hemagglutinin (HA) and neuraminidase (NA) proteins of influenza A viruses, we have characterized the epitopes of hundreds of mAbs targeting the H1 and H3 subtypes of HA and the N9 subtype of NA.

Since the first case of human infection in March 2013, continued reports of H7N9 cases highlight a potential pandemic threat. Highly immunogenic vaccines to this virus are urgently needed to protect vulnerable populations who lack protective immunity. In this study, an egg- and adjuvant-independent adenoviral vector-based, hemagglutinin H7 subtype influenza vaccine (HAd-H7HA) demonstrated enhanced cell-mediated immunity as well as serum antibody responses in a mouse model. Most importantly, this vaccine provided complete protection against homologous A/(H7N9) viral challenge suggesting its potential utility as a pandemic vaccine.

Current influenza vaccines induce strain-specific immunity to the highly variable hemagglutinin (HA) protein. It is therefore a high priority to develop vaccines that induce broadly cross-protective immunity to different strains of influenza. Since influenza A M2 proteins are highly conserved among different strains, five tandem repeats of the extracellular peptide of M2 in a membrane-anchored form on virus-like particles (VLPs) have been suggested to be a promising candidate for universal influenza vaccine. In this study, ferrets were intramuscularly immunized with 2009 H1N1 split HA vaccine (“Split”) alone, influenza split vaccine supplemented with M2e5x VLP (“Split+M2e5x”), M2e5x VLP alone (“M2e5x”), or mock immunized. Vaccine efficacy was measured serologically and by protection against a serologically distinct viral challenge. Ferrets immunized with Split+M2e5x induced HA strain specific and conserved M2e immunity. Supplementation of M2e5x VLP to split vaccination significantly increased the immunogenicity of split vaccine compared to split alone. The Split+M2e5x ferret group showed evidence of cross-reactive protection, including faster recovery from weight loss, and reduced inflammation, as inferred from changes in peripheral leukocyte subsets, compared to mock-immunized animals. In addition, ferrets immunized with Split+M2e5x shed lower viral nasal-wash titers than the other groups. Ferrets immunized with M2e5x alone also show some protective effects, while those immunized with split vaccine alone induced no protective effects compared to mock-immunized ferrets. These studies suggest that supplementation of split vaccine with M2e5x-VLP may provide broader and improved cross-protection than split vaccine alone.

Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro , we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. IMPORTANCE Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses.

Marek׳s disease virus (MDV) is a widespread α-herpesvirus of chickens that causes T cell tumors. Acute, but not latent, MDV infection has previously been shown to lead to downregulation of cell-surface MHC class I (Virology 282:198–205 (2001)), but the gene(s) involved have not been identified. Here we demonstrate that an MDV gene, MDV012, is capable of reducing surface expression of MHC class I on chicken cells. Co-expression of an MHC class I-binding peptide targeted to the endoplasmic reticulum (bypassing the requirement for the TAP peptide transporter) partially rescued MHC class I expression in the presence of MDV012, suggesting that MDV012 is a TAP-blocking MHC class I immune evasion protein. This is the first unique non-mammalian MHC class I immune evasion gene identified, and suggests that α-herpesviruses have conserved this function for at least 100 million years.

Influenza virus causes widespread, yearly epidemics by accumulating surface protein mutations to escape neutralizing antibodies established from prior exposure. In contrast to antibody epitopes, T cell mediated immunity targets influenza epitopes that are more highly conserved and have potential for cross-protection. The extent of T cell cross-reactivity between a diverse array of contemporary and historical influenza strains was investigated in ferrets challenged with 2009 pandemic H1N1 influenza or the seasonal H3N2 strain, A/Perth/16/2009. Post-challenge cell-mediated immune responses demonstrated extensive cross-reactivity with a wide variety of contemporary and historical influenza A strains as well as influenza B. Responses in peripheral blood were undetectable by 36d post-challenge, but cross-reactivity persisted in spleen. The strongest responses targeted peptides from the NP protein and demonstrated cross-reactivity in both the CD4+ and CD8+ T cell populations. Cross-reactive CD4+ T cells also targeted HA and NA epitopes, while cross-reactive CD8+ T cells targeted internal M1, NS2, and PA. T cell epitopes demonstrated extensive cross-reactivity between diverse influenza strains in outbred animals, with NP implicated as a significant antigenic target demonstrating extensive cross-reactivity for both CD4+ and CD8+ T cells.

PB1-F2 is an accessory protein of most human, avian, swine, equine, and canine influenza A viruses (IAVs). Although it is dispensable for virus replication and growth, it plays significant roles in pathogenesis by interfering with the host innate immune response, inducing death in immune and epithelial cells, altering inflammatory responses, and promoting secondary bacterial pneumonia. The effects of PB1-F2 differ between virus strains and host species. This can at least partially be explained by the presence of multiple PB1-F2 sequence variants, including premature stop codons that lead to the expression of truncated PB1-F2 proteins of different lengths and specific virulence-associated residues that enhance susceptibility to bacterial superinfection. Although there has been a tendency for human seasonal IAV to gradually reduce the number of virulence-associated residues, zoonotic IAVs contain a reservoir of PB1-F2 proteins with full length, virulence-associated sequences. Here, we review the molecular mechanisms by which PB1-F2 may affect influenza virulence, and factors associated with the evolution and selection of this protein.

Avian influenza viruses of the H7 hemagglutinin (HA) subtype present a significant public health threat, as evidenced by the ongoing outbreak of human A(H7N9) infections in China. When evaluated by hemagglutination inhibition (HI) and microneutralization (MN) assays, H7 viruses and vaccines are found to induce lower level of neutralizing antibodies (nAb) than do their seasonal counterparts, making it difficult to develop and evaluate prepandemic vaccines. We have previously shown that purified recombinant H7 HA appear to be poorly immunogenic in that they induce low levels of HI and MN antibodies. In this study, we immunized mice with whole inactivated reverse genetics reassortant (RG) viruses expressing HA and neuraminidase (NA) from 3 different H7 viruses [A/Shanghai/2/2013(H7N9), A/Netherlands/219/2003(H7N7), and A/New York/107/2003(H7N2)] or with human A(H1N1)pdm09 (A/California/07/2009-like) or A(H3N2) (A/Perth16/2009) viruses. Mice produced equivalent titers of antibodies to all viruses as measured by enzyme-linked immunosorbent assay (ELISA). However, the antibody titers induced by H7 viruses were significantly lower when measured by HI and MN assays. Despite inducing very low levels of nAb, H7 vaccines conferred complete protection against homologous virus challenge in mice, and the serum antibodies directed against the HA head region were capable of mediating protection. The apparently low immunogenicity associated with H7 viruses and vaccines may be at least partly related to measuring antibody titers with the traditional HI and MN assays, which may not provide a true measure of protective immunity associated with H7 immunization. This study underscores the need for development of additional correlates of protection for prepandemic vaccines. IMPORTANCE H7 avian influenza viruses present a serious risk to human health. Preparedness efforts include development of prepandemic vaccines. For seasonal influenza viruses, protection is correlated with antibody titers measured by hemagglutination inhibition (HI) and virus microneutralization (MN) assays. Since H7 vaccines typically induce low titers in HI and MN assays, they have been considered to be poorly immunogenic. We show that in mice H7 whole inactivated virus vaccines (WIVs) were as immunogenic as seasonal WIVs, as they induced similar levels of overall serum antibodies. However, a larger fraction of the antibodies induced by H7 WIV was nonneutralizing in vitro . Nevertheless, the H7 WIV completely protected mice against homologous viral challenge, and antibodies directed against the HA head were the major contributor toward immune protection. Vaccines against H7 avian influenza viruses may be more effective than HI and virus neutralization assays suggest, and such vaccines may need other methods for evaluation.

The emergence of A(H7N9) virus strains with resistance to neuraminidase (NA) inhibitors highlights a critical need to discover new countermeasures for treatment of A(H7N9) virus-infected patients. We previously described an anti-NA mAb (3c10-3) that has prophylactic and therapeutic efficacy in mice lethally challenged with A(H7N9) virus when delivered intraperitoneally (i.p.). Here we show that intrananasal (i.n.) administration of 3c10-3 protects 100% of mice from mortality when treated 24 h post-challenge and further characterize the protective efficacy of 3c10-3 using a nonlethal A(H7N9) challenge model. Administration of 3c10-3 i.p. 24 h prior to challenge resulted in a significant decrease in viral lung titers and deep sequencing analysis indicated that treatment did not consistently select for viral variants in NA. Furthermore, prophylactic administration of 3c10-3 did not inhibit the development of protective immunity to subsequent homologous virus re-challenge. Taken together, 3c10-3 highlights the potential use of anti-NA mAb to mitigate influenza virus infection.

We infected mice with the 2009 influenza A pandemic virus (H1N1pdm09), boosted with an inactivated vaccine, and cloned immunoglobulins (Igs) from HA-specific B cells. Based on the redundancy in germline gene utilization, we inferred that between 72–130 unique IgH VDJ and 35 different IgL VJ combinations comprised the anti-HA recall response. The IgH VH1 and IgL VK14 variable gene families were employed most frequently. A representative panel of antibodies were cloned and expressed to confirm reactivity with H1N1pdm09 HA. The majority of the recombinant antibodies were of high avidity and capable of inhibiting H1N1pdm09 hemagglutination. Three of these antibodies were subtype-specific cross-reactive, binding to the HA of A/South Carolina/1/1918(H1N1), and one further reacted with A/swine/Iowa/15/1930(H1N1). These results help to define the genetic diversity of the influenza anti-HA antibody repertoire profile induced following infection and vaccination, which may facilitate the development of influenza vaccines that are more protective and broadly neutralizing. Importance: Protection against influenza viruses is mediated mainly by antibodies, and in most cases this antibody response is narrow, only providing protection against closely related viruses. In spite of this limited range of protection, recent findings indicate that individuals immune to one influenza virus may contain antibodies (generally a minority of the overall response) that are more broadly reactive. These findings have raised the possibility that influenza vaccines could induce a more broadly protective response, reducing the need for frequent vaccine strain changes. However, interpretation of these observations is hampered by the lack of quantitative characterization of the antibody repertoire. In this study, we used single-cell cloning of influenza HA-specific B cells to assess the diversity and nature of the antibody response to influenza hemagglutinin in mice. Our findings help to put bounds on the diversity of the anti-hemagglutinin antibody response, as well as characterizing the cross-reactivity, affinity, and molecular nature of the antibody response.

The association of seasonal trivalent influenza vaccine (TIV) with increased infection by 2009 pandemic H1N1 (A(H1N1)pdm09) virus, initially observed in Canada, has elicited numerous investigations on the possibility of vaccine-associated enhanced disease, but the potential mechanisms remain largely unresolved. Here, we investigated if prior immunization with TIV enhanced disease upon A(H1N1)pdm09 infection in mice. We found that A(H1N1)pdm09 infection in TIV-immunized mice did not enhance the disease, as measured by morbidity and mortality. Instead, TIV-immunized mice cleared A(H1N1)pdm09 virus and recovered at an accelerated rate compared to control mice. Prior TIV immunization was associated with potent inflammatory mediators and virus-specific CD8 T cell activation, but efficient immune regulation, partially mediated by IL-10R-signaling, prevented enhanced disease. Furthermore, in contrast to suggested pathological roles, pre-existing non-neutralizing antibodies (NNAbs) were not associated with enhanced virus replication, but rather with promoted antigen presentation through FcR-bearing cells that led to potent activation of virus-specific CD8 T cells. These findings provide new insights into interactions between pre-existing immunity and pandemic viruses.

Since the emergence of human H3N2 influenza A viruses in the pandemic of 1968, these viruses have become established as strains of moderate severity. A decline in virulence has been accompanied by glycan accumulation on the hemagglutinin globular head, and hemagglutinin receptor binding has changed from recognition of a broad spectrum of glycan receptors to a narrower spectrum. The relationship between increased glycosylation, binding changes, and reduction in H3N2 virulence is not clear. We evaluated the effect of hemagglutinin glycosylation on receptor binding and virulence of engineered H3N2 viruses. We demonstrate that low-binding virus is as virulent as higher binding counterparts, suggesting that H3N2 infection does not require either recognition of a wide variety of, or high avidity binding to, receptors. Among the few glycans recognized with low-binding virus, there were two structures that were bound by the vast majority of H3N2 viruses isolated between 1968 and 2012. We suggest that these two structures support physiologically relevant binding of H3N2 hemagglutinin and that this physiologically relevant binding has not changed since the 1968 pandemic. Therefore binding changes did not contribute to reduced severity of seasonal H3N2 viruses. This work will help direct the search for factors enhancing influenza virulence.

We used an improved ADCC assay to investigate HA-ADCC antibody responses in human sera following either seasonal or avian influenza vaccination. Our results suggest that detection of both neutralizing and non-neutralizing antibodies may better reflect protective capacity of HA-specific antibodies induced by avian influenza vaccines., Background. Detection of neutralizing antibodies (nAbs) to influenza A virus hemagglutinin (HA) antigens by conventional serological assays is currently the main immune correlate of protection for influenza vaccines However, current prepandemic avian influenza vaccines are poorly immunogenic in inducing nAbs despite considerable protection conferred. Recent studies show that Ab-dependent cell-mediated cytotoxicity (ADCC) to HA antigens are readily detectable in the sera of healthy individuals and patients with influenza infection. Methods. Virus neutralization and ADCC activities of serum samples from individuals who received either seasonal or a stock-piled H5N1 avian influenza vaccine were evaluated by hemagglutination inhibition assay, microneutralization assay, and an improved ADCC natural killer (NK) cell activation assay. Results. Immunization with inactivated seasonal influenza vaccine led to strong expansion of both nAbs and ADCC-mediating antibodies (adccAbs) to H3 antigen of the vaccine virus in 24 postvaccination human sera. In sharp contrast, 18 individuals vaccinated with the adjuvanted H5N1 avian influenza vaccine mounted H5-specific antibodies with strong ADCC activities despite moderate virus neutralization capacity. Strength of HA-specific ADCC activities is largely associated with the titers of HA-binding antibodies and not with the fine antigenic specificity of anti-HA nAbs. Conclusions. Detection of both nAbs and adccAbs may better reflect protective capacity of HA-specific antibodies induced by avian influenza vaccines.

We report on the genetic analysis of 213 highly pathogenic avian influenza (HPAI) H5N1 viruses isolated from poultry in Vietnam between 2007 and 2010. Phylogenetic analyses of the viral genomes revealed 38 distinct viral genotypes, 29 were novel and 9 were reported in Vietnam or neighboring countries in recent years. Viruses from only six genotypes persisted beyond one season or year. Thus, most reassortant viruses were transient, suggesting that such genotypes lacked significant fitness advantages. Viruses with clade 2.3.2.1 HA were re-introduced into Vietnam in 2009 and their prevalence rose steeply towards the end of 2010. Clade 2.3.4-like viruses (genotype V) were predominant in northern Vietnam and caused the majority of zoonotic infections, whereas clade 1.1 (genotype Z) viruses were only detected in the Mekong delta region, in southern Vietnam. Antigenic analysis of representative viruses from the four clades indicated substantial drift.

Influenza A virus reservoirs in animals have provided novel genetic elements leading to the emergence of global pandemics in humans. Most influenza A viruses circulate in waterfowl, but those that infect mammalian hosts are thought to pose the greatest risk for zoonotic spread to humans and the generation of pandemic or panzootic viruses. We have identified an influenza A virus from little yellow-shouldered bats captured at two locations in Guatemala. It is significantly divergent from known influenza A viruses. The HA of the bat virus was estimated to have diverged at roughly the same time as the known subtypes of HA and was designated as H17. The neuraminidase (NA) gene is highly divergent from all known influenza NAs, and the internal genes from the bat virus diverged from those of known influenza A viruses before the estimated divergence of the known influenza A internal gene lineages. Attempts to propagate this virus in cell cultures and chicken embryos were unsuccessful, suggesting distinct requirements compared with known influenza viruses. Despite its divergence from known influenza A viruses, the bat virus is compatible for genetic exchange with human influenza viruses in human cells, suggesting the potential capability for reassortment and contributions to new pandemic or panzootic influenza A viruses.

The importance of immunoproteasomes to antigen presentation has been unclear because animals totally lacking immunoproteasomes had not been available. Having now developed mice lacking the three immunoproteasome catalytic subunits, we found that the dendritic cells of these mice had defects in presenting several major histocompatibility complex (MHC) class I epitopes. During viral infection in vivo, the presentation of a majority of MHC class I epitopes was markedly reduced in immunoproteasome-deficient animals compared with wild-type animals, whereas presentation of MHC class II peptides was unaffected. According to mass spectrometry, the repertoire of MHC class I-presented peptides was ∼50% different from that in wild-type mice, and these differences were sufficient to stimulate robust transplant rejection of wild-type cells in mutant mice. These results indicated that immunoproteasomes were more important in antigen presentation than previously thought.

ER aminopeptidase 1 (ERAP1) customizes antigenic peptide precursors for MHC class I presentation and edits the antigenic peptide repertoire. Coding single nucleotide polymorphisms (SNPs) in ERAP1 were recently linked with predisposition to autoimmune disease, suggesting a link between pathogenesis of autoimmunity and ERAP1-mediated Ag processing. To investigate this possibility, we analyzed the effect that disease-linked SNPs have on Ag processing by ERAP1 in vitro. Michaelis–Menten analysis revealed that the presence of SNPs affects the Michaelis constant and turnover number of the enzyme. Strikingly, specific ERAP1 allele-substrate combinations deviate from standard Michaelis–Menten behavior, demonstrating substrate-inhibition kinetics; to our knowledge, this phenomenon has not been described for this enzyme. Cell-based Ag-presentation analysis was consistent with changes in the substrate inhibition constant Ki, further supporting that ERAP1 allelic composition may affect Ag processing in vivo. We propose that these phenomena should be taken into account when evaluating the possible link between Ag processing and autoimmunity.

All three members of the oxytocinase subfamily of M1 aminopeptidases, endoplasmic reticulum aminopeptidase 1 (ERAP1), ERAP2, and placental leucine aminopeptidase (PLAP), also known as insulin-regulated aminopeptidase, have been implicated in the generation of MHC class I-presented peptides. ERAP1 and 2 trim peptides in the endoplasmic reticulum for direct presentation, whereas PLAP has been recently implicated in cross-presentation. The best characterized member of the family, ERAP1, has unique enzymatic properties that fit well with its role in Ag processing. ERAP1 can trim a large variety of long peptide sequences and efficiently accumulate mature antigenic epitopes of 8–9 aa long. In this study, we evaluate the ability of PLAP to process antigenic peptide precursors in vitro and compare it with ERAP1. We find that, similar to ERAP1, PLAP can trim a variety of long peptide sequences efficiently and, in most cases, accumulates appreciable amounts of correct length mature antigenic epitope. Again, similar to ERAP1, PLAP continued trimming some of the epitopes tested and accumulated smaller products effectively destroying the epitope. However, the intermediate accumulation properties of ERAP1 and PLAP are distinct and epitope dependent, suggesting that these two enzymes may impose different selective pressures on epitope generation. Overall, although PLAP has the necessary enzymatic properties to participate in generating or destroying MHC class I-presented peptides, its trimming behavior is distinct from that of ERAP1, something that supports a separate role for these two enzymes in Ag processing.

The human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) potently restrict human immunodeficiency virus type 1 (HIV-1) replication, but they are neutralized by the viral protein Vif. Vif bridges A3G and A3F with a Cullin 5 (Cul5)-based E3 ubiquitin ligase and mediates their proteasomal degradation. This mechanism has been extensively studied, and several Vif domains have been identified that are critical for A3G and A3F neutralization. Here, we identified two additional domains. Via sequence analysis of more than 2,000 different HIV-1 Vif proteins, we identified two highly conserved amino acid sequences, 81 LGxGxSIEW 89 and 171 EDRWN 175 . Within the 81 LGxGxSIEW 89 sequence, residues L81, G82, G84, and, to a lesser extent, I87 and W89 play very critical roles in A3G/A3F neutralization. In particular, residues L81 and G82 determine Vif binding to A3F, residue G84 determines Vif binding to both A3G and A3F, and residues 86 SIEW 89 affect Vif binding to A3F, A3G, and Cul5. Accordingly, this 81 LGxGxSIEW 89 sequence was designated the 81 LGxGxxIxW 89 domain. Within the 171 EDRWN 175 sequence, all residues except N175 are almost equally important for regulation of A3F neutralization, and consistently, they determine Vif binding only to A3F. Accordingly, this domain was designated 171 EDRW 174 . The LGxGxxIxW domain is also partially conserved in simian immunodeficiency virus Vif from rhesus macaques (SIVmac239) and has a similar activity. Thus, 81 LGxGxxIxW 89 and 171 EDRW 174 are two novel functional domains that are very critical for Vif function. They could become new targets for inhibition of Vif activity during HIV replication.

Many MHC class I-binding peptides are generated as N-extended precursors during protein degradation by the proteasome. These peptides can subsequently be trimmed by aminopeptidases in the cytosol and/or the endoplasmic reticulum (ER) to produce mature epitope. However, the contribution and specificity of each of these subcellular compartments in removing N-terminal amino acids for Ag presentation is not well defined. In this study, we investigated this issue for antigenic precursors that are expressed in the cytosol. By systematically varying the N-terminal flanking sequences of peptides, we show that the amino acids upstream of an epitope precursor are a major determinant of the amount of Ag presentation. In many cases, MHC class I-binding peptides are produced through sequential trimming in the cytosol and ER. Trimming of flanking residues in the cytosol contributes most to sequences that are poorly trimmed in the ER. Because N-terminal trimming has different specificity in the cytosol and ER, the cleavage of peptides in both of these compartments serves to broaden the repertoire of sequences that are presented.

Previous experiments using enzyme inhibitors and RNA interference in cell lysates and cultured cells have suggested that tripeptidyl peptidase II (TPPII) plays a role in creating and destroying MHC class I-presented peptides. However, its precise contribution to these processes has been controversial. To elucidate the importance of TPPII in MHC class I Ag presentation, we analyzed TPPII-deficient gene-trapped mice and cell lines from these animals. In these mice, the expression level of TPPII was reduced by >90% compared with wild-type mice. Thymocytes from TPPII gene-trapped mice displayed more MHC class I on the cell surface, suggesting that TPPII normally limits Ag presentation by destroying peptides overall. TPPII gene-trapped mice responded as well as did wild-type mice to four epitopes from lymphocytic choriomeningitis virus. The processing and presentation of peptide precursors with long N-terminal extensions in TPPII gene-trapped embryonic fibroblasts was modestly reduced, but in vivo immunization with recombinant lentiviral or vaccinia virus vectors revealed that such peptide precursors induced an equivalent CD8 T cell response in wild-type and TPPII-deficient mice. These data indicate that while TPPII contributes to the trimming of peptides with very long N-terminal extensions, TPPII is not essential for generating most MHC class I-presented peptides or for stimulating CTL responses to several Ags in vivo.

Aminopeptidases in the endoplasmic reticulum (ER) can cleave antigenic peptides and in so doing either create or destroy MHC class I-presented epitopes. However, the specificity of this trimming process overall and of the major ER aminopeptidase ERAP1 in particular is not well understood. This issue is important because peptide trimming influences the magnitude and specificity of CD8 T cell responses. By systematically varying the N-terminal flanking sequences of peptides in a cell-free biochemical system and in intact cells, we elucidated the specificity of ERAP1 and of ER trimming overall. ERAP1 can cleave after many amino acids on the N terminus of epitope precursors but does so at markedly different rates. The specificity seen with purified ERAP1 is similar to that observed for trimming and presentation of epitopes in the ER of intact cells. We define N-terminal sequences that are favorable or unfavorable for Ag presentation in ways that are independent from the epitopes core sequence. When databases of known presented peptides were analyzed, the residues that were preferred for the trimming of model peptide precursors were found to be overrepresented in N-terminal flanking sequences of epitopes generally. These data define key determinants in the specificity of Ag processing.

The function of lentiviral Vif proteins is to neutralize the host antiviral cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F). Vif bridges a cullin 5-based E3 ubiquitin ligase with A3G and A3F and mediates their degradation by proteasomes. Recent studies have found that Vif uses different domains to bind to A3G and A3F. A 14 DRMR 17 domain binds to A3F, 40 YRHHY 44 binds to A3G, and 69 YxxL 72 binds to both A3G and A3F. Here, we report another functional domain of Vif. Previously, we demonstrated that human immunodeficiency virus type 1 (HIV-1) Vif failed to mediate A3G proteasomal degradation when all 16 lysines were mutated to arginines. Here, we show that K26, and to a lesser extent K22, is critical for A3G neutralization. K22 and K26 are part of a conserved 21 WxSLVK 26 (x represents N, K, or H) motif that is found in most primate lentiviruses and that shows species-specific variation. Both K22 and K26 in this motif regulated Vif specificity only for A3G, whereas the SLV residues regulated Vif specificity for both A3F and A3G. Interestingly, SLV and K26 in HIV-1 Vif did not directly mediate Vif interaction with either A3G or A3F. Previously, other groups have reported an important role for W21 in A3F and A3G neutralization. Thus, 21 WxSLVK 26 is a novel functional domain that regulates Vif activity toward both A3F and A3G and is a potential drug target to inhibit Vif activity and block HIV-1 replication.

Avian H7N9 influenza virus infection with fatal outcomes continues to pose a pandemic threat and highly immunogenic vaccines are urgently needed. In this report we show that baculovirus-derived recombinant H7 hemagglutinin protein, when delivered with RIG-I ligand, induced enhanced antibody and T cell responses and conferred protection against lethal challenge with a homologous H7N9 virus. These findings indicate the potential utility of RIG-I ligands as vaccine adjuvants to increase the immunogenicity of recombinant H7 hemagglutinin.

Long oligopeptides (>10 residues) are generated during the catabolism of cellular proteins in the cytosol. To be presented to T cells, such peptides must be trimmed by aminopeptidases to the proper size (typically 8–10 residues) to stably bind to MHC class I molecules. Aminopeptidases also destroy epitopes by trimming them to even shorter lengths. Bleomycin hydrolase (BH) is a cytosolic aminopeptidase that has been suggested to play a key role in generating MHC class I-presented peptides. We show that BH-deficient cells from mice are unimpaired in their ability to present epitopes from N-extended precursors or whole Ags and express normal levels of MHC class I molecules. Similarly, BH-deficient mice develop normal CD8+ T cell responses to eight epitopes from three different viruses in vivo. Therefore, BH by itself is not essential for the generation or destruction of MHC class I peptides. In contrast, when BH−/− mice are crossed to mice lacking another cytosolic aminopeptidase, leucine aminopeptidase, the resulting BH−/−leucine aminopeptidase−/− progeny show a selective increase in CD8+ T cell responses to the gp276 epitope from lymphocytic choriomeningitis virus, whereas the ability to present and respond to several other epitopes is unchanged. Therefore, BH does influence presentation of some Ags, although its role is largely redundant with other aminopeptidases.

Here we define the epitopes on HA that are targeted by a group of 9 recombinant monoclonal antibodies (rmAbs) isolated from memory B cells of mice, immunized by infection with A(H1N1)pdm09 virus followed by a seasonal TIV boost. These rmAbs were all reactive against the HA1 region of HA, but display 7 distinct binding footprints, targeting each of the 4 known antigenic sites. Although the rmAbs were not broadly cross-reactive, a group showed subtype-specific cross-reactivity with the HA of A/South Carolina/1/18. Screening these rmAbs with a panel of human A(H1N1)pdm09 virus isolates indicated that naturally-occurring changes in HA could reduce rmAb binding, HI activity, and/or virus neutralization activity by rmAb, without showing changes in recognition by polyclonal antiserum. In some instances, virus neutralization was lost while both ELISA binding and HI activity were retained, demonstrating a discordance between the two serological assays traditionally used to detect antigenic drift.

CD8+T cells respond to short peptides bound to MHC class I molecules. Although most antigenic proteins contain many sequences that could bind to MHC class I, few of these peptides actually stimulate CD8+T cell responses. Moreover, the T cell responses that are generated often follow a very reproducible hierarchy to different peptides for reasons that are poorly understood. We find that the loss of a single enzyme, endoplasmic reticulum aminopeptidase 1 (ERAP1), in the antigen-processing pathway results in a marked shift in the hierarchy of immunodominance in viral infections, even when the responding T cells have the same T cell receptor repertoire. In mice, ERAP1 is the major enzyme that trims precursor peptides in the endoplasmic reticulum and, in this process, can generate or destroy antigenic peptides. Consequently, when ERAP1 is lost, the immune response to some viral peptides is reduced, to others increased, and to yet others unchanged. Therefore, many epitopes must be initially generated as precursors that are normally trimmed by ERAP1 before binding to MHC class I, whereas others are normally degraded by ERAP1 to lengths that are too short to bind to MHC class I. Moreover, peptide trimming and the resulting abundance of peptide–MHC complexes are dominant factors in establishing immunodominance.

Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) appears to be specialized to produce peptides presented on class I major histocompatibility complex molecules. We found that purified ERAP1 trimmed peptides that were ten residues or longer, but spared eight-residue peptides. In vivo, ERAP1 enhanced production of an eight-residue ovalbumin epitope from precursors extended on the NH2 terminus that were generated either in the ER or cytosol. Purified ERAP1 also trimmed nearly half the nine-residue peptides tested. By destroying such nine-residue peptides in normal human cells, ERAP1 reduced the overall supply of antigenic peptides. However, after interferon-γ treatment, which causes proteasomes to produce more NH2-extended antigenic precursors, ERAP1 increased the supply of peptides for MHC class I antigen presentation.

Precursors to major histocompatibility complex (MHC) class I-presented peptides with extra NH2-terminal residues can be efficiently trimmed to mature epitopes in the endoplasmic reticulum (ER). Here, we purified from liver microsomes a lumenal, soluble aminopeptidase that removes NH2-terminal residues from many antigenic precursors. It was identified as a metallopeptidase named "adipocyte-derived leucine" or "puromycin-insensitive leucine-specific" aminopeptidase. However, because we localized it to the ER, we propose it be renamed ER-aminopeptidase 1 (ERAP1). ERAP1 is inhibited by agents that block precursor trimming in ER vesicles and although it trimmed NH2-extended precursors, it spared presented peptides of 8 amino acid and less. Like other proteins involved in antigen presentation, ERAP1 is induced by interferon-gamma. When overexpressed in vivo, we found that ERAP1 stimulates the processing and presentation of an antigenic precursor in the ER.

Mice are widely used for studying influenza virus pathogenesis and immunology because of their low cost, the wide availability of mouse-specific reagents, and the large number of mouse strains available, including knockout and transgenic strains. However, mice do not fully recapitulate the signs of influenza infection of humans: transmission of influenza between mice is much less efficient than in humans, and influenza viruses often require adaptation before they are able to efficiently replicate in mice. In the process of mouse adaptation, influenza viruses acquire mutations that enhance their ability to attach to mouse cells, replicate within the cells, and suppress immunity, among other functions. Many such mouse-adaptive mutations have been identified, covering all 8 genomic segments of the virus. Identification and analysis of these mutations have provided insight into the molecular determinants of influenza virulence and pathogenesis, not only in mice but also in humans and other species. In particular, several mouse-adaptive mutations of avian influenza viruses have proved to be general mammalian-adaptive changes that are potential markers of pre-pandemic viruses. As well as evaluating influenza pathogenesis, mice have also been used as models for evaluation of novel vaccines and anti-viral therapies. Mice can be a useful animal model for studying influenza biology as long as differences between human and mice infections are taken into account.